72 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(4):72-73 case report chondroid syringoma of the scalp: a rare skin adnexal tumor kuladeepa ananda vaidya 1 , raghunath prabhu 2 *, neha kumar 3 , sakshi sadhu 2 1 department of pathology, srinivas institute of medical sciences and research center, mukka ,surathkal, mangalore, india 2 dpartment of surgery, kasturba medical college, manipal, manipal university, india 3 foundation year 2, university hospital of north staffordshire, london, united kingdom abstract. chondroid syringoma is a rare skin adnexal tumor constituting 0.01% to 0.1% of all primary skin tumors. herein we report a case of benign chondroid syringoma in a 47-year-old male patient presenting with an asymptomatic, solitary swelling on the scalp. fine needle aspiration cytology of the lesion suggested a possibility of skin adnexal tumor. wide excision of the tumor was performed and histopathology revealed components of both epithelial and mesenchymal origin resembling pleomorphic adenoma of salivary gland. keywords: chondroid syringoma, skin, adnexal tumour, benign introduction chondroid synringomas (css) are mixed tumors of the skin, having both epithelial and mesenchymal constituents [1]. hirsch and helwig gave them the appellation chondroid syringoma, because of the presence of sweat gland elements which are set in a cartilaginous stroma. it is a rare primary skin tumor; the incidence is < 0.01% and affects middle aged and older men [2] case report a 47-year-old man presented with a skin colored nodular swelling on the scalp measuring around 3x2cm in size. it was present since 3 years and was gradually increasing in its size. physical examination revealed a palpable swelling in the right frontal aspect of the scalp in subcutaneous plane. the swelling was non-tender and was not associated with any other skin lesions with the same features. a provisional clinical diagnosis was made of a dermoid cyst. fine needle aspiration cytological examination revealed a possibility of a benign skin adnexal tumor. he underwent an excisional biopsy of the swelling. grossly tumor was unencapsulated measuring around 2.5x2.5x1.5cm, grey white to grey brown in color with focal cystic space filled with mucoid material [fig. 1]. formalin fixed paraffin embedded routine hematoxylin and eosin sections revealed a circumscribed tumor tissue in dermis, with tumor cells arranged in small nests, cords and tubules within a dense hyalinised stroma with focal chondromyxoid changes. cells were round to ovoid with small nuclei, inconspicuous nucleoli and eosinophilic to clear cytoplasm [figs. 2 and 3]. few large cysts showed eosinophilic material and cholesterol clefts. there was no evidence of atypical mitosis or necrosis. since microscopically there was no feature of malignant changes, final histopathological impression of benign chondroid syringoma was imparted. the postoperative course was uneventful. at the last follow-up visit 6 months after the surgery, there were no signs of recurrence. discussion css are quite rare skin tumors and account for only about 0.01% to 0.01% of primary adnexal tumors [3]. css are usually considered to be benign, but they may recur post resection and some malignant cases with metastases have been reported [4]. css are solitary asymptomatic insidiously growing nodules on the head and neck, with a higher affinity for males than females (ratio about 2-3:1). most clinicians find it hard to classify these lesions due to the asymptomatic nature and rare presentations. headington put forward the idea of two types of these lesions– apocrine (irregular branching tubules lined by epithelium) and eccrine (uniform, small tubules within a myxoid-chondroid matrix) [5]. fine needle aspiration cytology (fnac) is used for diagnostic purposes and may prove to be useful to determine the pathology before excision; however, examination of the excised tissue is most reliable in establishing a definitive diagnosis [2]. the apocrine css may show a variety of different adnexal differentiation (including follicular and sebaceous), while the eccrine css lack these [5]. extensive research has previously suggested an eccrine origin for these ___________________________________________________________ * corresponding author: raghunath prabhu, md (drraghu81@yahoo.co.in). mailto:drraghu81@yahoo.co.in 73 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org fig 1: (a) tumor was arising from skin measuring around 2.5x2.5x1.5cm, grey white to grey brown in colour with focal cystic spaces filled with mucoid material. (b) tumor cells seen arranged in small nests, cords and tubules within a dense hyalinised stroma. (c) nonbranching ducts and tubules set in chondromyxoid stroma. tumors however after much controversy within this area, they are now thought to be apocrine [6]. mills suggested that these mixed tumors are monoclonal neoplasms with replicating cells can differentiate into epithelial or mesenchymal entities, thereby accounting for the histological variability of these tumors [7]. hirsch and helwig described five histological criteria that make up this diagnosis: collections of cuboidal or polygonal cells, tubuloalveolar structures lined with cuboidal cells, ductal structures with rows of cuboidal cells, some keratinous cysts and a matrix of varying components [1]. the treatment modality of choice for benign css is surgical excision. fnac is useful for diagnosis but examination of the excised tissue is the best way to deduce a definitive diagnosis [8]. it is important to include a margin of the tumor during excision to reduce chances of recurrence. although some local recurrences have been reported, largely patients make an uneventful recovery like our case. conclusion chondroid syringoma is a rare and usually benign subcutaneous tumor made up of mesenchymal and epithelial elements. it is usually suspected in middle-aged men presenting with lesions in the head and neck regions. the malignant form is usually seen in women, in the extremities or trunk. magnetic resonance imaging can help identify the extent of the tumor but an fnac is the intervention of choice for diagnosis. excision is the best modality of management, with tissue biopsy then giving a clearer picture of the exact histological nature of the lesion. conflict of interest the authors declare no conflicts of interest. references 1. hirsch p, helwig eb. chondroid syringoma. mixed tumor of skin, salivary gland type. arch dermatol 84:835847, 1961. 2. bhasin t s, mannan r, bhatia p k, sharma m, bhalla a. fine needle aspiration cytology diagnosis of the eccrine variant of chondroid syringomacase report of a rare entity with review of literature. j clin diagn res 4:2641-2644, 2010. 3. bekerecioglu m, tercan m, karakok m et al. benign chondroid syringoma: a confusing clinical diagnosis. eur j plast surg 25:316-318, 2002. 4. tsoitis g, papdimitriou c, kanitakis j et al. malignant cutaneous mixed tumor. am j dermatopathol 22:347, 2000. 5. headington jt. mixed tumors of skin: eccrine and apocrine types. arch dermatol 84:989-996, 1961. 6. mandeville jt, roh jh, woog jj et al. cutaneous benign mixed tumor (chondroid syringoma) of the eyelid: clinical presentation and management. ophthal plast reconstr surg 20:110-116, 2004. 7. mills se. mixed tumor of the skin: a model of divergent differentiation. j cutan pathol 11:382-386, 1984. 8. agrawal v, gupta rl, kumar s et al. malignant chondroid syringoma. j dermatol 25:547-549, 1998. a b c 87 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(1):87-88 case report amyand’s hernia while repairing the bilateral inguinal hernia arif aslaner*, tuğrul çakır, umut rıza gündüz, burhan mayir, nurullah bülbüller department of general surgery, antalya training and research hospital, antalya, turkey abstract. amyand's hernia is the term used for inguinal hernia containing appendix. it is a rare condition and found in 1% of inguinal hernia repairs. here we report a case of amyand's hernia in a 61 years old male who was diagnosed with bilateral inguinal hernia. he underwent surgery and bilateral inguinal hernia repair with prosthetic meshes and without appendectomy. the patient was discharged uneventfully. keywords: amyand’s hernia, inguinal hernia, bilateral, repair introduction the presence of the appendix vermiformis inside an inguinal hernia sac was first described in 1736 by claudius amyand [1]. it has an incidence of 1% [2] and is complicated by acute appendicitis in 0.10 % of cases [3]. inguinal hernia repair is one of the most common surgical procedures of general surgery and we may encounter some rare situations such as the appendix vermiformis inside the hernia sac. appendectomy should or should not be performed at the same time with hernia repair. here we report a case of amyand's hernia occurring in a 61 years old man, who presented with bilateral inguinal hernia. case report a 61 years old male was admitted to our outpatient clinic with bilateral pain and swelling on both groins. he had operation for left inguinal hernia 6 years ago. on physical examination of inguinal region, there was bilateral reducible mass without scrotal involvement. a diagnosis was made as bilateral inguinal hernia with the left side recurrence. he underwent surgery and first the left recurrent inguinal hernia side was approached with a 5 cm left side old oblique incision parallel to the inguinal ligament. subcutaneous tissue through scarpa’s fascia was divided until aponeurotic fibers of the external oblique muscle were seen. after dividing the external oblique to the superficial inguinal ring, the contents of the inguinal canal were then circumscribed using blunt dissection. the hernia sac lateral to the inferior epigastric pedicle was dissected away from the spermatic cord to the deep inguinal ring. the hernia sac was opened, peritoneum was ligated and dissected then excessive hernia sac was excised. we performed a tension free repair with polypropylene mesh (7x10cm). next the right inguinal hernia side was approached in the same manner. the hernia sac was opened and the appendix vermiformis and distal ileum segments were seen inside (fig. 1). there were no inflammatory changes in the appendix, ileum or cecum. the hernia sac contents including appendix vermiformis and distal ileum segments were retracted into the peritoneal cavity. appendectomy was not performed. the peritoneum was ligated and dissected then excessive hernia sac was excised. a tension free repair with polypropylene mesh (7x10cm) was performed same as for the left side. the patient was discharged uneventfully at first day after operation. discussion this eponymous disease was named after claudius amyand (1680-1740) who performed the first successful appendectomy on an 11-year-old boy in 1735 [1]. the incidence of amyand’s hernia is less than 1% of inguinal hernias and male predominance with usually right side location is common [2]. as in our case, the involvement of appendix is seen mostly during operation incidentally. however, preoperative diagnosis can be made by ultrasonography, computerize tomography and magnetic resonance imaging. losanoff and basson classified amyand’s hernia to 4 types [4, 5]. in type 1, the inguinal hernia sac had a normal appendix inside, which was managed with a reduction and mesh repair [6]. in types 2, 3 and 4, there was appendicitis inside the hernia sac. appendix vermiformis was inflamed in type 2, perforated in type 3 and complicated in type 4. appendectomy and hernia repair without any synthetic ___________________________________________________________ * corresponding author: arif aslaner, md (arifaslaner@gmail.com). http://www.ajecr.org/ http://en.wikipedia.org/wiki/eponymous_disease http://en.wikipedia.org/wiki/appendectomy mailto:arifaslaner@gmail.com 88 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org figure 1 right spermatic cord, appendix vermiformis and distal ileum inside the hernia sac were seen. material such as polyprolene mesh was performed in types 2,3 and 4. our patient had bilateral inguinal hernia. left recurrent hernia and right inguinal hernia which had appendix vermiformis inside were both repaired tension free with polypropylene meshes. appendectomy was not performed. amyand’s hernia is a rare entity and hard to diagnose preoperatively. we conclude that type 1 amyand’s hernia can be repaired with polyprolene mesh without requiring any appendectomy. conflict of interest the authors declare no conflicts of interest. references 1. amyand c. of an inguinal rupture, with a pin in the appendix caeci, incrusted with stone; and some observations on wounds in the guts. philos trans r soc lond 39:329-336, 1736. 2. kaymakci a, akillioglu i, akkoyun i, guven s, ozdemir a, gulen s. amyand's hernia: a series of 30 cases in children. hernia 13:609-612, 2009. 3. gurer a, ozdogan m, ozlem n, yildirim a, kulacoglu h, aydin r. uncommon content in groin hernia sac. hernia 10:152-155, 2006. 4. losanoff je, basson md. amyand hernia: a classification to improve management. hernia 12:325-326, 2008. 5. losanoff je, basson md. amyand hernia: what lies beneath—a proposed classification scheme to determine management. am sur 73:1288-1290, 2007. 6. green j, gutwein lg. amyand's hernia: a rare inguinal hernia. j surg case rep 11:9, 2013. http://www.ajecr.org/ am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(1):81-82 brief review weekly missing dose (“6/7” ssu protocol): a rational approach in warfarin use sina owlia school of medicine, shahid sadoughi university of medical sciences (ssu), yazd, iran abstract. warfarin is still used as a standard drug for long term oral anticoagulation. we hypothesized that chronic warfarin use six days a week (“6/7” ssu protocol) is a safe and effective method in order to minimize the burden of frequent blood testing. our unpublished data indicated that weekly missing dose of warfarin a day per week could attain an ideal therapeutic goal with a need to less frequent blood samplings without the risk of warfarin toxicity (bleeding) or significant drop in therapeutic serum level. our rationale was rather a high half-life of warfarin (20-60 hours) with more than 97% protein bounding. so, disruption of daily oral prescription of warfarin by off-days (a day each week) can effectively halt the risk of bleeding without considerable impact on its anticoagulation effects. we hypothesized that due to unappreciated long elimination half-life, this mode of dosing (six days a week) could be more justified than the continuous daily oral prescription. this fact has been experienced for years regarding practice with digoxin (with 36-48 hours half-life). similarly this concept could be true for every drug with “more than a day” half-life like warfarin. keywords: warfarin, anticoagulation, weekly dose, “6/7” ssu protocol introduction thrombotic events are among the most frequent causes of morbidity and mortality worldwide even in young people [1]. abnormal tendency to coagulation and underlying comorbidities leading to hemostasis such as valvular or some conductive heart diseases or atraial fibrillation are the major indications for anticoagulation therapy. several agents are used to fight abnormal coagulation or block physiologic clotting processes. among them intravenous or subcutaneous heparin injections are the standard method of initial anticoagulation in most settings for decades [2]. however chronic heparin use has some burden such as heparin induced thrombocytopenia (hit) and negative bone mineral balance [3]. however except for pregnancy, long term use of heparin is never clinically indicated. instead, warfarin still is a standard drug for long term oral anticoagulation in most series. effect of warfarin on bone loss is a matter of debate [4]. the most dread complications of warfarin use are unsteady blood level and risk of fatal and non-fatal bleeding. however, the clinical advantage of this drug has been partially offset by its very high individual variability in the dosing in order to attain ideal results [5]. this variability in part is due to strong individual pharmacogenetics of warfarin. two major problems with clinical use of warfarin are warfarin resistance and the other, warfarin overdose. although the issue of warfarin resistance is sometimes a clinical problem, however, the thrombotic events secondary to under-therapeutic level of warfarin could be as fatal as warfarin overdose. albeit not so rare in clinical practice, there are limited data on the frequency of warfarin resistance due to diverse etiologies [6]. warfarin overdose on the other hand, may be secondary to drug intake or interaction with other drugs. to prevent this, serial daily blood tests for inr is recommended especially during first days. considering life-long need for anticoagulation therapy in almost all cases and fluctuations in serum therapeutic level, the issue of exhausting serial blood sampling is also a big problem [7]. d showed that low-dose warfarin protocol (5 mg per day without loading dose) with infrequent blood testing is a safe and effective alternative in addition data show that the burden of blood testing is high, even in cases of rather good blood control to maintain a safe state [8]. this dilemma has led to searching new drugs (oral direct thrombin inhibitors such as ximelagatran or dabigatran) with better safety margin, however, the issue of cost and effectiveness and global availability are still their major disadvantages [9]. many clinicians try to minimize warfarin overdose/ adverse reactions using “low-dose protocols” with less frequency of blood samplings [8]. in this protocol, starting daily dose of 5 mg warfarin is followed by daily blood tests for inr during first days of treatment. several studies showed that warfarin induction with low oral doses could be safe even in the absence of concurrent heparin use [6]. ___________________________________________________________ * corresponding author: sina owlia (sinowlia@gmail.com). http://www.ajecr.org/ mailto:sinowlia@gmail.com am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org this approach may prevent paradoxical thrombosis as may be seen in high-dose loading protocols. as initial “low-dose” warfarin (without loading dose) has “anticoagulant” activity so it may boost the standard “anti-thrombotic” effects of chronic warfarin use [10]. this favorable event could be more amplified if prescription intervals could prevent warfarin overdose and bleeding. we hypothesized that chronic warfarin use six days a week (“6/7” ssu protocol) is a safe and effective method in order to minimize the burden of frequent blood testing. our unpublished data indicated that weekly missing doses of warfarin a day per week (after 3-5 times the half-life period) could attain an ideal therapeutic goal with a need to less frequent blood samplings without the risk of warfarin toxicity (bleeding) or significant drop in therapeutic serum level. our rationale was rather a high half-life of warfarin (2060 hours) with more than 97% protein bounding [11]. so, disruption of daily oral prescription of warfarin by off-days (a day each week away from warfarin) can effectively halt the risk of bleeding without considerable impact on its anticoagulation effects. we hypothesized that due to unappreciated long elimination half-life, this mode of dosing (six days a week) could be more justified than the continuous daily oral prescription. this fact has been experienced for years regarding practice with digoxin (with 36-48 hours half-life). considering the fact that continuous daily oral dosing is associated with a great risk of digoxin toxicity, many clinicians use under-dose protocol or offdays to prevent digoxin toxicity [12]. similarly this concept could be true for every drug with “more than a day” halflife like warfarin. acknowledgment we greatly appreciate dr. mona ghasemian (pharmacologist) for her useful comments. conflict of interest the author declares no conflicts of interest. references 1. montiel-manzano g, de la pena-diaz a, majluf-cruz a, et al. national evaluation of the diagnosis of activated protein c resistance. rev invest clin 55:358-369, 2003. 2. linhardt, r., heparin: an important drug enters its seventh decade. chemist indust 2:45-50, 1991. 3. nelson-piercy c. hazards of heparin: allergy, heparin-induced thrombocytopenia and osteoporosis. baillieres clin obstet gynaecol 11: 489-509, 1997. 4. rezaieyazdi z, et al. reduced bone density in patients on long-term warfarin. int j rheum dis 12:130-135, 2009. 5. takahashi h, echizen h. pharmacogenetics of warfarin elimination and its clinical implications. clin pharmacokinet 40:587-603, 2001. 6. osinbowale o, al malki m, schade a, bartholomew jr. an algorithm for managing warfarin resistance. cleve clin j med 76:724-730, 2009. 7. stewart s, murphy nf, walker a, mcguire a, mcmurray jj. cost of an emerging epidemic: an economic analysis of atrial fibrillation in the uk. heart 90:286-292, 2004. 8. harper p, monahan k, baker b. warfarin induction at 5 mg daily is safe with a low risk of anticoagulant overdose: results of an audit of patients with deep vein thrombosis commencing warfarin. intern med j 35:717-720, 2005. 9. pink j, pirmohamed m, lane s, hughes da. costeffectiveness of pharmacogene-tics-guided warfarin therapy vs. alternative anticoagulation in atrial fibrillation. clin pharmacol ther 95:199-207, 2014. 10. jaffer a, bragg l. practical tips for warfarin dosing and monitoring. cleve clin j med 70:361-371, 2003. 11. palareti g, legnani c. warfarin withdrawal. pharmacokinetic-pharmacodynamic considerations. clin pharmacokinet 30:300-313, 1996. 12. ahmed a, waagstein f. low-dose digoxin and reducti on in mortality and morbidity in heart failure. int j cardiol 136:91-92, 2009. http://www.ajecr.org/ am j exp clin res, vol. 1, no. 1, 2014 http://www.ajecr.org american journal of experimental and clinical research j exp clin res 2014;1(1):1 editorial american journal of experimental and clinical research it is my great pleasure to announce the launch of the american journal of experimental and clinical research [issn 2330-9237 (print) and issn 2330-9245 (online) http://www.ajecr.org]. we have worked tremendously in getting this journal ready to launch and are confident that the journal will rapidly progress during a short period of time to reach a highly competitive level. we welcome you to this new journal! the journal was founded by a group of scientists who are devoted to the advancement of experimental and clinical medical and dental research and their clinical applications. during the past decades, considerable progress has been made in basic and clinical medical researches with the ultimate aim of understanding and unraveling the mechanism(s) underlying the development of human diseases. these achievements have provided challenges in eliminating or alleviating the effects of human illnesses and thus helping to prolong the life spans of human being. it was only several decades ago that people frequently died from general diseases which nowadays are considered highly treatable. such diseases as influenza, tuberculosis or diarrhea no longer pose a significant mortality threat in contemporary society. thanks to the discovery of powerful vaccines and also increased public health education level to prevent contagious or communicable diseases. although other diseases such as heart diseases and cancers still account for primary cause of death, considerable improvements in the early diagnosis and treatment of such disorders have enabled increased survival and prolonged life of the patients. with the invention of new technologies, medications and procedures, the effectiveness of the treatment of previously lethal diseases has dramatically increased. the availability of ultrasound, computerized tomography, magnetic resonance imaging, positron emission tomography (pet) and so on that are less invasive techniques has also increased the effectiveness of early diagnosis and thus more effective therapy. all of these achievements have been brought about through extensive experimental and clinical researches. the impacts of experimental and clinical research on human life expectancy are undeniable and will be more pronounced with rapid advances in biomedical research. currently, cellular and molecular research scientists are rigorously engaged in the development of methodologies and pharmaceuticals that will eradicate human diseases. the american journal of experimental and clinical research provide a platform for these studies to be shared with the research community at large. the american journal of experimental and clinical research is an open-access, peer-reviewed online and print journal that encompasses all aspects of experimental and clinical research in the field of medicine and dentistry. the development of open-access online journals has created new opportunities and new challenges for publishers, authors, and readers. the american journal of experimental and clinical research aims to publish articles contributing to the development of basic and clinical medical and dental science researches and also encourages submissions considering the new methodologies to facilitate experimental approaches. the journal is at present a quarterly published journal that is dedicated to be a high quality platform to facilitate rapid publication and circulation of novel discoveries in all aspects of basic and clinical medical and dental researches. the american journal of experimental and clinical research welcomes original and review articles on both clinical investigation and basic medical research. there are also categories such as case reports, short communications, methods/techniques, letter to the editor/correspondences and editorials. we believe that the success and reputation of the american journal of experimental and clinical research depend on the quality of the articles published and therefore encourage you to submit your important research work to this journal. in addition, we believe that providing a fair and rapid peer-review and manuscript process to publication is of utmost importance to our authors. we also continuously improve our journal standing by publishing high standard articles. we would like to take this opportunity to thank our editorial board members, authors and editorial staff for their support and active participation in making this journal possible. in addition, we would like to invite all of our colleagues to consider the american journal of experimental and clinical research as a future venue for their research works. m. ghazizadeh, md, phd editor-in-chief american journal of experimental and clinical research http://www.ajecr.org http://www.ajecr.org/ http://www.ajecr.org/ 393 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2020;7(1):393-395 case report sjögren syndrome complicated by acute lymphoblastic leukemia fariba binesh1,2, mohammad bagher owlia3, mohammad reza vahidfar4, pegah kamal1, sara mirhosseini5 1department of pathology, shahid sadoughi university of medical sciences, yazd, iran 2hematology and oncology research center, shahid sadoughi university of medical sciences and health services, yazd, iran 3department of rheumatology, shahid sadoughi university of medical sciences, yazd, iran 4hematooncologist, yazd, iran 5 medical student, shahid sadoughi university of medical sciences, yazd, iran abstract. malignant lymphoma is a well-recognized occurrence in sjögren syndrome; however, progression to acute lymphoblastic leukemia is extremely uncommon. here we present a patient with sjögren syndrome complicated with acute lymphoblastic leukemia. a 41-year-old male, who was diagnosed as primary sjögren syndrome 3 months ago was admitted to our hospital due to hematuria and weakness. the complete blood count showed bicytopenia. bone marrow biopsy and aspiration cytology confirmed the diagnosis of acute lymphoblastic leukemia. he received combination chemotherapy with prednisolone, doxorubicin, cyclophosphamide, vincristine and intrathecal methotrexate. sjögren syndrome and other autoimmune diseases may build a portion of a continuum of lymphoproliferative disorders which have joined with b-cell neoplasms. keywords: sjögren syndrome, acute lymphoblastic leukemia, malignant lymphoproliferative disorder introduction sjögren’s syndrome (ss) is one of the most prevalent autoimmune disorders. its prevalence is between 0.21% and 0.72% [1]. cardiovascular diseases followed by malignancies and infections [2] display major reasons of death in patients with pss. there are case reports of some malignancies including solid organ neoplasms among patients with ps [3]. particularly, a raised risk for thyroid, lip, oral cavity, stomach, and ovarian cancer has been reported [4]. the increased occurrence of malignant lymphoma in primary pss was first described in 1963 by bunim and talal [5] and then has been ascertained by others [6]. nonhodgkin’s lymphomas (nhl) happen in about 2.7–9.8% of pss patients and the latter data revealed that nhl risk augments 2.2% per year of age with a 4.3-fold excess risk in pss in comparison with the other people [7]. albeit most cells permeating the salivary glands of patients with pss are t cells [8] the bulk of malignant lymphomas developed are of b-cell type. here, we report one case of pss complicated by acute lymphoblastic leukaemia (all). case presentation a 41-year-old male, a known case of ss, visited our hospital 3 months ago with complaints of epigastric pain, frequency and hematuria which he had had for the past one week. pain was radiating to the hypogastric area but was not associated with nausea and vomiting. the patient had experienced severe asthenia for the previous two weeks. in his past medical history there were xerostomia, xerophthalmia and swelling of the neck. that time ultrasound (us) studies had shown thyroid and bilateral submandibular gland enlargements. he was diagnosed with primary ss and was treated with oral prednisolone, hydroxychloroquine, methotrexate and oral care. at the time of new admission he looked ill and pale. a clinical examination revealed dry mouth, with no other functional signs. sternal tenderness was obvious. thyroid gland and both submandibular salivary glands were palpable and enlarged. there was no evidence of peripheral lymphadenopathy or hepatosplenomegaly. blood test showed wbc =1200/ mm3, hb =13.3 g/dl,plt= 20000/ mm3, urea =141mg/dl, cr = 2.8 mg/dl, p= 9.1mg/dl, ast=107 iu/l, alt=128 iu/l, ldh=3397u/l and k=4.3meq/l. a peripheral blood smear revealed no abnormal cell. other laboratory data were unremarkable. chest x-rays and abdominal us were all normal. renal us revealed normal kidney size with increased parenchychymal echogenicity but without hydronephrosis. considering bicytopenia and after oncologist consultation, bone marrow examination was done. histologic sections showed marrow interstitial ___________________________________________________________ * corresponding author: mohammad bagher owlia, md (bagherowlia@gmail.com) ). http://www.ajecr.org/ mailto:bagherowlia@gmail.com 394 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org figure 1. histologic sections show marrow interstitial and paratrabecular infiltration by immature lymphoid cells (hematoxylin-eosin stain; left panel x10 and right panel x40 magnification). figure 2. neoplastic lymphoid cells were positive for tdt, cd20 and cd10 and negative for cd3 (immunohistochemical stain; upper panels and lower left panel x10, lower right panel x40 magnification). and paratrabecular infiltration by immature lymphoid cells (fig. 1). ihc stains of the bone marrow revealed that neoplastic lymphoid cells were positive for tdt, cd20 and cd10 and negative for cd3 and cd 56 (fig. 2). the patient was diagnosed with all (b cell type) associated with ss. then he referred to oncology ward and received combination chemotherapy with prednisolone, doxorubicin, cyclophosphamide, vincristine and intra-thecal methotrexate. now, the patient is in good condition after chemotherapy and is going to have a bone marrow transplant. discussion the association of cancer with connective tissue diseases is well known. the oldest well-known association recorded was that of dermatomyositis with stomach adenocarcinoma [9]. but association with other malignancies has also been reported [10]. one third of the malignancies seen in pss are lymphomas. one research found amongst the 112 patients with pss, 25 suffered from malignancy (before or after pss), with malignant lymphoma developed in 11 cases. it is said that the severity of disease activity at the time of diagnosis is associated with the likelihood of http://www.ajecr.org/ 395 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org hematologic malignancies in pss [11]. pilar brito-zerón [12] got a raised risk for the development of thyroid and gi malignancies in these patients. in addition, they revealed that the patients with primary ss had an 11-fold higher risk of malignant hematological neoplasms than the general population [12]. although there have been numerous studies [9-12] of increased risk of malignancy in patients with pss, we have found just two cases of acute lymphoblastic leukemia in setting of pss [11, 13]. the third published patient could be the present case. despite these observations the cause is unknown. it is claimed that genetic changes such as mutation, amplification, deletion, chromosomal translocations and defects in dna repair may be causative. the impact of oncogenes and certain viruses should not be overlooked [14]. on the other hand, patients with pss are potentially subject to some degree of immunosuppression [15] therefore, these patients are rather susceptible to secondary malignancies such as hematologic neoplasms. the use of disease-modifying medication in autoimmune diseases may be associated with an increased risk of malignancy. due to factors not well known. it is probable that polyclonal b cell proliferation may be transformed to monoclonal ones and finally transform to malignant lymphoproliferative disease. however, this possibility should be interpreted with caution as the use of these drugs is limited to cases with severe illness. lazarus et al. did not find any significant differences in the use of immunomodulatory drugs consumed to treat severe forms of ss when comparing these patients with or without malignancy [11]. the question that arises here is whether or not sjögren syndrome is a paraneoplastic syndrome in our patient? short gap between onset of pss symptoms and lymphoblastic leukemia is in favor of paraneoplastic syndrome. another study suggested the possible paraneoplastic nature of ss [16]. it is likely that ss and other autoimmune diseases build portion of a continuum of lymph proliferative disorders which have joint genesis with b-cell neoplasms. in this regard, development of acute lymphoblastic leukemia may be the result of genesis of an eternal clone. however, the concordance of these two diseases is indicative of a possible causal relation. conclusion patients with pss should be closely monitored for the possibility of hematologic and solid malignancies. conflict of interest the authors declare no conflicts of interest. references 1. patel r and shahane a. the epidemiology of sjögren’s syndrome. clin epidemiol 6:247-255, 2014 2. chiu yh, chung ch, lin kt, lin cs, chen jh, chen hc, et al. predictable biomarkers of developing lymphoma in patients with sjögren syndrome: a nationwide population-based cohort study. oncotarget 8:50098-50108, 2017. 3. molad y, okon e, stark p, prokocimer m. sjö gren’s syndrome associated t cell large granular lymphocyte leukaemia: a possible common etiopathogenesis. j rheumatol 28:2551-2552, 2001. 4. brito-zerón p, kostov b, fraile g, caravia-durán d, maure b, rascón fj, et al. characterization and risk estimate of cancer in patients with primary sjögren syndrome. j hematol oncol 10:90, 2017. 5. bunim jj, talal n. development of malignant lymphoma in the course of sjögren’s syndrome. trans assoc am physicians 76:45-56, 1963. 6. valesini g, priori r, bavoillot d et al. differential risk of non-hodgkin’s lymphoma in italian patients with primary sjogren’s syndrome. j rheumatol 24:2376-2380, 1997. 7. alunno a, maria comasia leone, roberto giacomelli r, gerli r, carubbi f. lymphoma and lymphomagenesis in primary sjögren’s syndrome. front med 5:102, 2018. 8. nocturne g, mariette x. sjögren syndromeassociated lymphomas: an update on pathogenesis and management. br j haematol 168:317-327, 2015. 9. talbot jh. acute dermatomyositis-polymyositis and malignancy. semin arthritis rheum 6:305-360, 1997. 10. bernatsky s, boivin jf, jospeh l et al. an international cohort study of cancer in systemic lupus erythematosus. arthritis rheum 52:1481-1490, 2005. 11. m. n. lazarus, d. robinson1, v. mak1, h. mّller1 and d. a. isenberg, incidence of cancer in a cohort of patients with primary sjögren’s syndrome, rheumatology 45:1012-1015, 2006. 12. brito-zerón p, kostov b, fraile g, et al. characterization and risk estimate of cancer in patients with primary sjögren syndrome. j hematol oncol 10:90, 2017. 13. blanes a, villar a, amigo v, monzo e and gonzalez sal m. primary sjögren’s syndrome in men: evolution to acute lymphoblastic leukemia. ann med int 8:605-608, 1991. 14. i. s. lossos and d. morgensztern. prognostic biomarkers in diffuse large b-cell lymphoma. j clin oncol 24:995-1007, 2006. 15. kamel ow, van de rijn m, hanasono mm, warnke ra. immunosuppression-associated lymphoproliferative disorders in rheumatic patients. leuk lymphoma 16:363-368, 1995. 16. bartoloni e, alunno a and gerli r. primary sjögren’s syndrome as paraneoplastic disorder: a case report. clin exp rheumatol 30:454. 2012. http://www.ajecr.org/ 451 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2022;9(1):451-453 original article vitamin d and human journey shakiba m1*, shakiba sh2 1children growth disorder research center, shahid sadoughi university of medical sciences, yazd, iran 2medical pharmacist abstract. herein, we propose a hypothesis for the low level of vitamin d as a sign of untimely relocation of humans during history. this improper displacement made the human species prone to infectious and noninfectious diseases during the life journey. the low level of vitamin d is a sign that needs to be addressed as a marker of an unsafe journey in a lifetime, not the cause for associated diseases, and the replacement of vitamin d is the least performed in this regard. keywords: vitamin d deficiency, movement, vitamin d supplementation, indoor activity introduction life has begun to evolve in the oceans over 1 billion years ago. phytoplankton, as an earlier habitant of the earth, took advantage of sunlight and used it as an energy source to generate carbohydrates. in order to protect the important components of cells, such as deoxyribonucleic acid, ribonucleic acid, and amino acids with double bonds from the phototoxic wavelength of ultraviolet b (290 nm), in this tiny factory of photosynthesis, vitamin d was produced to act as a sunscreen [1]. on the other hand, the adequate level of this the hormone was a photochemical signal encouraging organisms to start a "safe journey" from the surface to the deep ocean in order to prevent excessive exposure to the phototoxic dosage of ultraviolet b (i.e., a signal for timely relocation and protection) [2]. vertebrates and vitamin d all vertebrates need vitamin d for movement and healthy skeleton and produce vitamin d through skin exposure to the sun or consumption of precursors from sea staff eating phytoplankton in their food chain or from plants [3]. this skeleton gives the ability to vertebrates that move freely and sometimes settle in places that are not optimal for vitamin d production. humans and vitamin d vitamin d has had a huge impact on the evolution of human skin color. by gradual movement toward higher latitude from the equatorial area, only females who had evolved to lighter skin were able to produce enough vitamin d and have normal pelvis anatomy to normally deliver a healthy neonate; females with a low level of vitamin d (probably dark skin with a low level of vitamin) were excluded from the reproductive cycle due to maternal complications [4]. this is the reason that the residents of northern latitudes have lighter skin color at present. skin pigmentation is determined by one favoring photoprotection close to the equator and another favoring vitamin d production closer to the pole. two other histories from human movement and vitamin d go back to the industrial revolution. in the 16th century, the majority of large cities in europe hosted people who migrated from rural to urban areas. this movement was accompanied by living in homes close to each other. however, coal consumption made cities heavily polluted. reports showed that more than 80 % of children in these cities had rickets [5]. in 1822, a century before hess’s discovery of sunlight exposure as a treatment of rickets (1923), sniadecki in warsaw, poland, said that a lack of sunlight was the reason for diseases taking a hundred years to be accepted [5]. in the 1980s, when tuberculosis was regarded as an incurable and noninfectious disease and killed more than 20% of individuals in britain, george bodington (1799-1882) had stated that "those, such as shepherds and farmers, working in rural areas did not usually get the disease". he advised individuals to follow the lifestyle of the individuals who were immune to the disease [6]. the forced movement of blacks is another history of human movement and vitamin d. the slave trade took blacks out of africa and settled them, among other places, across north america, canada, and northern europe, where for months of the year, sun rays stroke less directly, drastically reducing the amount of sunlight to those whose skin was not adapted and unable to make adequate vitamins d. at that time, medical reports described a disease that is ___________________________________________________________ *corresponding author: mehrdad shakiba (shakiba@ssu.ac.ir) http://www.ajecr.org/ mailto:shakiba@ssu.ac.ir 452 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org similar to hypocalcemia of vitamin d deficiency, which was called "negro diseases" observed in black slaves after a period of settling; with neurological symptoms and psychosis, they were also prone to many infectious diseases [7]. the reason was the untimely relocation and fast displacement of blacks from one continent to another continent that i would call "slavery diseases". through time, human adaptations to different solar beams have become more cultural than natural. rapid human migrations with the industrial revolution, increase in urbanization in modern life, and changes in lifestyle have created mismatches between skin pigmentation and environmental conditions [8]. although vitamin d deficiency was noticed more commonly in blacks and immigrants, it is not currently limited to them and includes all races of black, white, and yellow. moreover, it is not limited to one continent as it is pandemic. can vitamin d deficiency again be a sign of inappropriate human displacement from one place to another places? can vitamin d deficiency be an alarming signal for an unsafe mankind journey from outdoor to indoor? the evidence has shown that indoor activities and lack of sun exposure are the risks for premature deaths, such as obesity, smoking, and inactivity [9, 10]. it will be more important when we realize that during sun exposure, several other byproducts are produced in the skin, such as lumisterol and tachysterol, with antiproliferative and anti-inflammatory effects, and regulate epidermal growth. in other words, vitamin d production is only 15% of byproducts that are produced during solar exposure, and other photoproducts have other benefits which will turn out in the future [11]. the prescription of a vitamin d supplement to normalize 25-hydroxyl vitamin d is a very simplistic approach to the problem that has been occurred several times in history. during the last 30 years, the association of low level of vitamin d with several non-skeletal diseases from migraine to cancers was observed; however, in trials of vitamin d supplementation, there were minimal observed effects or still doubts over its benefit; this evidence may demonstrate the low level of vitamin d as a marker and indicative of association, not the major cause for the above-mentioned diseases [12, 13]. hundred years ago, the world had the experience of a viral pandemic costing the lives of more than 50 million individuals. a brilliant physician in the massachusetts state defense force observed that the outdoor treatment of patients with fresh air and plenty of exposure to the sun decreased mortality and medication consumption. the low level of vitamin d is now believed as a risk factor for the aforementioned disaster [14, 15]. new experience with coronavirus disease 2019 (covid-19) again highlighted "the low level of vitamin d as a risk factor for severe diseases and mortality"; nevertheless, patients need the modality of treatment only available in places in which there is no direct sun exposure. in addition, the governmental policy for lockdown limits outdoor activities and sun exposure [16]. it is worthy of mentioning that a lack of outdoor activities is the main reason for the low level of vitamin d synthesis. the countries, such as uganda, with a high level of outdoor physical activities, had a low prevalence of the disease and a very low level of mortality from covid-19. the data from the world health organization has also shown a trend of low levels of physical activities and severity of covid19 [17-20]. at the beginning of life, adequate vitamin d was a signal for a safe journey in phytoplankton, enabling vertebrates to move freely. history reveals that this movement and displacement of humans from one continent to another, rural to urban, was accompanied by a low level of vitamin d making humans prone to some diseases. if humans have adequate sun exposure and do not receive any vitamin d supplement, then an adequate level of 25-hydroxyl vitamin d (>30 ng/dl) would be a marker of the optimal benefit of sun exposure and adequacy of outdoor activity. the low level of vitamin d is a signal that the life journey is not safe and humans have been improperly displaced. furthermore, humans are prone to the diseases that slaves get. vitamin d deficiency should make humans think again and wonder where they have moved earlier in their daily lives, although taking vitamin d is the least that should be performed. huge human costs might be the result of ignoring this message of nature and history. conflict of interest the authors declare no conflicts of interest. references 1. holick m. phylogenetic and evolutionary aspects of vitamin d from phytoplankton to humans. in: pkt pang and mp schreibman (eds), vertebrate endocrinology: fundamentals and biomedical implications academic press, inc (harcourt brace jovanovich) orlando, fl 1989;3:7-43. 2. holick mf. vitamin d: a millennium perspective. j cell biochem 88:296–307, 2003. 3. holick mf. evolution and function of vitamin d. recent results cancer res 164:3-28, 2003. 4. merewood a, mehta sd, chen tc, bauchner h, holick mf. association between vitamin d deficiency and primary cesarean section. j clin endocrinol metab 94:940945, 2009. 5. mozołowski w. jędrzej sniadecki (1768-1838) on the cure of rickets. nature 143:121, 1939. 6. bodington g. an essay on the treatment and cure of pulmonary consumption, on principles natural, rational and successful. london, england: simpkin, marshall, hamilton, and kent; 1906. 7. kiple, kenneth, and virginia kiple. "the african connection: slavery, disease, and racism." phylon (1960), vol. 41, no. 3, 1980, pp. 211–222. jstor, www.jstor.org/ stable/274784. [accessed 3 may 2021]. 8. wacker m1, holick mf1. sunlight and vitamin d a global perspective for health dermato endocrinol 5:51-108, 2013. 9. alfredsson l, armstrong bk, butterfield da, et al. insufficient sun exposure has become a real public health problem. int j environ res public health 17:5014, 2020. http://www.ajecr.org/ 453 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org 10. cho y, ryu sh, lee br, kim kh. effects of artificial light at night on human health: a literature review of observational and experimental studies applied to exposure assessment. chronobiol int 32:1294-1310, 2015. 11. holick mf, maclaughlin ja, doppelt sh. regulation of cutaneous previtamin d3 photosynthesis in man: skin pigment is not an essential regulator. science 211:590593, 1981. 12. kenneth w. vitamin d screening and supplementation in primary care: time to curb our enthusiasm. am fam physician 97:226-227, 2018. 13. kuwaiti lc, leblanc e screening for vitamin d deficiency in adults: updated evidence report and systematic review for the us preventive services task force. jama 325:1443-1463, 2021. 14. brooks wa. the open air treatment of influenza. am j public health 8:746-750, 1918. 15. d slusky. sunlight and protection against influenza-national bureau of economic research, economics & human biology, 2018. 16. pugach iz, pugach s. strong correlation between the prevalence of severe vitamin d deficiency and population mortality rate from covid-19 in europe. wien klin wochenschr133:403-405, 2021. 17. world health organization. gho by category. prevalence of insufficient physical activity among adults data by world bank income groups. who 2017. 18. who coronavirus disease (covid-19) dashboard. who 2020. 19. chesnut wm, macdonald s, wambier cg. could diet and exercise reduce risk of covid-19 syndemic? med hypotheses 148: 110502, 2021. 20. ghelani d, alesi s, mousa a. vitamin d and covid-19: an overview of recent evidence. int j mol sci 22:10559, 2021. http://www.ajecr.org/ 33 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(2):33-34 case report virchow’s node: a look beyond gut carcinoma bharath c*, komala h m department of pathology, vijayanagar institute of medical science, bellary, karnataka, india abstract virchow’s node, left supraclavicular lymph node contains metastasis of many abdominal visceral malignancies. urothelial carcinoma comprises 90% of all primary bladder cancer with metastases usually limited to the pelvic nodes. though distant lymph node involvement is rare but cannot be entirely overlooked. presence of virchow’s node with bladder tumor is considered as incurable metastatic disease. a 40 year old male presented with history of fever, intermittent hematuria and burning micturition since one week. incidentally left supraclavicular lymph node was found to be enlarged. fnac showed the features of urothelial carcinoma. patient was subjected to further relevant investigations. ct-scan showed well defined enhancing lesion with intra-luminal extension. biopsy was done which revealed primary tumor to be urothelial carcinoma of bladder. supraclavicular lymph node metastases are rare in this case and indicate widespread disease with poor prognosis. conclusion: this case is a rare presentation of urothelial carcinoma metastases to virchow’s node. picking up nodal metastases may influence therapeutic decisions and fnac may be a useful tool in diagnosing such metastases with certainty. keywords: virchow’s node, urothelial carcinoma, fnac, metastases introduction virchow’s node (left supraclavicular lymph node) may contain metastases of many thoracic and abdominal visceral malignancies such as lung, breast, esophageal, gastric, pancreatic, gynecologic, and prostate cancers [1]. urothelial carcinoma accounts for 90% of cases of bladder cancer with metastases usually limited to the regional pelvic nodes [2]. metastasis to non-regional lymph nodes especially cervical lymph nodes is extremely rare presentation [3]. only few reports have been published so far and with poor prognosis [3]. though distant lymph node involvement is rare but cannot be entirely overlooked. case report a 40 year old male presented with history of fever, intermittent hematuria and burning micturition since one week. incidentally left supraclavicular lymph node was found to be enlarged. patient was subjected for fine needle aspiration cytology (fnac). cytology showed cellular smears consisting of atypical epithelial cells in papillary fragments, monolayered sheets and loose clusters with both squamous and glandular differentiation. these cells showed stratification of the nuclei within the fragments. cells with eccentrically placed nucleus, spindle cells, racquet like cells, pyramidal cells, and atypical stripped nuclei were also seen. it was diagnosed as metastasis of urothelial carcinoma (fig.1 and fig.2). patient was subjected to further relevant investigations. ct scan showed well defined enhancing mass lesion in the bladder measuring 4.5×4.8 cm arising from anterior wall with intraluminal extension. hypodense lesions in both lobes of liver and right iliac fossa were seen suggestive of metastases. biopsy of the bladder mass was done which revealed primary tumor to be urothelial carcinoma (fig. 3). supraclavicular lymph node metastases are rare in this case and indicate widespread disease with poor prognosis. discussion bladder cancer is the most common malignant disease of the urinary tract [2]. it is commonly a disease of older age and is more prevalent among men than women [2]. it is the second most prevalent cancer for men and 10th most prevalent cancer for women [4]. it has variable metastatic potential and almost any organ can be involved. data on its metastatic pattern are limited [2]. the pattern of recurrence and metastases are not dependent on the features of the primary tumor [5]. common sites of metastatic spread of bladder carcinoma are regional lymph nodes (90%), liver (47%), lung (45%), bone (32%), peritoneum (19%), pleura (16%), kidney (14%), adrenal gland (14%), and the intestine (13%) [1]. most common lymph nodes involved are external iliac, internal iliac and obturator (20%-45%) as the primary lymphatic drainage of the bladder, and the common iliac sites as the secondary drainage [3]. the possible route of spread to head and neck region is by haematogenous route through vertebral veins and by lymphatic route [3]. the presence of virchow’s node with ___________________________________________________________ * corresponding author: bharath c, md (bhar5anu@yahoo.co.in). mailto:bhar5anu@yahoo.co.in 34 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org figure 1 fnac showing atypical epithelial cells arranged in papillary fragments (hematoxylin and eosin stain 10x) muscle by haematogenous through vertebral veins and by lymphatics [3]. presence of virchow’s node with muscle invasive bladder tumor is considered as incurable metastatic disease as the pathological retrograde tumour cell deposition against the normal drainage of the node (towards the thoracic duct) imply extensive tumour occupation of the retro peritoneum. study done by hessan et al. among 207 patients with metastasis to the head and neck area lymph nodes showed only 3 cases having metastasis with urothelial origin [5]. ferlito et al. reported a series of genitourinary tumors and found this group to be the third most frequent tumor site to metastasize to the supraclavicular fossa [4]. figure 2 cluster of pleomorphic cells showing nuclear overlapping with coarse chromatin (h&e 45x) conclusion this case is a rare presentation of urothelial carcinoma metastases to virchow’s node. identification of nodal involvement is important because the presence of nodal metastasis advances the disease to stage iv [2]. picking up figure 3 atypical tumor cells in varied pattern diagnosed as urothelial carcinoma (h&e 40x) nodal metastases may influence therapeutic decisions and fnac can be used as first line investigation in diagnosing such metastases with certainty. conflict of interest the authors declare no conflicts of interest. references 1. seneviratne ln, jayasundare jmnrk, perera nd. virchow’s node: an unheard site of metastatic bladder cancer. sri lanka j urol 10:28-30, 2009. 2. shinagarel ab, ramaiya nh, jagannathan jp, fennessy fm, taplin me, van den abbeele ad. metastatic pattern of bladder cancer: correlation with the characteristics of the primary tumor. ajr am j roentgenol 196:117-122, 2011. 3. tunio ma, alasiri m, bayoumi y, fareed m, ahmad s. cervical lymph node metastasis from transitional cell carcinoma of urinary bladder: case report and review of literature. j solid tumors 2:59-62, 2012. 4. ogunyemi o, rojas a, hematpour k, rogers d, head c, bennett c. metastasis of genitourinary tumors to the head and neck region. eur arch otorhinolaryngol 267:273-279, 2010. 5. hessan h, strauss m, sharkey fe. urogenital tract carcinoma metastatic to the head and neck. laryngoscope 96:1352–1356, 1986. 91 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(1):91-92 case report malignant fibrous histiocytoma of the abdominal wall arif aslaner*, burhan mayir, tuğrul çakır, umut rıza gündüz, nurullah bülbüller department of general surgery, antalya training and research hospital, antalya, turkey abstract. malignant fibrous histiocytoma (mfh) or undifferentiated pleomorphic sarcoma is a type of malignt neoplasm that arises from any soft tissue and bone involving extremities, abdomen and retroperitoneum. mfh of the external oblique abdominis muscle is rare. surgical resection of the mass is the treatment of choice depending on the stage of the disease and the invasion depth of the tumor. radiotherapy, chemotherapy and immunotherapy are the other treatment methods. we present a case of a 71-year old man with the diagnosis of mfh on external oblique muscle which was completely resected. we believe that adjuvant chemoradiotherapy following surgical resection of the tumor was the most appropriate treatment for this disease. keywords: malignant fibrous histiocytoma, abdomen, magnetic resonance imaging introduction malignant fibrous histiocytoma (mfh) was first introduced in 1961 and described as a tumor rich in histiocytes with a storiform growth pattern [1]. mfh was the most common sarcoma appearing during the 6th and 7th decades with male predominance. it involves the 30% of all soft tissue sarcomas [2], and occurs most frequently in the limbs, retroperitoneum, trunk and abdomen [3, 4]. we report a case of a 71-year-old man with the diagnosis of mfh in the left external oblique abdominis muscle, treated by removal of the tumor followed by radiochemotherapy. case report a 71-year-old male was admitted to our clinic complaining with a palpable mass in the abdominal wall. on his clinical examination, a large, semi mobile, painless mass was palpated at the level of the left lomber margin of external oblique abdominis muscle growing slowly in 6 months. laboratory findings included normal leucocyte: 9.500/mm3, hb: 12.2 g/dl afp: 6.11 ng/ml, ca-15.3:6.0 u/ml, ca-19.9:18.5 u/ml and cea:2.93 ng/ml. the abdominal ultrasound revealed a mass limited to the abdominal wall. the abdominal magnetic resonance imaging (mri) revealed a large mass of 85 × 75 mm in diameter, arising from the left external oblique abdominis muscle with sarcomatous characteristics (fig. 1, a, b). transient needle biopsy revealed the masss to be a mesenchymal tumoral lesion and he underwent total excision of the mass for the exact diagnoses. through an oblique incision, a total resection of the tumor with the external oblique abdominis muscle and sheath was performed (fig. 2). two suction drains were placed laterally between the internal oblique muscle and the subcutaneous tissue, and the skin was closed with 3/0 prolene suture continuously. the patient’s postoperative course was uneventful. drainage tubes were removed on the third postoperative day and he discharged without any complaints with recommendation of outpatient control. pathology diagnosis established the specimen as mfh. macroscopically the encapsulated tumor specimen measured as 85mm ×75 mm in diameter size, and the cut surface of the tumor was pinky-white in color with hemorrhage and necrosis. the tumor microscopically showed spindle cells arranged in a storiform pattern with nuclear atypia and mitoses. to further clarify the diagnosis of the tumor, immunoperoxidase staining was performed. the tumor cells were negative for desmin, cd34 and s100; focal positive for vimentin, cd68, lysozyme, α-1antitrypsin ct and factor xiiia. the patient was referred to medical and radiation oncology for further treatment. discussion mfh can appear at any age but commonly seen at 50 to 70 years of age with male sex dominance. mfh can arise in any part of the body especially in lower limb. the other localizations were upper limb and retroperitoneum. patients usually complaining of abdominal pain, fatigue, weight loss, and a palpable mass arisen within a short period of time. the mass doesn’t usually cause any pain as in our case, unless it is compressing a nearby nerve. mfh consists of histiocyte like and fibroblast like cells arranged in a storiform pattern, with other pleomorphic cells and ___________________________________________________________ * corresponding author: arif aslaner, md (arifaslaner@gmail.com). http://www.ajecr.org/ http://www.nlm.nih.gov/medlineplus/ency/article/002374.htm http://commons.wikimedia.org/wiki/file:storiform_pattern_-_high_mag.jpg mailto:arifaslaner@gmail.com 92 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org figure 1 the abdominal mri showing a mass with 85 × 75 mm in diameters. a. axial view b. transvers view multinucleated giant cells [5]. mfh manifests a broadrange of histopathologic features; storiform-pleomorphic, myxoid, giant cell, inflammatory and angiomatoid type. the storiform pleomorphic pattern is the most common type [6] and it accounts for approximately two-third of figure 2 the total resected specimen with external oblique abdominis muscle mfh [7]. our case belonged to the storiform pleomorphic type. the histologic pleomorphism, degree of vimentin staining, absence of the smooth muscle marker desmin, and the macroscopic and microscopic findings of the resected tumors from our patient favored the diagnosis of mfh. most mfhs are locally invasive, but distant metastasis may spread via the blood or lymphatics. mfhs have a high likelihood of recurrence and metas tasis. the risk correlates with the size and depth of the primary tumor. the overall 2 year survival rate is 60% and 20% will die or suffer with local recurrence [3, 8]. extensive surgical resection is the main treatment. concurrent radiotherapy, chemotherapy, and immunetherapy reduce the possibility of metastasis and local recurrence. prognosis depends on the tumor size, histological grade, disease stage, invasion depth and the resection margin of the tumor [7]. it has been reported that the average 5 year survival rate of patients is 59-66.7% and the local recurrence rate is 16-31% [7]. radical treatment usually results in abdominal wall defects that may need reconstruction. different options on abdominal wall reconstruction exist from primary closure to pedicled or myocutaneous flaps. in our case, abdominal wall primarily closed without any need for reconstruction. two suction drains were placed laterally between the mesh and the subcutaneous tissue while the patient’s postoperative course was uneventful. we conclude that adjuvant chemoradiotherapy following total surgical resection of the tumor was the most appropriate treatment for this disease. conflict of interest the authors declare no conflicts of interest. references 1. kauffman sl, stout ap. histiocytic tumors (fibrous xanthoma and histiocytoma) in children. cancer 14: 469482, 1961. 2. sternheim a, jin x, shmookler b, jelinek j, malawer mm. 'telangiectatic' transformation in soft tissue sarcomas. a clinicopathology analysis of an aggressive feature of high grade sarcomas. ann surg oncol 15: 345354, 2008. 3. weiss sw, enzinger fm. malignant fibrous histiocytoma: an analysis of 200 cases. cancer 41: 22502266, 1978. 4. peiper m, zurakowski d, knoefel wt, izbicki jr. malignant fibrous histiocytoma of the extremities and trunk: an institutional review. surgery 135: 59-66, 2004. 5. murphey md. world health organization classification of bone and soft tissue tumors: modifications and implications for radiologists. semin musculoskelet radiol 11: 201-214, 2007. 6. meister p. malignant fibrous histiocytoma: history, histology, histogenesis. pathol res pract 183: 1-7, 1988. 7. allen kb, skandalakis lj, brown bc, gray sw, skandalakis je. malignant fibrous histiocytoma of the pancreas. am surg 56: 364-368, 1990. 8. kobayashi k, narita h, morimoto k, hato m, ito a, sugiyama k. primary malignant fibrous histiocytoma of the ileum: report of a case. surg today 31: 727–731, 2001. a b http://www.ajecr.org/ 149 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(1):149-151 case report ewing’s sarcoma of ilium, a diagnostic dilemma case report with review of literature usama saleh alshaya, munzir abbashir, mubashir maqbool wani* department of orthopedics, division of surgical specialities, king fahad medical city, riyadh, saudi arabia abstract. ewing’s sarcoma is a highly malignant tumor of bone and is more common in children in the age group of 10 to 20 years. sometimes the classic clinical and radiological presentation of ewing’s sarcoma may not be the norm and patient may have an atypical presentation leading to diagnostic confusion. this is especially true for ewing’s sarcoma involving iliac bone. we present such a case of ewing’s sarcoma involving the right ilium in a patient presenting as right lower quadrant pain and non-specific radiological changes. to the best of our knowledge, this scenario has not been reported in literature. we recommend early magnetic resonance imaging and computed tomography to diagnose the disease early when there is slightest suspicion of the disease. keywords: ewing’s sarcoma, ilium, diagnosis introduction ewing’s sarcoma is known for being highly malignant. named after james ewing [1], it is the second most common primary malignant tumor of bone in children with a characteristic predilection for an age group between 1020 years [2]. although the exact etiology is not known, it has been found that in 85% of es patients, the pathognomonic t (11:22) chromosomal translocation is found. the location of es is most often in the pelvis and lower extremity [3]. in 12.5% cases of es, iliac bone is the site of origin. clinically, the most common presenting symptom is pain (90%), followed by swelling (70%) [4]. radiologically, it is described as a central, diaphyseal, lytic tumor, which is often permeative and has a lamellated or ‘onion skin’ periosteal reaction affecting a long bone, and associated with softtissue mass. the bone lesions are usually lytic, but may be sclerotic or mixed. most of the lesions are diaphyseal or metadiaphyseal. diagnosing ewing’s sarcoma of ilium remains a challenge in its early stages. this is partly because of the myriad of symptoms it presents with and the very nonrecognizable subtle changes radiographically when in its initial stage. we describe one such case who had ewing’s sarcoma of right ilium and presented with pain in the right lower quadrant of the abdomen. we also reviewed the literature regarding the diagnostic dilemma of ewing’s sarcoma of ilium and the different clinical presentations it can have. none of the patients with ewing’s sarcoma to the best of our knowledge has ever presented with right lower quadrant pain case report a girl 13 years of age, saudi in origin presented to us with a history of pain right lower quadrant of 6 months duration. she had been visiting many clinics for her pain with no relief to her symptoms. in the initial stages of her disease, she had undergone multiple ultrasound examinations with no clue of the diagnosis. the patient already had undergone appendectomy 2 years back, so appendicitis was not kept as a differential diagnosis. in one of the latest ultrasound examination of the abdomen, the sonologist picked up a mass in her right iliac fossa arising from pelvis suspecting an abscess or a tumor. patient was referred to our clinic for further evaluation and treatment. on our examination, there was no tenderness in the right iliac fossa nor any palpable swelling could be palpated. esr, cbc, crp were normal. x ray pelvis ap was done which did not show any significant bony changes (fig. 1 a). mri of the pelvis was done which showed a soft tissue mass arising along the right iliac bone most probably soft tissue sarcoma with focal bony involvement (figs. 1 b and 1 c). this was followed by technetium -99m mdp scintigraphy. this was negative for multifocal skeletal metastatic disease and negative for local invasion of the osseous structures. ct scan of thorax and abdomen was done which ruled out any metastasis. under sterile conditions, ultra sound -guided fine needle aspiration of the right iliac fossa mass was done using 22 g needle. about 15 ml hemorrhagic fluid was aspirated. this showed presence of malignant cells. it was followed by an open biopsy of the mass which was ___________________________________________________________ * corresponding author: mubashir maqbool wani, mbbs (mmwani@kfmc.med.sa). mailto:mmwani@kfmc.med.sa 150 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org figure 1 (a) x-ray anteroposterior view of the pelvis showing hard to pick changes in the right ilium. (b and c) mri images of the patient before initiation of neo-adjuvant chemotherapy, showing mass arising along the right iliac bone most probably soft tissue sarcoma with focal bony involvement. reported by the histopathologists as ewing’s sarcoma. patient was put on neo adjuvant chemotherapy which included vincritine, cyclophosphamide, doxorubicin and etoposide , ifosfamide (vcd/ie 3 weekly). following neoadjuvant chemotherapy, the mass had shrunk in size and internal hemipelvectomy was undertaken excising the tumor mass along with the involved bone spearing the sacroiliac joint and hip joint (fig. 2 a). the margins were negative for any residual tumor. the patient was again given chemotherapy after surgery and radiotherapy was added to minimize the chances of recurrence. presently patient is doing fine, is ambulatory and pain free. the last mri done for the patient does not show any evidence of recurrence (figs. 2 b and 2 c). the patient will follow us regularly. written informed consent was obtained from the patient for publication of this case report and accompanying images. discussion early diagnosis of ewing’s sarcoma of ilium remains challenge partly because of the subtle difficult to pick changes in the radiographs and the clinical symptoms patients have. after reviewing the literature by searching pubmed with terms like ‘ewing’s sarcoma of ilium’,’ diagnosis of ewing’s sarcoma of ilium’, ‘clinical presentations of ewing’s sarcoma’, we could find few case reports regarding the atypical presentation of ewing’s sarcoma. the delay in diagnosis can partly be due to the the radiologic appearance of inflammatory and tumorous lesions in the iliac bone which is characterized by destructive alterations and consolidations simultaneously. this pattern is nonspecific. the value of plain films of this area is compromised by the anatomy of the iliac bone and by overlying structures [5). there have been instances where authors have diagnosed ewing’s sarcoma of the ilium as sacroilietis [6, 7, 8] and in some cases even septic arthritis [9] possibly because of transarticular spread in these patients. the authors of these cases mainly relied on clinical presentation and plain radiography initially which lead to the diagnostic confusion. there have also been cases reported of ewing’s sarcoma of ilium mimicking juvenile rheumatoid arthritis [10] and pain in the hip [11]. in our case the patient’s presentation as right lower quadrant pain again lead to diagnostic confusion and delayed treatment till a definite diagnosis could be reached to. patients of ewing’s sarcoma need a multidisciplinary approach to treatment which includes involvement of oncologists, radiation oncologists, surgeons and radiologists. haematogeneous spread to lungs and bone is very common in ewing’s sarcoma, so neo adjuvant chemotherapy has an important role in treatment of ewing’s sarcoma. while surgery is effective and appropriate for patients who can undergo complete resection with acceptable morbidity, children who have unresectable tumors or who would suffer loss of function are treated with radiation therapy alone. we were fortunate enough to have clear margins at the time of surgery in our patient without causing any functional disability to the patient. prognosis depends on extent of the disease, size and location of the tumor, presence or absence of the tumor metastasis, tumor response to therapy, age, and disease relapse. most centers today report long-term survival of 60% to 70%. the worst prognostic factor is the presence of distant metastasis. even with aggressive treatment, patients with metastasis have only a chance of 20% long-term survival. histological grades are of no prognostic significance. conclusion we believe that ewing’s sarcoma should be kept as one of the rare differential diagnosis for lower quadrant pain. ultrasonography should not be relied on much and x-rays may not add more to the knowledge. lower threshold should be kept for investigations like ct and mri to pick the disease early and start the appropriate treatment. conflict of interest the authors declare no conflicts of interest. references 1. huvos ag. james ewing: cancer man. ann diagn pathol 2:146-148, 1998. 2. grier he: the ewing family of tumors: ewing’s sarcoma and primitive neuroectodermal tumors. pediatr clin north am 44:991-1004, 1997. a b c 151 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org figure 2 (a) x-ray image of the patient showing right internal hemipelvectomy. (b and c) mri images of the patient showing no evidence of tumor. 3. kissane jm, askin fb, foulkes m et al.: ewing’s sarcoma of bone clinic pathologic aspects of 303 cases from the intergroup ewing’s sarcoma study. hum pathol 14:773-779, 1983. 4. vlasak r, sim fh: ewing’s sarcoma. orthop clin north am 27:591-603, 1996. 5. appell rg, oppermann hc. differential diagnosis of tumorous changes in the iliac bone region in children. case reports and review of the literature. radiologe 24:6067, 1984. 6. adelman hm, wallach pm, flannery mt: ewing’s sarcoma of the ilium presenting as unilateral sacroiliitis. j rheumatol 18:1109-1111, 1991. 7. pouchot j, barge j, marchand a, carbonnier j, vinceneux p: ewing’s sarcoma of the ilium mimicking an infectious sacroiliitis. j rheumatol 19:1318-1320, 1992. 8. al-adsani a, niazy mn, mohd m. ewing's sarcoma of the ilium mimicking sacroiliitis. rheumatology (oxford). 38:792-793, 1999. 9. halwai ma 1 , mir ba, wani mm, bashir a, hussain a. ewing's sarcoma of the ilium mimicking inflammatory arthritis of the hip: a case report. cases j 2:6487, 2009. 10. wang xn, su gx, wu fq. report of a child with ewing's sarcoma who was misdiagnosed as juvenile idiopathic arthritis. zhonghua er ke za zhi 50:866-867, 2012. 11. de maddi f, rigante d, de ritis r, et al. misdiagnosis of ewing's sarcoma of the ilium at the pelvis x-ray. rheumatol int 32:1109-1110, 2012. a b c 387 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2020;7(1):387-389 new prospect covid-19 and sarcoidosis: a two-way possible association hamidreza bashiri1, mohammadreza mirjalili2, fahimeh rashidi maybodi3* 1rheumatology department, shahid sadoghi university of medical sciences, yazd, iran 2internal medicine department, shahid sadoughi university of medical sciences, yazd, iran 3periodontology department, faculty of dentistry, shahid sadoughi university of medical sciences, yazd, iran abstract. immunological impairments such as lymphopenia in patients with sarcoidosis and their treatment by glucocorticoids and cytotoxic drugs may increase the risk of severe bacterial and viral infections, so these people may be more susceptible to covid-19 as a disease with viral source. on the other hand, many previous studies pointed to viral trigger in sarcoidosis but no specific virus was mentioned. a recent french cross sectional study on 482 patients, suggests that daily active tobacco smokers have a lower r isk of developing severe or symptomatic covid-19 as compared to nonsmokers. it should be mentioned that our primary observations during the first covid-19 outbreak (february-april 2020) on 206 covid-19 patients admitted in intensive care units in shahid sadoughi hospital, yazd, iran, showed a possible negative association between smoking and coronavirus-2 infection. an observation that was addressed in the literature for sarcoidosis. some previous studies mentioned that current smokers also have less probability for developing sarcoidosis. according to all these comparable features in patients with covid-19 and sarcoidosis, may be one day in future, it will be reported that a sarcoidosis-like disease found in patients with covid-9 history in 2020. who knows? keywords: covid-19, sarcoidosis, association coronavirus disease 2019 (covid-19), caused by coronavirus-2 (cov-2), is a global happening. as of march twenty first, 2020 infected patients were present in 167 countries and there have been quite 285,000 cases worldwide with nearly 12,000 death [1]. at the end of the first month of 2020, the world health organization stated that covid-19 is a public-health emergency of worldwide importance [2]. so, it is critical to take steps to stop transmission and save lives. in this regard, it is important to spot the individuals which are at higher risk for this illness. age over 70 and underlying systemic conditions such as cardiovascular disease, diabetes and high blood pressure were all associated with an increased risk of death among covid-19 patients [3].the possible mechanism for these associations will be explained hereinafter. people with autoimmune or rheumatologic disease such as rheumatoid arthritis have also an increased risk due to the immune system impairment or to the immunosuppressive effects of corticosteroids and synthetic or biological diseasemodifying drugs, although chloroquine and hydroxychloroquine have now been included in covid19 pneumonia treatment protocols [4]. studies showed that coronavirus-2 requires the angiotensin-converting enzyme 2 receptor (ace2) as a port to enter host cells [5]. ace2 is highly expressed in the lung, heart and vascular endothelium, counteracting the effects of angiotensin ii in states with excessive activation of the renin-angiotensin system such as hypertension and heart failure [1]. consistent with a hypothesis, angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may also increase the danger of severe covid19. angiotensin-converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs) are commonly used medications for patients with cardiovascular diseases such as refractory high blood pressure and heart failure. these two families of drugs are also prescribed for the management of cardiovascular diseases in elderly patients and in patients with diabetes. intravenous infusions of aceis and arbs in animal models increase the numbers of angiotensin converting enzyme 2 (ace2) receptors in the cardiopulmonary circulation. patients taking aceis or arbs chronically are assumed to have increased numbers of ace2 receptors throughout their cardiopulmonary circulations which also results in an upregulation of ace2 as observed in experimental animals [6]. a further point that should be noticed is the genetic predisposition for an increased risk of covid-19, which might be due to ace2 polymorphisms that have been linked to diabetes mellitus and hypertension, specifically in asian populations. so, the predisposition of a patient might result from a combination of both drug therapy and ace2 polymorphism [7]. an ___________________________________________________________ * corresponding author: dr. fahimeh rashidi maybodi (f_rashidi63@yahoo.com) ). http://www.ajecr.org/ mailto:f_rashidi63@yahoo.com 388 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org opposing hypothesis has suggested that increasing of ace2 or infusion of human recombinant ace2 might protect against cov-2 infections. however, it has to be emphasized that the role of ace2 in this protection, are not clear. furthermore, it should be considered that aceis have been reported to modify the adaptive immune response, suggesting that long-term periods of aceis’ usage might suppress the adaptive immunity, which has a key role against viral infections. available published data indicate briefly that ace2 is a double-edged sword, specifically when considering patients with covid-19 and comorbidities of hypertension, diabetes and cardiovascular disease [8]. despite all of these controversies, during reading these articles, a question that comes to mind is: could there be a link between other systemic diseases such as sarcoidosis and the increased risk of covid-19 and vice versa? angiotensin-converting enzyme (ace) is elevated in sarcoidosis patients and may also be elevated in other certain pulmonary granulomatous disorders (silicosis and tuberculosis), hyperthyroidism and diabetes [9, 10] and covid-19 as mentioned before. polymorphism in the ace gene that affects the ace enzyme level in metabolic diseases like hypertension and sarcoidosis might be a genetic marker for predisposition of these diseases in some populations [11] similar to what mentioned recently for covid-19 [7]. on the other hand, several studies mentioned an important role for th17 cells besides th1 in sarcoidosis, as the frequency of il-17–producing t cells is increased in peripheral blood and lungs of subjects with sarcoidosis compared with controls [12]. peripheral blood of patients with severe covid-19 had a strikingly high number of th17 cells, further supporting a th17 type cytokine storm in this disease which results in lung tissue damage. elevated th17 as the same of th-1 responses or enhanced il-17-related pathways are also discovered in mers-cov and sars-cov patients [13] according to the novelty of covid-19 disease, genetic predisposing assessments has not been investigated yet but in mers-cov infection, mhc ii molecules, such as hla-drb1*11:01 are associated with the susceptibility to this infection [5]. hla-drb1*11:01 (or, 1.69; 95% ci) is also associated with susceptibility to sarcoidosis [14]. the presence of lymphopenia in sarcoidosis has long been proven with scientific studies [15]. it is also been reported that lymphocytes’ count was reduced to lower than 5% within 2 weeks after covid-19 disease onset [16]. lung involvement with a peripheral predominance is a presentation in covid-19 patients and was previously seen in patients with sars-cov and mers-cov infections and the chest ct showed that disease progressed with ground-glass opacities, which is similar to that of sars-cov-2 infection [5]. nodules in sarcoidosis also tend to be more abundant around broncho-vascular structures and sub-pleural along the chest wall [17]. immunological impairments such as lymphopenia in patients with sarcoidosis and their treatment by glucocorticoids and cytotoxic drugs may increase the risk of severe bacterial and viral infections [18], so these people may be more susceptible to covid-19 as a disease with viral source. on the other hand, many previous studies pointed to viral trigger in sarcoidosis [19] but no specific virus was mentioned. a recent french cross sectional study on 482 patients, suggests that daily active tobacco smokers have a lower risk of developing severe or symptomatic covid-19 as compared to nonsmokers [20]. it should be mentioned that our primary observations during the first covid-19 outbreak (february-april 2020) on 206 covid-19 patients admitted in intensive care units in shahid sadoughi hospital, yazd, iran, showed a possible negative association between smoking and coronavirus-2 infection. an observation that was addressed in the literature for sarcoidosis. some previous studies mentioned that current smokers also have less probability for developing sarcoidosis [21, 22]. according to all these comparable features in patients with covid-19 and sarcoidosis, may be one day in future, it will be reported that a sarcoidosis-like disease found in patients with covid-19 history in 2020. who knows? conflict of interest the authors declare no conflicts of interest. references 1. clerkin jk, fried ja, raikhelkar j, sayer j, griffin jm. covid-19 and cardiovascular disease. circulation 141:1648-1655, 2020. 2. wu d, wu t, liu q, yang z. the sars-cov-2 outbreak: what we know. int j infect dis 94:44-48, 2020. 3. jordan re, adab p, cheng kk. covid-19: risk factors for severe disease and death. brit med j 368: m1198, 2020. 4. favalli eg, ingegnolia f, de luciaa o, cincinelli g. covid-19 infection and rheumatoid arthritis: faraway, so close! autoimmun rev 2020; https://doi.org/10.1016/j.jpha. 2020.03.001. 5. li x, geng m, peng y, meng l, lu s. molecular immune pathogenesis and diagnosis of covid-19. j pharmaceut anal 10:102-108, 2020. 6. diaz jh. hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19. j travel med 27: taaa041, 2020. 7. fang l, karakiulakis g, roth m. are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? lancet respir med 8:e21, 2020. 8. fang l, karakiulakis g, roth m. antihypertensive drugs and risk of covid-19? lancet respir med 8:e32e33, 2020. 9. bergantini l, bianchi f, cameli p, mazzei ma. prognostic biomarkers of sarcoidosis: a comparative study of serum chitotriosidase, ace, lysozyme, and kl-6. dis markers 2019; id 8565423. 10. ungprasert p, carmona em, crowson cs, matteson el. diagnostic utility of angiotensin converting enzyme in sarcoidosis: a population-based study. lung 194:91-95, 2016. http://www.ajecr.org/ 389 am j exp clin res, vol. 7, no. 1, 2020 http://www.ajecr.org 11. sarı g, kurt e, saydam f. association between i/d polymorphism in the ace gene and sarcoidosis in turkish patients. cytotechnology 67:1067-1072, 2015. 12. wu d, yang xo. th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib. j microbiol immunol infect 53:368-370, 2020. 13. georas sn, chapman tj. sarcoidosis and t-helper cells th1, th17, or th17.1? am j respir crit care med 193:1198-1200, 2016. 14. levin am, adrianto i, datta i, iannuzzi mc. association of hla-drb1 with sarcoidosis susceptibility and progression in african americans. am j respir cell mol biol 53: 206-216, 2015. 15. doğan c, cömert ss, çağlayan b. is lymphopenia detected in sarcoidosis associated with the disease activity? south. clin. ist. euras 30:151-157, 2019. 16. tan l, wang q, zhang d. lymphopenia predicts disease severity of covid-19: a descriptive and predictive study. signal transduct targeted ther 5:33. doi: 10.1038/s41392-020-0148-4. 17. nunes h, uzunhan y, gille t. imaging of sarcoidosis of the airways a nd lung parenchyma and correlation with lung function. eur respir j 40: 750-765, 2012. 18. duréault a, chapelon c, biard l. severe infections in sarcoidosis incidence, predictors and long-term outcome in a cohort of 585 patients. medicine 96:49(e8846), 2017. 19. rossides m, kullberg s, askling j. are infectious diseases risk factors for sarcoidosis or a result of reverse causation? findings from a population‑based nested casecontrol study. european j epidemiol 2020; https://doi.org/ 10.1007/s10654-020-00611-w. 20. miyara m, tubach f, pourcher v. low incidence of daily active tobacco smoking in patients with symptomatic covid-19. open access 2020. doi: 10.32388/wpp19w. 21. ungprasert p, crowson cs, matteson el. smoking, obesity and risk of sarcoidosis: a population-based nested case-control study. respir med 120: 87-90, 2016. 22. urbankowski t, knyziak-medrzycka i, domagałakulawik j, chazan r. sarcoidosis and tobacco smokingclinical picture, diagnostic tests results and bronchoalveolar lavage fluid composition. pol merkur lekarski 32:298-301, 2012. http://www.ajecr.org/ 35 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(2):35-37 case report rarely seen rectum tumor with bad prognosis: a case of large cell neuroendocrine carcinoma rojbin karakoyun, 1 mani habibi, 1 * seyda gunduz, 2 dinc suren, 3 nurullah bülbüller 1 1 department of surgery, antalya training and research hospital, antalya, turkey 2 department of medical oncology, antalya training and research hospital, antalya, turkey 3 department of pathology, antalya training and research hospital, antalya, turkey abstract. large cell neuoendocrine carcinoma forms an extremely aggressive and poor prognostic subset of neuroendocrine tumors with a tendency to early metastasis. however palliative chemotherapy in patients with metastatic disease may provide longer progression-free survival but the important issue is to detect and surgically treat the disease at an early stage. in this article, we present a case of large cell neuroendocrine carcinoma of the rectum which was admitted to our hospital with a complaint of rectal bleeding for about a month and multiple dsitant metastases at the time of diagnosis. the patient received palliative chemotherapy with cisplatin plus etoposide and zoledronic acid and an overall 6 month survival time was achieved. generalizing the screening programs for colorectal cancers and facilitating access to colonoscopy are essential for early diagnosis and therapy of rectal neuroendocrine tumors. keywords: rectal neoplasms, carcinoma, large cell, neuroendocrine introduction neuroendocrine tumors originate from the neuroendocrine cells which exist almost in any part of the body. although these cells are present most commonly in gastrointestinal system, neuroendocrine tumors are rather rare in colon and rectum. large cell neuroendocrine carcinoma constitutes an extremely aggressive subgroup with poor prognosis and it has a tendency to early metastasis [1]. we present a case of large cell neuroendocrine rectum tumor with multiple distant metastases at the time of diagnosis. case report a seventy-one year old male patient admitted to our hospital with a complaint of rectal bleeding that started about one month earlier. hemogram, biochemical analysis and tumor markers were all within normal limits. a colonoscopy was performed upon palpation of a mass on the anterior rectum in rectal examination. colonoscopy revealed a tumoral mass initiating from 3rd cm of the rectum and multiple biopsies were obtained. pathologic examination revealed glands with normal appearance as well as infiltrating tumor as solid areas and trabecular structures. tumor cells had large, hyperchromatic, pleomorphic nuclei with large cytoplasm. mitotic and apoptotic activity was marked in tumor cell nuclei (fig1). in immunohistochemical studies, a wide positivity was observed with synaptophysin (fig 2) and focal positivity with cd56 (fig.3), both of which being neuroendocrine markers. these findings were considered as consistent with large cell neuroendocrine carcinoma. metastasis screening by positron emission tomography (pet) imaging revealed widespread multiple metastases in lungs, liver and bone. with the diagnosis of metastatic large cell neuroendocrine carcinoma, palliative chemotherapy was initiated at the oncology clinic. cisplatin with a dosage of 75mg/m 2 and etoposide with a dosage of 100mg/m 2 were administered. due to widespread bone metastasis, zoledronic acid 4 mg iv was given. disease progression developed in the patient following 3 courses of chemotherapy treatments. overall a 6 month of survival could be achieved. discussion neuroendocrine tumors are rare neoplasms that have a rather wide clinical presentation. classifications of neuroendocrine tumors have been done according to organ systems. therefore many recent classifications and revisions caused inconsistencies and confusions on defining and reporting these rare tumors. in order to provide standardization, world health organization (who) and european neuroendocrine tumor society (enets) carried out studies. gastrointestinal system neuroendocrine tumors were divided into 3 main groups in a classification done by who in 2010; grade 1, 2 and 3 neuroendocrine carcinomas. neuroendocrine carcinomas ___________________________________________________________ * corresponding author: mani habibi, md (manihabibi@gmail.com). mailto:manihabibi@gmail.com 36 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org were further divided into two subgroups as large cell and small cell carcinomas [2]. grading was done according to the number of mitosis; less than 3, between 3 and 20 and above 20, reflecting grade 1, 2 and 3, respectively. in cases where biopsy material was small, ki 67 index should be used because mitosis count can be problematic [3]. neuroendocrine tumor cells are characterized by presence of neurosecretory granules and neurosecretory markers in cytoplasm that can be observed with electron microscope. these markers are specific neuron enolase (nse), chromogranin, synaptophysin and cd56 [4]. figure 1 glandular structures belonging to the normal-appeared rectum showing tumor islands with large hyperchromatic and pleomorphic nuclei and large cytoplasm on the right side (hematoxylin and eosin stain x200). approximately half of the patients with diagnosis of rectal neuroendocrine tumor are asymptomatic and they were incidentally diagnosed during colonoscopies done for other indications and for colorectal cancer screening. change in bowel habits, hematochezia and rectal pain are the major symptoms [5]. size of the tumor, depth of inva figure 2 widespread positivity for synaptophysin (immunohistochemical stain x200) sion and lymph node positivity are the important factors indicating malignant behavior. in a study, patients with rectal tumor less than 1 cm had 10% metastasis while those with tumors between 1 and 2 cm had 10-15% and those with tumors larger than 2 cm had 60% metastasis [6]. similar to adenocarcinomas, liver is the most common distant organ where metastasis occurs [7]. when metastasis develops, these tumors manifest rather an aggressive course. figure 3 focal positivity for cd56 (immunohistochemical stain x200) the term “large cell neuroendocrine carcinoma” was first used by bernick et al. [8]. in their studies, neuroendocrine carcinoma was detected in only 0.6% of the patients with colorectal cancer, whereas 0.2% was evaluated as large cell neuroendocrine carcinoma. most of these patients were in advanced stage at the time of diagnosis and average survival period was reported as 10.4 months. one year survival rate was 46% and our patient had an overall survival period of 6 months [8]. surgery and systemic chemotherapy is the main treatments for local disease. case series include cisplatin and florourasil, cisplatin and etoposide based combinations along with oxaliplation, 5-fluorouracil and leucovorin combinations (folfox) in systemic chemotherapies [9-11]. we used cisplatin and etoposide treatment regimen in our patient; however we could not achieve a response because he had already widespread metastasis at admission and his performance was poor. in conclusion large cell neuroendocrine malignancies are rather rare in rectum. they have a rather aggressive and poor prognosis because of tendency for early metastasis. limited number of cases in literature challenge to establish recommendations about the treatment. although palliative chemotherapy options could provide longer survivals without progression in the patients, diagnosing the illness at early stage and surgical treatment are of prime importance. generalizing the screening programs for colorectal cancers and facilitating access to colonoscopy are crucial for diagnosing rectal neuroendocrine tumors at early stage. conflict of interest the authors declare no conflicts of interest. 37 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org references 1. pascarella m, mccloskey d, jenab-wolcott j, vala m, rovito m, mchugh j. "large cell neuroendocrine carcinoma of the colon: a rare and aggressive tumor." j gastrointest oncol 2: 250-253, 2011. 2. bosman f t, camerio f, hruban r, et al. who classification of tumors of the digestive system. lyon, france: iarc press; 2010. 3. klimstra ds, modlin ir, coppola d, lloyd rv, suster s: the pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. pancreas 39:707-712, 2010. 4. simon sr, fox k. neuroendocrine carcinoma of the colon: correct diagnosis is important. j clin gastroenterol 17:304-307, 1993. 5. merg a, wirtzfeld d, wang j, cheney r, dunn kb, rajput a. viability of endoscopic and excisional treatment of early rectal carcinoids. j gastrointest surg 2007; 11: 893-897, 2007. 6. mani s, modlin im, ballantyne gh, ahlman h, west ab. carcinoids of the rectum. j am coll surg 179: 179: 231-248, 1994. 7. vilallonga r, espin basany e, lopez cano m, landolfi s, armengol carrasco m. neuroendocrine carcinomas of the colon and rectum: a unit's experience over six years. rev esp enferm dig 100:11–16, 2008. 8. bernick pe, klimstra ds, shia j, minsky b, saltz l, shi w, et al. neuroendocrine carcinomas of the colon and rectum. dis colon rectum. 47:163-169, 2004. 9. kim, h g, lee ji, jeong s, lee jw, kwon ks, kim, h et al. a case of large cell neuroendocrine carcinoma of the colon. korean j gastroenterol 54: 46-49, 2009. 10. park js, kim l, kim ch, bang bw, lee dh, jeong s, et al. synchronous large-cell neuroendocrine carcinoma and adenocarcinoma of the colon. gut liver 4:122–125, 2010. 11. moertel cg, kvols lk, o'connell mj, rubin j. treatment of neuroendocrine carcinomas with combined etoposide and cisplatin: evidence of major therapeutic activity in the anaplastic variants of these neoplasms. cancer 68:227, 1991. 136 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(4):136-137 case report acute leukemia case presented with hypercalcemia mehmet selçuk bektaş 1, 2 , murat doğan 1 , lokman üstyol 2 , mehmet açikgöz 2 , sevil ari yuca 1 , yaşar cesur 1 , a. faik öner 1 1 department of pediatric health and disease, faculty of medicine, yüzüncü yıl university turkey 2 van lokman hekim hospital, van, turkey abstract. an 8-year-old girl patient referred to our emergency clinic with articular pain, stomachache and fever complaints. past history revealed that she was suffering from pain in both knees and ankle joints for 8 days. the joint temperature increased and swelling did not accompany articular pain. family history was unremarkable. in the physical examination, there was sensitivity in the knees, elbows and ankles during movement. the patient had normal complete blood cell count, and no blast or atypical cells were observed in peripheral smear. serum electrolytes, liver and kidney function tests were normal except for hypercalcemia. the 25 (oh) vitamin d and 1-25 (oh)2 vitamin d levels were within normal range. in bone marrow aspiration, infiltration of cells with lymphoblastic and homogenous cellular features was observed. with positivity of ccd79, cd19, cd45, the case was considered as preb cell leukemia. body bone scintigraphy performed for bone metastasis was normal. after the chemotherapy, hydration and furosemid treatment, the calcium level returned to normal. this case emphasized on the fact that, children with hypercalcemia should undergo a detailed examination for malignancies even though no blast or atypical lymphocyte are observed in their peripheral blood smear before steroid treatment is applied and if necessary, bone marrow aspiration should be taken into account. keywords: acute leukemia, hypercalcemia, childhood malignancy introduction hypercalcemia and its presentation are rare situations among malignancies of childhood. in children, leukemia, rabdomyosarcomas, malignant rabdoid tumors, hodgkin and non-hodgkin tumors, hepatoblastoma, neuroblastoma and ewing sarcoma may be mentioned among malignancies presenting hypercalcemia as complications [1]. in acute leukemia, hypercalcemia is more frequent than in solid tumors as a cause of presentation. while symptomatic hypercalcemia in acute leukemia is usually not resistant to treatment, hypercalcemia that develops more often in the later phases of the disease in solid tumors is resistant to treatment [2]. the treatment includes intravenous hydration, loop diuretics, calcitonine, mitramycine and the subjacent treatment. in the cases where no clue is obtained, biphosphonates are used [1]. we present a case of an 8-year-old girl admitted to our emergency unit for articular pain, stomachache and fever and presented with hypercalcemia and acute leukemia. case presentation an 8-year-old girl patient applied to our emergency clinic for articular pain and stomachache and fever complaints. her past history showed that she was suffering from pain in both knees and ankle articulations for 8 days. it was determined that joint temperature increase and swelling do not accompany articular pain. the patient suffered from stomachache like cramps associated with intermittent fever three days later. in the physical examination there was no particular feature in personal and familiar history. her body weight was 24 kg (25-50 p), her height was 123 cm (25-50 p). a sensibility was observed in knees, elbows and ankles during movement, with stable vital observations. the other physical examination features were normal. the complete blood cell counts were as follows: hg: 13.5 g/dl, leucocytes: 14400/mm 3 , hematocrit 37.4%, mcv: 76.1 fl, platelet 208.000/mm 3 . in the results of the peripheral blood smear were as follows: 34% lymphocyte, 63% neutrophiles, 2% monocytes, 1% eosinophiles, trombocytes were in mass and no blast or atypical cell have been observed. biochemical evaluations showed that serum calcium (ca) was 14.23 mg/dl, phosphor (p) was 4.97 mg/dl, alkaline phosphatase (alp) was 305 u/l, lactic dehydrogenase was 1000 u/l. other biochemical examinations (serum electrolytes, liver and renal function test, pre-prandial blood glucose level) were ___________________________________________________________ *corresponding author: m. selçuk bektaş, md (selcukbektas008@gmail.com). http://www.ajecr.org/ mailto:selcukbektas008@gmail.com 137 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org within normal ranges. while the erythrocyte sedimentation rate was 40 mm/hour, c-reactive protein level was 41 mg/l (n:< 3 mg/l). prothrombin time (14.4 sec), activated partial tromboplastin time (29.4 sec), fibrinogen (395 mg/dl) levels were normal. moreover, parathormone (pth) level was 9.7 u/l, 25 (oh) vitamin d level was 30 ug/l (n:10-60 ug/l) and 1-25 (oh)2 vitamin d level was 57pg/ml (30-50 pg/ml). homogenous cellular with lymphoblast type infiltration was observed in bone marrow aspiration. this case with positivity of ccd79, cd19, cd45 (respectively 94.6%, 37.9%, 100%) was considered as preb cell leukemia. body bone scintigraphy performed for bone metastasis was normal. after the chemotherapy and hydration (3000 ml/m 2 /day), prednisolone 2 mg/kg/day and furosemid treatment (1 mg/kg) were administered and the calcium levels returned to the normal level. disscussion hypercalcemia is rare but has severe complications that should be treated immediately. in malignancies, the incidence of hypercalcaemia was reported to be between 0.2% and 0.7% [1-4]. childhood acute lymphoblastic leukemia is one of the most frequent malignancies presenting with hypercalcemia. the incidence is known as 0.39% [2, 3, 5]. hypercalcemia associated with malignancy develops as a result of excessive secretion of interleukin-1 (il-1), il-2, il-6, transforming growth factor beta, 1,25(oh)2 vitamin d, direct bone invasion, parathormone associated proteins (pth-rp) and prostaglandin e2 and rarely excessive secretion of parathormone [6, 7]. pth and pth-rp can directly be secreted from lymphoblasts. pth and pth-rp leads to hypercalcemia by causing bone resorption. in these cases, hypercalcemia is present in peripheral blood frequently [1]. in our case, in the studies performed for the determination of hypercalcemia etiology, parathormone, 25 (oh)2 and 1,25 (oh)2 vitamin d levels, total body scintigraphy and bone survey were normal. no blast was present in peripheral blood. the absence of bone resorption showed that in hypercalcemia etiology, pth-rp and pth had no role. i̇n our case, as il-1, il-2, il-6 and tgf-beta have not been studied, these factors are supposed to be efficient in hypercalcemia etiology. in the treatment of hypercalcemia associated with malignancy, the classic treatment of hypercalcemia has been used. the scheme of this treatment includes intravenous hydration, furosemid, corticosteroids and calcitonine. when the treatment is not efficient or when it cannot be applied due to cardiac or renal problems, biphosphonates should be taken into consideration as an alternate treatment possibility [1]. biphosphonates are quite efficient in hypercalcemia associated with malignancy. there are few studies about the use of biphosphonates in the treatment of hypercalcemia associated with malignancy in children. after iv administration of biphosphonates, a temporary hypocalcemia, hypophosphatemia and hypomagnesemia may develop [8]. moreover, fever and grip–like symptoms are among the other side effects [9]. in our case, steroid, iv hydration and furosemid treatment and bfm all treatment protocol were used to ensure normocalcemia. biphosphonates were not used. in conclusion, this study emphasizes on the fact that children with hypercalcemia should undergo a detailed examination for malignancies even no blast or atypical lymphocyte are observed in peripheral smear, before steroid treatment is performed and if necessary bone marrow aspiration should be taken into account. conflict of interest the authors declare no conflicts of interest. references 1. efthymiadou s a, gkentzi d, varvarigou a. hypercalcemia and osteolytic lesions as presenting symptoms of acute lymphoblastic leukemia in childhood: the use of zoledronic acid and review of the literature. j pediatr endocrinol metab 27:349-354, 2014. 2. jamal cy, islam mm, rahman sa. acute lymphoblastic leukaemia presenting with severe hypercalcaemia. mymensingh med j 20:134-137, 2011. 3. rajagopal r, lum sh, jalaludin my, krishnan s, abdullah wa, ariffin h. hypercalcaemia: an unusual presenting feature of childhood acute lymphocytic leukemia. br j haematol 163:147, 2013. 4. trehan a, cheetham t, bailey s. hypercalcemia in acute lymphoblastic leukemia: an overview. j pediatr hematol oncol 31:424-427, 2002. 5. n. alikaşifoğlu a, küpeli s, yetgin s. use of bisphosphonates for resistant hypercalcemia in children with acute lymphoblastic leukemia: report of two cases and review of the literature. turkish j pediatr 48: 248-252, 2006. 6. miyamura t, taguchi f, ishikura h, et al. production of parathyroid hormone-related peptide in a patient with acute lymphocytic leukemia with extensive osteolytic lesions and hypercalcemia. am j hematol 50:150-151, 1995. 7. bhat gm. a child with acute lymphoblastic leukemia (all) presenting with symptomatic hypercalcemia and multiple osteolytic lesions. indian j med paeditr oncol 28: 46-47, 2007. 8. mathur m, sykes ja, saxena vr, rao sp, goldman gm. treatment of acute lymphoblastic leukemiainduced extreme hypercalcemia with pamidronate and calcitonin. pediatr crit care med 2003; 4: 252-255, 2003. 9. adami s, bhalla ak, dorzzi r, et al. the acute phase response after bisphosphonate administration. calcif tissue int 41: 326-331, 1987. http://www.ajecr.org/ 89 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(1):89-90 case report acute suppurative parotitis: a rare entity in early infancy hosein heydari 1 , bahareh abtahi-naeini 2 , mohsen akhavan sepahi 1 , ali saffaei 3 , mohsen pourazizi 4 * 1 pediatric medicine research center, qom university of medical sciences, qom, iran 2 department of dermatology, skin diseases and leishmaniasis research center, students’ research committee, isfahan university of medical sciences, isfahan, iran 3 pharmacy students’ research committee, school of pharmacy, isfahan university of medical sciences, isfahan, iran 4 students’ research committee, semnan university of medical sciences, semnan, iran. abstract. acute suppurative parotitis (asp) is a rare entity in early infancy. the clinical presentation may be non-specific. here, we describe a 70-day-old breast-fed female infant with massive purulent drainage from the mouth. diagnostic workup such as ultrasound revealed purulent exudate from stensen’s duct consistent with parotitis. culture of the exudate showed growth of staphylococcus aureus. based on the clinical presentations and ultrasound findings, the patient was diagnosed with right asp. the patient responded well to a 7day antibiotic therapy and supportive measures. thus asp should be considered in the differential diagnosis of a neonatal parotid swelling and purulent drainage from the mouth, since early and prompt diagnosis prevents morbidity and complications. to the best of our knowledge it is the first documented case of asp in early infancy in iran. keywords: parotitis, infant, infection introduction acute suppurative parotitis (asp) is rarely encountered in the neonatal and early infancy age group [1-3]. the peak incidence of this disease is between the age of 2 and 14 [4]. due to the rarity of acute suppurative parotitis, it is important to gain exact and extensive insight into the general and specific aspects of the pathological changes of salivary glands, in these age groups for prompt diagnosis to prevent complications. here, we describe a female infant with acute suppurative parotitis, due to the rarity and unusual presenting signs i.e. massive purulent drainage from the mouth. a study by spiegal et al identified only 32 cases over four decades in the english literatures, and found a 72% male prevalence [2]. however, to the best of our knowledge it is the first documented case in iran. the clinical characteristics and treatment options of this rare infection are also reviewed. case report a 70-day-old breast-fed female infant presented with a 1-day history of fever and purulent drainage from the mouth. she was born at 38 weeks gestation via natural vaginal delivery with a birth weight of 2960 grams. perinatal history was unremarkable. her postnatal course was further complicated by the development of neonatal poor feeding and sepsis. on the day of admission, she presented with purulent drainage from the mouth associated with low grade fever and irritability. her weight was 4600 g and axillary temperature was 38.5 degree centigrade. the parents reported no history of trauma to the infant's face or head, and the mother denied any history suggestive of mastitis or recent skin infection. general examination revealed an irritable, non-toxic looking, febrile infant. she was in a state of normal hydration and perfusion. the anterior fontanelle was normotensive and the occipito-frontal circumference was 38.5 centimetre. examination of the head and neck revealed a diffuse, tender, firm 3.0×3.0 centimetre area of induration with mild enlargement of the right mandibular and right preauricular region (the zone of the parotid gland) (fig. 1). the overlying skin was mildly inflamed with no obvious erythema or warmness. pus exuded from the right inflamed stenson's duct especially when pressure was applied to the gland. the rest of the physical examination was unremarkable. parotid pus, blood, and urine cultures were obtained. initial laboratory tests showed a hemoglobin level of 9.7 g/dl and total white blood cell count of 15200 /mm3 with 57% neutrophils, 40% lymphocytes. blood culture for bacterial growth was egative. the erythrocyte sedimentation rate was 77 mm/h and c-reactive protein, 54/3 mg/dl. the renal and liver function tests, serum electrolytes, and ___________________________________________________________ * corresponding author: mohsen pourazizi, md (m.pourazizi@yahoo.com). http://www.ajecr.org/ mailto:m.pourazizi@yahoo.com 90 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org figure 1 mild enlargement of the right mandibular and right preauricular region (arrow). urine analysis were normal. direct smear from stensen's duct showed gram positive cocci with 6-8 white blood cells and the culture showed growth of staphylococcus aureus. in sensitivity studies, the organism was sensitive to vancomycin, cephalexin, novobiocin, ceftriaxone, ciprofloxacin, cefazolin andtrimethoprim/sulfamethoxazole. ultra-sonography of the parotid glands revealed an enlarged right parotid gland with heterogeneous echogenicity compatible with asp, without any abscess formation. cervical and intraparotid lymph nodes were normal. based on the clinical presentations and ultrasound findings, the patient was diagnosed with acute right asp. she was treated with a 7-day course of parenteral clindamycin at 10 mg/kg/doses every 6 hours and amikacin at 15mg/kg/day. after 3 days of parenteral antibiotics therapy, the fever resolved and on the 5th day of treatment the parotid swelling gradually resolved. follow-up examination demonstrated no residues or abnormalities of the gland and she did not show chronic recurrent parotitis. discussion the most common presentation of asp is fever, swelling and erythema in the pre-auricular area [5]. purulent drainage from stenson's duct is pathognomonic of this condition, and culture of the exudate will both confirm the diagnosis and is of great help in the treatment [5]. the diagnostic criteria of suppurative parotitis include: a combination of parotid swelling, purulent exudation from stenson's duct, and growth of pathogenic bacteria in the pus culture [6, 2]. our patient fulfilled all these criteria. although the diagnosis of asp is primarily based on the patient’s clinical findings, but examination with ultrasound as a noninvasive and useful option may help confirm the diagnosis (as was applied in our patient), differential diagnosis to exclude other predisposing factors such as the anatomical abnormalities of stenson's duct, mechanical salivary duct obstruction secondary to sialolithiasis, and infection related to parotid gland, and neoplasms are also of great importance [5]. advanced imaging studies may be considered when the diagnosis is in doubt to rule out other congenital and inflammatory disorders of the parotid gland [5]. bacterial seeding of the parotid can occur hematogenously, but infection is more common from oral flora tracking in a retrograde fashion into the gland [5]. although, several risk factors for the development of asp have been identified, but our patient did not show any such risk factors. these risk factors include: low birth weight, oral trauma, immune suppression, and congenital variations in the ductal structure. sepsis and malnutrition are also frequently observed in infants with parotitis. dehydration is another risk factor as it causes salivary stasis leading to bacterial ascent from the oral cavity [7, 5]. breastfeeding or contaminated formula can transmit bacteria and potentially cause sialadenitis [8]. in our case, the infant was breastfed but her mother did not show any signs of mastitis as it is reported by sekhon et al [9]. the differential diagnosis of facial swelling that may be confused with parotid enlargement includes: maxillary infections, trauma, lymphangiomas, hemangiomas, lipomas, adenomas, extrapulmonary manifestations of tuberculosis and the human immunodeficiency virus in susceptible populations [5, 10]. in conclusion, although asp is rare, it should be strongly considered in cases of neonatal and infantile sepsis associated with facial swelling with or without any predisposing factors, as septic parotitis could be easily missed without careful examination. conflict of interest the authors declare no conflicts of interest. references 1. leake d, leake r. neonatal suppurative parotitis. pediatrics 46:202-207, 1970. 2. spiegel r, miron d, sakran w, horovitz y. acute neonatal suppurative parotitis: case reports and review. ped infect dis j 23:76-78, 2004. 3. ellies m, laskawi r. diseases of the salivary glands in infants and adolescents. head face med 6:1, 2010. 4. g. seifert am, haubrich j, chilla r. virussialadenitis. in: speicheldrüsenerkrankungen, pathologie, klinik, therapie, fazialischirurgie. thieme, stuttgart, ny, s.131-136, 1984. 5. schwab j, baroody f. neonatal suppurative parotitis: a case report. clin ped 42:565-566, 2003. 6. david rb, o'connel ej. suppurative parotitis in children. am j dis child 119:332-335, 1970. 7. d’souza jn, geary c, mukerji s. neonatal parotid gland enlargement: is it suppurative parotitis? a case report. am j case reports 13:41, 2012. 8. ozdemir h, karbuz a, ciftci e, fitoz s, ince e, dogru u. acute neonatal suppurative parotitis: a case report and review of the literature. international journal of infectious diseases. int j infect dis 15:e500-502, 2011. 9. sekhon p, williams d, sara jds, mcculloch na. acute bacterial suppurative parotitis of the neonate: a case report and review. int j ped otorhinolaryngol 7:132-133, 2012. 10. stiller m, golder w, doring e, biedermann t. primary and secondary sjogren's syndrome in childrena comparative study. clin oral invst 4:176-182, 2000. http://www.ajecr.org/ 64 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(4):64-67 original article variations in adrenal hormones in law enforcement servicemen during a mission to local armed conflict roman koubassov*, yury barachevsky department of medicine catastrophe, northern state medical university, archangelsk, russia abstract. in a previous study, we reported changes in the adrenocorticotropic hormone (acth) and cortisol secretion in blood samples from law enforcement personnel during the mission to local armed conflict region. in the present study, we demonstrate those changes collectively with additional data on changes in the adrenaline and noradrenaline in the urine samples of the same individuals. the study was conducted on 48 male officers who were deployed to an army conflict teritory for a duration of 4 months. at the onset of the mission, there was a modest increase in all hormones corresponding to the general adaptation syndrome theory. as the mission started, significant increases were observed in the mean levels of the hormonal parameters in both serum and urine at different time points as compared to those before the mission. at first week of deployment, a sharp increase in the secretory activity of medulla and cortical adrenal gland was found and at the termination of the mission a dysfunction of hypophysis-adrenal gland regulation system was identified. these findings might lead to disturbances in interhormonal relationships and cause decreased stress tolerance in the relevant individals. keywords: law enforcement officer, adrenaline, noradrenaline, adrenocorticotropic hormone, cortisol, emergency condition introduction the principal responsibility of any government is to secure safety of its citizens. accordingly, every type of life activity has to be protected from potential conflict hazards. the world community has entered into a new era in the 21st century, creating intensification of different political, ideological, religion, or economical conflicts and crises. some factors with much impact on social transformation includes technological progresses and global environment changes 1-3. in order to maintain law enforcement in different territories and secure the safety of citizens, special police squads are needed. the professional task of law enforcement officers occurs in extreme conditions and often in emergency situations. the service duties are in the range of medium security with hardware assistance, special equipment and different weapons [4, 5]. in addition, besides professional detrimental factors affecting an armed personnel who has been trans-located from another region, other factors such as specific climatologic and geographical environments of a combat territory are pivotal [6, 7]. these factors based on their severity and duration may create considerable health problems ranging from functional disorders to pathological conditions with permanent impairments [8, 9]. in such individuals, various functional changes occur in order to provide adaptation to those conditions. in fact, the endocrine system plays a major role in forming a compensatory regulatory mechanism to counter extreme impacts. in such response, activation of sympathoadrenal system plays a pivotal role [10, 11]. in a previous study, we reported changes in the adrenocorticotropic hormone (acth) and cortisol secretion in blood samples from law enforcement officers during the mission to local armed conflict area [12]. subsequently, the aim of the present study was a comprehensive assessment of changes in acth, cortisol, adrenaline, and noradrenaline, secretions in both blood and urine samples from the same group of servicemen during a mission to the local armed conflict territory. materials and methods we studied 48 male officers (mean age: 28.28±0.51) from the ministry of home affairs who were law enforcement servicemen. all subjects had a mission to an army conflict territory (north caucasus) for the purpose of maintaining law enforcement. the duration of their mission was 4 months. in all cases, we measured the blood serum acth by radioimmunoassay (cis bio international, cedex, france) and cortisol by enzyme immunoassey (monobind inc, california, usa). in addition, the levels of adrenaline and noradrenaline in urine samples of all cases were measured using gass chromatography method with mass spectrometer detector. statistical analysis of the data was performed using the spss 15.0 software. the mean and ___________________________________________________________ * corresponding author: roman v koubassov, phd ( romanas2001@gmail.com). http://www.ajecr.org/ mailto:romanas2001@gmail.com 65 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org standard deviation (sd) was calculated for each measurement. to assess the universal distribution, shapiro-wilk normality test was applied. comparative analysis of means was performed by wilcoxon rank test. a p value less than 0.05 was considered as significant. results overall the results of our study showed that in all cases, the levels of the hormones examined were in the normal physiological range before the mission. however, significant increases were found in the mean levels of the hormonal parameters at different time points during the mission as compared to those before the mission. the mean serum acth level was increased more than two-fold in the individuals at 14 days after mission as compared to before mission (table 1). at 1 month after mission, the acth level was increased four-fold (p<0.001). at 2 months after mission, we found a subsequent acth rise but it was less significant when compared with the measurement at 1 month (p=0.005). at the time of termination of the mission, the mean acth level was decreased but it still remained higher than the level before mission (p<0.001). the serum cortisol level changed during the combat mission with a different pattern from acth dynamics (table 1). particularly, at 14 days after the mission, the mean cortisol level increased as compared with before mission (p=0.002). however, it was decreased at 1 month (p=0.006) and 2 months (p<0.001) as compared with 2 weeks after the mission. the mean cortisol level at 1 and 2 months showed no statistical difference as compared to before mission (p>0.05). at the end of the mission (after 4 month), the cortisol level increased to a maximum level. the level was similar to that of the two weeks measurement but better than before the mission and those at 1 and 2 months (p=0.05 and p=0.003 respectively). the dynamics of urinary catecholamine levels in law enforcement servicemen during combat mission had analogous features with serum acth and cortisol changes but with less statistical significance (table 2). urinary excretion of adrenaline in 2 weeks combatants was increased twice as compared with before the mission (p<0.001). at 1 month, this parameter remained similar to that at 2 weeks (p=0.65). at 2 months, urinary adrenaline concentration reached the highest level as comared with 2 weeks and 1 month measurements (p=0.002 and p=0.02 respectively). when the combat mission was over, the mean adrenaline level decreased, but it was higher than that of before the mission (p=0.04). the analyses of urinary noradrenaline excretion showed an increase at 2 weeks after the mission (table 2). at 1 month after the mission, unlike adrenaline, the noradrenaline level increased as compared to that at 2 weeks (p=0.05) and continued at a similar level at 2 months. when the combat mission was over, noradrenaline concentration decreased, but it was higher than that of before the mission (p=0.003). discussion suprarenal hormones play a leading role in the formation of adaptation response to environmental factors in humans [13]. abnormality of adrenal regulatory function occurs during excessive and long-term exposure to harmful envir onmental agents and results in a decline in human performance and physical and mental suffering (distress). the principal manifestations of these abnormalities are hormonal hypersecretion, target cell resistance, and failure of feedback regulation mechanism [14, 15]. various investigations have shown that combatants with first time experience of deployment to a mission have allostasis laboratory markers of increased acth and cortisol levels after the mission. the level reaches to a maximum after 2 weeks. at the end of combat mission, these parameters decrease but remain higher than those at the beginning of the mission. this is indicative of a homeostasis imbalance retention [16]. a number of studies demonstrate that persistence of high levels of catecholamine, acth and cortisol in combatants for more than 6 months predetermine a triggering pathogenic mechanism of posttraumatic stress disorder [17-19] in our study, the serum acth level at 2 months after the combat mission was constantly increased. at the time of termination of the mission (after 4 month), it decreased, but still exceeded twice when compared with that of before the mission. with regard to the serum cortisol changes, in spite of a sharp increase after the first two weeks, a decrease of this hormone was obserrved, although acth table 1 serum adrenocorticotropic hormone (acth) and cortisol changes at different durations of mission duration acth (pg/ml) cortisol (nmole/l) before mission a 20.07±3.20 404.81±124.54 2 weeks b 55.80±15.68 489.25±112.46 1 month c 86.43±17.56 426.80±102.54 2 months d 4 months e 96.60±17.18 55.37±10.70 407.82±101.66 471.16±117.78 values are mean±sd. statistical significances: acth (a vs. b,c, d,e, and b vs. c,d, and c vs. e and d vs. e, p<0.001; b vs. e, p< 0.81; c vs. d, p<0.005. cortisol (a vs b, p<0.002; a vs. c, p<0.35; a vs. d, p<0.89; a vs. e, p<0.003; b vs. c, p<0.006; b vs. d, p<0.001; b vs. e, p<0.44; c vs. d, p<0.37; c vs e, p<0.05; d vs e, p<0.006 . table 2 urinary adrenaline and noradrenaline changes at different durations of mission duration adrenaline (nmole/day) noradrenaline (nmole/day) before mission a 83.01±18.63 160.35±38.85 2 weeks b 117.12±55.68 229.30±102.34 1 month c 122.80±67.56 275.68±121.58 2 months d 4 months e 161.55±77.18 91.32±57.62 268.32±111.67 193.61±66.05 values are mean±sd. statistical significances: adrenaline (a vs. b,c,d, and d vs. e, p<0.001; a vs. e and a,c vs. e, p<0.04; b vs. c, p<0.65; b,c vs. d, p<0.002; b vs. e, p<0.03). noradrenaline (a vs. b,c,d, and c,d vs. e, p<0.001; a vs. e, p<0.03; b vs. c,e, p<0.05; b vs. d, p<0.07; c vs. d, p<0.76). http://www.ajecr.org/ 66 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org was increased. therefore, hormonal disbalance could be the first sign of disturbances in hypophysis-adrenal gland regulation system. at the end of mission, a dramatic cortisol increase was found, comparable with the first 2 weeks. in fact, this may predestine a derangement of adaptation process. catecholamine (adrenalitermine or noradrenaline) generated in adrenal medulla produces a short-term adaptation process. it has a catabolic effect and involves in almost all metabolisms. catecholamine secretion is increased in stress conditions and extreme situations. the target cells and action mechanisms for adrenaline and noradrenaline are different though of common physiological effect. thus, adrenaline (anamed as “fear hormone”) improves individual’s weakness resistance in initial stress time at high speed. the noradrenaline (named as “fury hormone”) act after adrenaline. its blood secretion corresponds with aggression and promote muscle strength. noradrenaline is potent as adrenaline effect [20, 21]. in our study, we found that at first two weeks after the combat mission, the urinary adrenaline and noradrenaline levels were increased in the military servicemen. this observation could be atttibuted to the natural adaptation process to environmental changes. furthermore, at two months, the adrenaline level was increased but when the mission was over it was decreased. however, the adrenaline level remained higher than that before the combat mission. this observation could be due to a retardation of adaptation process and probably first sign of distress. concerning the noradrenaline changes, there was a significant increase and prolonged duration of this hormone. from the physiologic point of view, this appears to be a normal individual’s response required for elongation of survival probability in stress situation. similar results have been obtained in other investigations. taken together, in the military servicemen during the armed mission a specific feature of catecholamine dynamics was observed which was attributable to adaptation process but in some cases with signs of distress. these hormonal imbalances could remain up to 6 month or more [22]. in conclusion, in combatants particularly special police squads that have acquired professional skills in extreme conditions and during the mission to local armed conflict territory, the secretory function of adrenal gland undergoes changes corresponding to the principles of general adaptation syndrome theory. at first week after the mission to military zone, a sharp increase is seen in the secretory activity of medulla and cortex of adrenal gland. at the termination of combat mission, signs of dysfunction in hypophysis–adrenal gland regulation system appear that may lead to disturbances in interhormonal relationships and thus weakening of stress tolerance. the disturbance of endocrine regulation requires establishment of special measures to reduce it. the ultimate aim of these precautions is to provide increased resistance and capability to deal with extreme conditions in emergency case and prevention of mortality risk. these measures should include a long-range and clear-cut planning of combat missions, early diagnostics including laboratory tests for armed personnels to be deployed to harmful locations, special training (physical, psychological, preventive, etc.) for armed personnels who are going to work in extreme conditions and emergency situatiuons, and special medical rehabilitation measures that provide rapid restoration of the individual’s health after the termination of combat mission. . conflict of interest the authors declare no conflicts of interest. references 1. iverson t, perrings c. precaution and proportionality in the management of global environmental change. glob environ chang 22:161-177, 2012. 2. kovats rs, butler cd global health and environmental change: linking research and policy. curr opin environ sustain 4:44-50, 2012. 3. schulze-makuch d, irwin l.n, fairén a.g. drastic environmental change and its effects on a planetary biosphere. icarus 225:775-780, 2013. 4. brisebois r, hennecke p, kao r, mcalister v, po j, stiegelmar r, tien h. canadian forces health services research consortium. the role 3 multinational medical unit at kandahar airfield 2005-2010. can j surg 54:124129, 2011. 5. koubassov rv, barachevsky ye, lupachev vv. problems of professional safety of local armed conflict servicemen. medico-biological and socio-psychological problems of safety in emergency situations. 1:39-46, 2014. (in russian). 6. shellman sm, hatfield c, mills m.j. disaggregating actors in international conflict. j peace res 47:83-90, 2010. 7. herrell rk, bliese pa, hoge cw. effect of combat intensity, depression, alcohol misuse, and family history of depression and alcohol misuse on ptsd in a sample of post-deployment us soldiers. compr psychiatry 54:e4-e5, 2013. 8. artiss k. the combat soldier. mil med 165:33-40, 2000. 9. dobson m. combat stress reaction. in encyclopedia of stress (fink g. ed). usa: academic press, pp. 524-529, 2007. 10. dunn aj, swiergiel ah. the role of corticotropinreleasing factor and noradrenaline in stress-related responses, and the inter-relationships between the two systems. eur j pharmacol 583:186-193, 2008. 11. kino t., charmandari e, chrousos g.p. disorders of the hypothalamic-pituitary-adrenocortical system. in handbook of neuroendocrinology (fink g., pfaff dw, levine j. eds). usa, ny: academic press, pp. 639-657, 2012. 12. koubassov rv, barachevsky ye, lupachev vv. adrenocorticotropic hormone and cortisol secretion changes among the law enforcement personnel during the mission to the area of armed conflicts. int j biomed 4:7678, 2014. 13. selye h. stress without distress. philadelphia, usa: lippincott, p. 171, 1974. 14. thrivikraman k.v, nemeroff cb, plotsky pm. http://www.ajecr.org/ 67 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org sensitivity to glucocorticoid-mediated fast-feedback regulation of the hypothalamic-pituitary-adrenal axis is dependent upon stressor specific neurocircuitry. brain res 870:87-101, 2000. 15. buckingham jc. glucocorticoids, role in stress. in encyclopedia of stress (fink g. ed). usa: academic press, pp. 210–217, 2007. 16. morgan ca, wang s, rasmusson a, hazlett g, anderson g, charney, ds. relationship among plasma cortisol, catecholamines, neuropeptide y, and human performance during exposure to uncontrollable stress. psychosom med 63:412-422, 2001. 17. yehuda r. current status of cortisol findings in post-traumatic stress disorder. psychiatr clin north am 25:341-368, 2002. 18. bremner jd, vythilingam m, vermetten e, adil j, khan s, nazeer a, afzal n, mcglashan t, elzinga b, anderson gm, heninger g, southwick sm, charney ds. cortisol response to a cognitive stress challenge in posttraumatic stress disorder (ptsd) related to childhood abuse. psychoneuroendocrinology 28:733-750, 2003. 19. auxemery y. etiopathogenic perspectives on chronic psycho traumatic and chronic psychotic symptoms: the hypothesis of a hyperdopaminergic endophenotype of ptsd. med hypotheses. 79:667-672, 2012. 20. emerson aj, kappenman dp, ronan pj, renner kj, summers ch. stress induces rapid changes in serotonergic activity: restraint and exertion. behav brain res 111:83-92. 21. eiden le. neuropeptide–catecholamine interactions in stress. in: a new era of catecholamines in the laboratory and clinic. usa, elsevier inc. 68:399-404, 2013. 22. murburg mm, mcfall me, ko gn, veith rc. sympathoadrenal response to combat-related versus combat-unrelated stressors in combat veterans with posttraumatic stress disorder (ptsd) and controls. biol psychiatry 25:a33-a34, 1989. http://www.ajecr.org/ 258 am j exp clin res, vol. 5, no. 1, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(1):258-262 original article frequency of genitourinary tract disorders in a tertiary hospital in isfahan, iran hamid mazdak 1, 2 , zahra tolou-ghamari 1 * 1 isfahan kidney transplantation research centers, alzahra research centers, isfahan university of medical sciences, isfahan, iran 2department of urology, isfahan university of medical sciences, isfahan, iran abstract. for advanced planning related to health issues for hospitalized patients, we aimed to categorize genitourinary tract disorders in a large, representative sample of patients' population in a referral teaching hospital in isfahan, iran. in this retrospective study, we enrolled 3721 patients of both genders with a mean age of 45 (ranged from 1 to 93) years old. clinical and demographical data were recorded in excel. the statistical analyses processed using spss for windows. 79% of total population were males and in the 58% age ranged from 20 to 60 years old that was correlated to employed years of life. in the 78% complications related to genitourinary tract disorders ranked as: urolithiasis (n = 27%), prostate (n = 17%), kidney (n = 14%), testis (n = 11%) and bladder (n = 9%). in the residual 22% of population classified disorders were as: penis, urethral stricture, inguinal hernia, vesicoureteral reflux, urinary incontinence, cystocele, renal colic and adrenal gland. in the 528 patients classified kidney disorders were ranked as: esrd (51%), unknown kidney disease (38%), malignant neoplasm (7%), and cystic kidney (6%). testis disorders classified as varicocele (40%), testicular torsion (23%), cryptorchidism (16%), and hydrocele (10%). this study confirmed that among a group of genitourinary tract disorders, the frequency of urolithiasis and prostate were higher than others. the percentage of males with genitourinary tract disorders was higher than females. among testis disorders varicocele and testicular torsion was high. in the more than of half studied patients’ age was related to active and working years of lifetime. finally as the occurrence of urolithiasis and prostate disorders is increasing in isfahan, further research in these directions seems to be advantageous. keywords: urolithiasis, prostate, clustering, hyperplasia, genitourinary, malignant, penis introduction according to previous reports, urological disorders account for approximately 830,000 deaths (ranked as 12th) and 18,467,000 disability-adjusted life years (ranked as 17-th) annually. individuals with chronic kidney disease (ckd) often suffered from cardiovascular or cerebrovascular disease [1, 2]. in developed countries the fourth most common causes of end-stage renal disease (esrd), reported to be as autosomal dominant polycystic kidney disease, which affects 10 million people worldwide [3]. stengel b. et al., in 2003 investigated that after diabetes, glomerulonephritis places as the leading reason for esrd in european countries [4]. a recent study in 2016 recognized 48283 women with episode uncomplicated repeated urinary tract infection (uti); accounting for a total frequency of 102 per 100000 women, that were highest in women between the ages of 18-34 and 55-64 years old [5]. bladder infections and catheterization are the most important cause of uncomplicated and complicated uti respectively [6]. obstructive or reflux nephropathy is often attributed to urinary schistosomiasis in developing countries as a pattern of uti [7]. morton ar. et al., in 2002 confirmed that kidney stones seem to be a communal struggle in industrial countries [8]. the disease affects all age groups from less than 1 year old to more than 70 [9]. a recent publication in 2016 confirms that over last two decades the prevalence of hyperoxaluria in stone forming patients has been increased. there is may be the existence of geographical display between asian and western countries [10]. in compare to general population, in pediatric patients with unilateral renal agenesis, the prevalence of hypercalciuria and/or hypercitraturia seems to be greater. similarly the prevalence of urolithiasis in the families of these children is also higher than that in the general population [11]. related to lower ut symptoms, benign prostatic hypertrophy could lead to obstructive renal failure and esrd [12] the three variants including prncr1, pcat1, and pcat2 that are situated within or near a number of prostate cancerrelated long noncoding rnas at the 8q24 area, could specified as multiple risk ___________________________________________________________ * corresponding author: prof. zahra tolou ghamari (toloeghamari@pharm.mui.ac,ir). http://www.ajecr.org/ mailto:toloeghamari@pharm.mui.ac,ir 259 am j exp clin res, vol. 5, no. 1, 2018 http://www.ajecr.org alternatives [13]. moreover, the study was designed to classify genitourinary tract disorders in order to provide reference data of surgical relevance. materials and methods ethical approval this study was approved by ethics committee of isfahan kidney transplantation research center (iktrc) and supported by isfahan deputy of research with the code number of 295126. design and data collection a retrospective survey associated to 3721 patients (females; n = 797 and males; n = 2924) was carried out from 2013 to 2015. all patients attended urology ward located at the alzahra hospital, conducted to isfahan kidney transplantation research center iktrc. there was no induction in treatment procedure. demographical data, patients' hospital records' no, the exact date of (admission, discharge, and hospital stay) and the reason for urology ward attending were noted in excel. statistical analysis all collected data were analyzed by spss v. 20 (chicago, il, usa). distribution analysis showed that most of the parameters follow non-gaussian distribution. mean, minimum and maximum were calculated for the values of age. results out of total population (n= 3721) studied 79% were males. as shown in figure 1, the mean age of patients was 45 years old, which was ranged from 1 to 93 years old. the lowest and highest rate of age belong to the patients with < 20 years old (12%) and patients with 20 to 60 years old (58%) respectively. studied population related to the age groups of 60 to 93 year old were involved in 30%. figure 2 shows that in 78% of patients with genitorurinary tract complications that ended to surgery treatment were ranked as highest to lowest such as: urolithiasis (n = 992; 27%) > prostate (n = 614; 17%) > kidney (n = 528; 14%) > testis (n = 411;11%) and bladder (n =349; 9%). analysis of data in 992 patients with stone showed that 69% were males. with the mean age of 44, the minimum and maximum age was from 1 to 88 years old. there were 33 patients under the age of 10 years old as ranged from the age of 1 (n = 5), age 5 (n = 5), age 2 (n = 9) and so on. in these children the location of stones was ranked as; ureter (67%) > kidney (24%) and bladder (9%). in the 959 patients that aged from more than 10 to 93 years old, the location of stones recorded as follow: kidney (50%, plus 1% in pelvis and calyx) > ureter (44%) and bladder 5%. the frequency of disorders related to prostate was 614 out of 3721 (17%). figure 3 shows the distribution of age in patients with prostate disorders. with a minimum of 22 and a maximum of 91 year old the mean age of patients with prostate complications' was 65 year old. in 528 patients with the mean age of 43 (ranged from 3-89 year old) due to disorders of kidneys ended to surgery treatment, figure 1. distribution of age in population studied (n = 3721). figure 2. frequency of urological complications in 78 % of population studied (n = 2894). figure 3. distribution of age in patients with prostate disorders. 76% were males (figure 4). the frequency of end stage renal disease (esrd) or kidney donors was 267 (51%) that was more than 198 that was recorded as unknown kidney disease (38%). malignant neoplasm of kidneys and cystic kidneys was diagnosed in 36 (7%) and 27 (5%) patients respectively. the mean age of patients with testis disorders was 26.2 years old (ranged from 1 to 83 years old). as shown in figure 5, age related testis disorders in 26 and 59% were between 10 to 20 and 20 to 60 years old respectively. figure 6 shows the reason for testis surgery ranked as: varicocele (40%) > testicular torsion (23%) > cryptor http://www.ajecr.org/ 260 am j exp clin res, vol. 5, no. 1, 2018 http://www.ajecr.org figure 4. the distribution of patients with kidney disorders (n = 528). figure 5. distribution of age in patients with testis disorders (n = 411). figure 6. reasons for surgery related to testis (n = 411). chidism (16%)> hydrocele (10%)> inflammation of testicles and epididymis (7%) > testicular shrinkage (2%) > male sterilization (1%) > urethral diverticulum (1%). the mean age related to bladder disorders was 54.8 year old (ranged from 3 89 years old). the cause related to bladder disorders in 81 % was not clear. in the remaining population of patients with genitourinary tract complications those treatments ended to surgery could be ranked as: penis > urethral stricture > inguinal hernia > vesicoureteral reflux > urinary incontinence > cystocele > renal colic > adrenal gland). discussion the results of this investigation showed that the highest rate of patients (64%), those needed surgical treatment due to genitourinary tract disorders, belonged to the age group which was associated to employ years of life from 20 to 70 years old. in this study the frequency of calculi was positioned as first place with a value of 27% (n = 3721) in which 69% were males. the incidence of nephrolithiasis is increasing worldwide, particularly in women and with increasing age [14]. about 5% of american women and 12% of men will develop a kidney stone at some time in their life, and prevalence has been rising in both sexes [15]. previous publication confirmed that nephrolithiasis can have different clinical presentations, ranging from asymptomatic to large obstructing calculi in the upper urinary tract that can severely impair renal function and lead to esrd [9]. a recent study by pickard r et al., in 2015 showed that a combination of tamsulosin and nifedipine seem not to be effective in decreasing the need for further treatment to achieve stone clearance in 4 weeks in patients with ureteric colic [16]. urinary tract stone in our population affected all age groups from 1 to 88 year old. this is in agreement with previous publication by hussain et al., in 1996 related to the afro-asian geoepidemiological study of stone. they confirmed that the disease affects all age groups from less than 1 year old to more than 70, with a male to female ratio of 2 to 1. they reported also that the prevalence of calculi ranges from 4 to 20 percent [8]. in this study, urinary tract stones in small number of children (n = 33) and adult (n = 959) were noted as ureter (67%), kidney (24%) and kidney (51 %) and ureter (44%) respectively. according to previous report, prostate cancer is the second mainly detected cancer as well as the sixth most cause of death in males with cancer worldwide [17]. the rates of incidence and mortality are also increasing in asian and european countries.18 in agreement with previous studies, the attendance of urology ward due to prostate disorders was placed in second order. regarding to risk factors old age, race, family history and chronic inflammation (benign prostate tissue) seem to be important; as men over the age of 65 are at higher risk for prostate cancer [17-19]. in this study in 96% of population age was more than 50 year old. regarding to kidney disorders, patients with esrd have an exceedingly high morbidity and mortality compared to the general population [20]. in this study in the 51 % of patients with disorders of kidneys that ended to surgery treatments, the final recorded cause was mentioned as esrd. serrano t, et. al., in 2013 stated that there is data for a number of associations between rates of four male reproductive disorders such as hypospadias, cryptorchidism, testicular cancer and low sperm concentration at an international scale [21]. in agreement with previous publication, varicocele, testicular torsion and cryptorchidism ranked as the highest causes of testis disorders’. in a minor group of patients disorders of pelvic, penis and other complications were recorded. according to report published in 2013, in the unites stated of america, the incidence of micropenis was malignant neoplasm of kidneys esrd or kidney donors unknown kidney disease cystic kidneys 0 50 100 150 200 250 300 1 2 3 4 n u m b e r o f p a ti e n ts kidney disorders http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/pubmed/?term=pickard%20r%5bauthor%5d&cauthor=true&cauthor_uid=25998582 http://www.ncbi.nlm.nih.gov/books/nbk11791/ http://www.ncbi.nlm.nih.gov/pubmed/?term=serrano%20t%5bauthor%5d&cauthor=true&cauthor_uid=23670171 261 am j exp clin res, vol. 5, no. 1, 2018 http://www.ajecr.org reported as 1.5 in 10 000 male children born between 1997 and 2000. in some turmoil related to penis, a fibrous scar in the tunica albuginea can result in multiple penile deformities. torsion of the penis is also not uncommon but rarely provokes a complaint. regarding the pelvic floor disorders' urinary and fecal incontinence and pelvic organ prolapse, it affects population of most women [22-27]. finally given the burden associated to the iranian health care system, for a big population of patients with genitourinary tract disorders which treatment ended to surgery, advanced research is recommended for further understanding toward improvement of future surgical management. conclusion the result of this study confirmed that further attempt for an adjustable strategy could have beneficial direction related to pharmacological and surgical care toward patients with prostate disorders. also further attention toward the study of urolithiasis seems to be important. finally, the outcome of this research suggests a classification of health scope outcome and economic design by vigilant manipulative tactics (based on: iranian evidence-problem-solve) in the field of medicine. acknowledgement this study was supported by grant no. 295126 from isfahan university of medical sciences. conflict of interest the authors declare no conflicts of interest. references 1. hostetter th. chronic kidney disease predicts cardiovascular disease. new engl j med 351:1344–1346, 2004. 2. tolou-ghamari z. investigation of final causes of death in 5360 deceased patients within a teaching hospital in isfahan, iran. am j exp clin res 3:161-164, 2016. 3. grantham j. pathogenesis 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lee jp. early referral to a nephrologist improved patient survival: prospective cohort study for end-stage renal disease in korea. plos one 8:e55323, 2013. 21. serrano t, chevrier c, multigner l, cordier s, jégou. international geographic correlation study of the prevalence of disorders of male reproductive health. hum reprod 2013 28:1974-1986, 2013. 22. levine l, rybak j, corder c, farrel mr. peyronie's disease plaque calcification-prevalence, time to identification, and development of a new grading classification. j sex med 10:3121-3128, 2013. 23. shaeer o. torsion of the penis in adults: prevalence and surgical correction. j sex med 5:735-739, 2008. 24. hatipoğlu ni, s kurtoğlu. micropenis: etiology, diagnosis and treatment approaches. j clin res pediatr endocrinol 5: 217-223, 2013. http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/pubmed/?term=suskind%20am%5bauthor%5d&cauthor=true&cauthor_uid=26825489 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http://www.ncbi.nlm.nih.gov/pubmed/?term=chevrier%20c%5bauthor%5d&cauthor=true&cauthor_uid=23670171 http://www.ncbi.nlm.nih.gov/pubmed/?term=multigner%20l%5bauthor%5d&cauthor=true&cauthor_uid=23670171 http://www.ncbi.nlm.nih.gov/pubmed/?term=cordier%20s%5bauthor%5d&cauthor=true&cauthor_uid=23670171 http://www.ncbi.nlm.nih.gov/pubmed/?term=j%c3%a9gou%20b%5bauthor%5d&cauthor=true&cauthor_uid=23670171 http://www.ncbi.nlm.nih.gov/pubmed/?term=rybak%20j%5bauthor%5d&cauthor=true&cauthor_uid=24119147 http://www.ncbi.nlm.nih.gov/pubmed/?term=corder%20c%5bauthor%5d&cauthor=true&cauthor_uid=24119147 http://www.ncbi.nlm.nih.gov/pubmed/?term=farrel%20mr%5bauthor%5d&cauthor=true&cauthor_uid=24119147 http://www.ncbi.nlm.nih.gov/pubmed/?term=kurto%26%23x0011f%3blu%20s%5bauth%5d 262 am j exp clin res, vol. 5, no. 1, 2018 http://www.ajecr.org 25. nygaard i, barber md, burgio k. prevalence of symptomatic pelvic floor disorders in us women jama 300: 1311-1316, 2010. 26. yazdani m, tolou-ghamari z, asadi-samani m, mazdak h, yazdani e. investigation of prostate disorders in 614 patients attended alzahra urology ward between the years 2013 to 2015 in isfahan. int j pharm clin res 8: 1543-1547, 2016. 27. tolou-ghamari z, mehavari-habibabadi j, palizban aa. evidence based pharmacotherapy of epilepsy. arch neuroscience 2: e18468. 28. tolou-ghamari z, najafi mr, mehavari habibabadi j, zare m. preliminarily analysis of carbamazepine (cbz) c0 in patients visited isfahan epileptic clinics. int j prev med s343-346, 2013. http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=18799443 408 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2021;8(1):408-413 original article introduction of the yeast strain cryptococcus uzbekistanensis as a melatonin producer: evaluation of the effect of different growth media on melatonin production ali nosrati* school of allied medicine, tehran university of medical science (tums), tehran, iran abstract. melatonin (n-acetyl-5-methoxytryptamine) is a neuro-hormone produced in the pineal gland. it is found in animals, plants and fungi. its biological properties contribute to the circadian rhythm, reproductive physiology and antioxidant activity. melatonin is known to help reduce the effects of the jet lag by cultivating the necessary re-set of the body's circadian rhythm. natural production of melatonin in humans depends on the amount of light as well as age; in fact, natural melatonin production declines with age. the present study was conducted to evaluate melatonin expression in yeast species. one hundred and thirty yeast species were screened and analyzed for melatonin production. it was found that cryptococcus uzbekistanensis produced 46 ppm of melatonin in the optimized condition. the box-behnken methodology was used to identify significant factors in terms of the carbon source, temperature, growth incubation, and tryptophan concentration for optimization of melatonin production. the response surface methodology (rsm) was applied to optimize the levels of these factors. in the presence of 3 ppm of tryptophan in the growth media, a growth incubation time of 72 hours, glucose as carbohydrate, and a temperature of 308 kelvin, cryptococcus uzbekistanensis produced the maximum concentration of melatonin. this is among few instances that a microorganism is found to produce melatonin and thus the results can be utilized for industrial production of melatonin. keywords: melatonin, c. uzbekistanensis, media engineering, experimental design method, box-behnken, rsm. introduction melatonin is a major neuro-hormone secreted during the dark hours of the night by the human pineal gland. melatonin synthesis is inhibited when the retina detects light. first melatonin production in humans goes back to age 3–6 month. the maximum production of melatonin occurs at the age of 1 to 5 years [1]. melatonin secretion by the pineal gland progressively declines by age. a significant reduction of circulating melatonin is also observed in numerous disorders including neurological conditions such as alzheimer’s disease and metabolic disorders particularly type 2 diabetes [2], which results in sleep problems. melatonin has an effective role in preventing jet lag symptoms and therefore it could be used for treatment of the travelers experiencing the jet lag [3]. melatonin also has anti-inflammatory and anti-oxidant effects. a study recently assessed the effects of melatonin on the covid 19 patients and found that anti-inflammatory effects of melatonin could help the patients [4]. using microorganisms to produce drugs and hormones, biotechnology can help ease the symptoms of human diseases. melatonin is found in some yeast species. according to a study, melatonin is a bioactive compound present in wine since it is synthesized by saccharomyces uvarum and some strains of saccharomyces cerevisiae during alcoholic fermentation. rodriguez naranjo et al. found melatonin in wine and introduced saccharomyces strains as the source [5]. another study found melatonin not only in saccharomyces strains but also in non-saccharomyces strains of wine yeasts [6]. moreover, torulaspora delbrueckii, hanseniaspora uvarum, starmeralla bacillaris and metschnikowia pulcherrima also produce melatonin [7]. microorganisms such as fungi could be used for expression and production of melatonin. among fungi, yeasts are the best choices since they are safe; single-cell organisms; moreover, considering the aforementioned studies, there is strong evidences of the presence and production of melatonin in yeasts. the aim of the present study was to find yeasts that produce melatonin. one hundred and thirty species of yeasts from the collection of environmental biotechnology laboratory of university of tehran were ___________________________________________________________ * corresponding author: ali nosrati, ms (a-nosrati@student.tums.ac.ir) http://www.ajecr.org/ 409 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org screened to investigate melatonin production. materials and methods collection of soil samples and isolation in this study, 130 yeast isolates were collected from different areas of iran and analyzed for isolation of potent yeasts. identification and phylogenic analysis for identification of the selected yeast, pcr amplification of the its dna was performed. the yeast was grown overnight in potato dextrose broth and the cells were harvested for dna extraction using an extraction kit. primers its1 (5′-tcc gta ggt gaa cct gcg g-3′) and its4 (5′-tcc tcc gct tat tga tat gc-3′) were used to amplify its and the obtained fragment was then sequenced and compared with the genbank data-base. phylogenic analysis of the yeast was also performed using the mega software. yeast isolation a collection of 130 yeast strains was available from previous work of yeast isolation. soil samples were collected from various regions of iran and enriched and cultured in ypg containing 5 g/l yeast extract, 10 g/l peptone, 20 g/l glucose with 100 mg/l added chloramphenicol and 500 mg/l rose bengal to isolate indigenous yeasts. the obtained isolates were purified on ypg agar and stored at 4˚c. sample preparation and evaluation of melatonin production in the next step 1×106 cell/ml of each yeast was cultured in a 50-ml flask containing 100 ml czapek dox broth (30 g/l sucrose, 3 g/l sodium nitrate, 1 g/l dipotassium phosphate, 0.5 g/l magnesium sulfate, 0.5 g/l potassium chloride and 0.01 g/l ferrous sulfate) supplemented with 0.59 g/l standard tryptophan (trp) (rodriguez-naranjo et al. 2012). the flasks were shaken at 120 rpm at 28˚c. after 24 hours, 1 ml of the culture medium was taken for melatonin production examination. it should be noted that all of the flasks and samples were protected from light during incubation since melatonin is a light-sensitive compound. melatonin production was assayed using the hplc method (hamase et al. 2000). selection of spectrophotometric positive species saccharomyces cerevisiae is a melatonin producer. our method for spectrophotometric detection of positive species was to culture saccharomyces cerevisiae in czapek dox broth medium in the presence of 1% of tryptophan by shaking at 120 rpm for two days. five milliliters of growth medium was then separated and centrifuged for 5 minutes at 400rpm.next, 15ml of dichloromethane was injected as solvent to separate melatonin after shaking the mixture. then, 3ml was separated from the top phase in which melatonin existed, and analyzed using a spectrophotometer with ʎ=285nm, and its absorption was 0.0713. using this method, all 130 yeast isolates were cultured. the species table 1 with the highest absorption and melatonin production were selected. hplc method a high performance liquid chromatography system with a uv detector was used for the determination of melatonin (shimadzu, japan). the hplc unit consisted of a pump (lc-10advp), injector (7725a), fluorescence detector (254 nm), integrator (scl10avp) and analytical column (clcods (25 cm) pn: 228-17873-92. the eluent included 70% acetonitrile and 30% h2o with a flow rate of 1 ml/min (hamase et al. 2000). optimization of culture condition various factors were evaluated for determining the ideal condition for melatonin production including the carbon source, four different concentrations of tryptophan, temperature and growth incubation. the aforementioned factors went subjected to response surface methodology (rsm). results hplc positive species cryptococcus uzbekistanesis (absorption: 2.634), cryptococcus aerius (absorption: 2.609) and cryptococcus adelinsis (absorption: 1.118) were spectrophotometry positive species analyzed using the hplc to check their melatonin production. the hplc results showed that; c. uzbekistanesis was a melatonin-producer yeast. considering the absorption of melatonin solute using hplc, the following equation was obtained for melatonin concentration: y=4851.5x+15013; where y is optical density and x is melatonin solution (ppm). the amount of melatonin that was produced was 45ppm. optimization by response surface methodology the results indicated that the indigenous yeast cryptococcus uzbekistanensis was able to produce melatonin in the fermentation broth. a statistical experiment design was used to engineer production media for different carbon sources, temperatures, growth incubation times, and tryptophan concentrations. response surface methodology (rsm) was applied to determine and optimize the effective factors in melatonin production. optimization of process parameters was carried out using the box-behnken design with the parameters found, including time, carbon source, tryptophan concentration code variables 1 carbone source indole glucose starch 2 tryptophan concentration(ppm) 1 2 3 3 growth time (hour) 24 48 72 4 temperature(k) 301 304.5 308 levels http://www.ajecr.org/ 410 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org fig. 1 melatonin production by c. uzbekistanesis and temperature. table 3 presents the design table. interpretation of the results collected by regression analysis of the plots of tables 4, 5, 6, 7, 8 and 9 using the box-behnken tool showed, the following results: indole melatonin (ppm) = 10.8 8.5 tryptophan + 1.676 time + 8.06 tryptophan*tryptophan 0.02175 time*time + 0.698 tryptophan*time. glucose melatonin (ppm) = -22.1 + 17.0 tryptophan + 2.530 time + 8.06 tryptophan*tryptophan 0.02175 time*time + 0.698 tryptophan*time. starch melatonin (ppm) = 7.6 + 2.5 tryptophan + 2.041 time + 8.06 tryptophan*tryptophan 0.02175 time*time + 0.698 tryptophan*time. anova of the quadratic regression model demonstrated that tryptophan concentration and growth incubation time were highly significant factors (p ≤ 0.005) (table 4,5, and 6). the goodness of fit of the model was checked using the determination coefficient (r2). the r2 value was 0.9667and the adjusted r2 value was 95.64. it was in reasonable agreement with the predicted r2 (90.82). graphical representation provides a method to visualize the relationship between the response and experimental levels of each variable and the type of interactions between test variables in order to deduce the optimum conditions. the interaction effects and optimal levels of the variables were determined by plotting three-dimensional (3d) response surface curves [8, 9] (table 7, 8, and 9). the response surface curves, which represent the interactions between variables, showed that a tryptophan concentration of 3 ppm, growth incubation time of 72 hours, glucose as the carbon source, and a temperature 308 k yielded the maximum melatonin concentration. the shape of the response surface curves showed a strong positive interaction between these tested variables. morphological identification the gene sequence similarity search in the eztaxon database showed that the sampled yeast belonged to cryptococcus uzbekistanis with 100 % similarity. the isolate was deposited in university of tehran microorganisms collection and its sequence was registered in the genbank under accession number kx347452. discussion about 94% long-haul travelers experience jet-lag due to changes in the circadian rhythm. the body's internal clock is controlled by the release of a hormone called melatonin. this pineal gland hormone is regulated by light. the levels of melatonin are highest in children and diminish with age. [10] the functional repertoire of melatonin makes it a medical ‘wonder drug’. naturally, it is prescribed for people with sleep problems like insomnia or disrupted circadian rhythms. it is also used to treat problems related to the central nervous system like depression or to prevent damages in neurons [11]. melatonin is also administered as a drug to ameliorate diseases like parkinson's or alzheimer's disease due to its ability to reduce oxidative stress through its free radical scavenging effect as well as indirect enhancement of the antioxidant defense systems. several experiments were performed using melatonin to probe its oncostatic effect on different cancer cells, and so its use as a drug is extended to other disorders like hypertension, diabetes, and urinary incontinence. furthermore, melatonin shows regenerative properties in tissues like the bone, muscle, and cartilage as well as the ability to sustain a successful in vitro embryo development in some animals because it promotes the development of blastocysts and increases the rates of embryo implantation, pregnancy, and postnatal survival of offspring. [12] in europe, around 1,700,000 people die solely due to http://www.ajecr.org/ 411 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org table 2 response surface methodology experiments. have indicated the high potential of melatonin as an anticancer drug against breast cancer. [13, 14, 15] since it is a natural antioxidant hormone produced in the human body, it can be administered to the elderly cancer patients who have lower levels of melatonin. the use of melatonin and other natural hormones is http://www.ajecr.org/ 412 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org fig. 2 interaction plot for melatonin (ppm). data means. fig. 3 residual plot for melatonin (ppm). fig. 4 fig. 5 surface plot of melatonin (ppm) vs. tryptophan (ppm). time (hour). regulated in europe and it can be only purchased as a medication with a prescription. the pharma grade of melatonin is expensive; hence, many people purchase cheap and crude forms. however, these unregulated cheap products contain impurities and are considered unhealthy fig. 6 surface plot of melatonin (ppm) vs. temperature (k). time (hour). fig. 7 surface plot of melatonin (ppm) vs. temperature (k), tryptophan (ppm). and toxic by european scientists. many people import melatonin capsules from other countries like the uk as otc (over-thecounter) drugs. the german [16] and dutch [17] markets have supported the regulated use of melatonin given the positive effects backed by scientific research. in the u.s., people spent 32 billion usd on the sleep market in 2012 [18] to which melatonin is closely linked because of the stressful lifestyle and increased life expectancy. that huge amount of money persuaded pharma companies to work in melatonin production using chemical synthesis methods but they did not achieve a high level of purity, which could be dangerous for the patient since it can trigger some secondary reactions. [19] a wonder drug like melatonin with its multi-functional properties needs to be made available to the general population of europe to curb cancer, autism, multiple sclerosis, depression, rare sleepwake disorders, insomnia, heart disease, high blood pressure, and aging. the project described in the present study may facilitate attaining this goal. chemically produced melatonin contains impurities that sometimes cause fatal secondary effects. highlypure melatonin is also available, but its price makes it unprofitable. this paper reports a biologic source for producing melatonin. the results showed that c. uzbekistanesis as a biologic source could produce melatonin. media engineering was used to improve melatonin production in the studied yeast species. acknowledgement the author wishes to thank the microbiology department of university of tehran, especially doctor http://www.ajecr.org/ 413 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org hamid moghimi, ms. raheleh bizhani for financial aid, and mr. kourosh bizhani for statistical assistance. conflict of interest the authors declare no conflicts of interest. references 1. fauteck jd, schmidt h, lerchl a, kurlemann g, wittkowski w. melatonin in epilepsy: first results of replacement therapy and first clinical results. biologic signals recept 1999 jan-apr;8(1-2):105-101. 2. hardeland r. melatonin in aging and disease -multiple consequences of reduced secretion, options and limits of treatment. aging dis. 2012 apr;3(2):194-225. 3. herxheimer a, petrie kj. melatonin for preventing and treating jet lag. cochrane database syst rev. 2001;(1):cd001520. 4. acuña-castroviejo d, escames g, figueira jc, de la oliva p, borobia am, acuña-fernández c. clinical trial to test the efficacy of melatonin in covid-19. j pineal res 2020;00: e12683. 5. rodriguez-naranjo, m. i., torija, m. j., mas, a., cantos-villar, e., and garciaparrilla, m. d. c (2012). production of melatonin by saccharomyces strains under growth and fermentation conditions. j. pineal res 53, 219– 224. doi: 10.1111/j.1600-079x.2012. 00990.x 6. fernandez-cruz, e., gonzález, b., muñiz-calvo, s. et al. intracellular biosynthesis of melatonin and other indolic compounds in saccharomyces and non-saccharomyces wine yeasts. eur food res technol 245, 1553–1560 (2019). 7. parra, mª ángeles & gonzalez, beatriz & beltran, gemma & mas, albert & torija, maria. (2019). melatonin and glycolytic protein interactions are related to yeast fermentative capacity. food microbiol 87. 103398. 10.1016/j.fm.2019.103398. 8. minitab 17 statistical software (2010). [computer software]. state college, pa: minitab, inc. (www.minitab.com) 9. d. c. montgomery, john wiley & sons, new york, ny, usa, 1991. 10. pandi-perumal, s. r., trakht, i., srinivasan, v., spence, d. w., maestroni, g. j., zisapel, n., &cardinali, d. p. (2008). physiological effects of melatonin: role of melatonin receptors and signal transduction pathways. progr neurobiol 85(3):335-353. 11. polimeni, g., esposito, e., bevelacqua, v., guarneri, c., & cuzzocrea, s. (2014). role of melatonin supplementation in neurodegenerative disorders. front biosci (landmark ed), 19, 429-446. 12. rivara, s., pala, d., bedini, a., & spadoni, g. (2015). therapeutic uses of melatonin and melatonin derivatives: a patent review (2012-2014). expert opin ther pat 25(4), 425-441. 13. el moneim, n. a. a., el masry, h., sorial, m. m., hewala, t. i., embaby, a., & sheweita, s. a molecular case-control study on the association of melatonin hormone and rs#10830963 single nucleotide polymorphism in its receptor mtnr1b gene with breast cancer. middle east j cancer 2015;6(1), 11-20. 14. hill sm, belancio vp, dauchy rt, xiang s, brimer s, mao l, hauch a, lundberg pw, summers w, yuan l, frasch t, blask de. melatonin: an inhibitor of breast cancer. endocr relat cancer. 2015 jun;22(3):r183-204. 15. hevia, d., gonzález‐menéndez, p., quiros‐ gonzález, i., miar, a., rodríguez‐garcía, a., tan, d.x., & sainz, r. m. melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer. j pineal res 2015;58(2), 234-250. 16. rivendell.eu. rivendell news the netherlands. [online] available at: http://www.rivendell.eu/internet/articles/articles.asp? pageid=276 [accessed 29 oct. 2015]. 17. public assessment report of the medicines evaluation board in the netherlands melatonin tiofarma 1 mg, 3 mg and 5 mg tablets tiofarma b.v., the netherlands melatonin. 29 june 2012. 18. mackey, m. sleepless in america: a $32.4 billion business. [online] the fiscal times. available at: http://www.thefiscaltimes.com/articles/2012/07/23/sleepl ess-in-america-a-32-4-billion-business [accessed 29 oct. 2015]. 19. laforce, r., rigozzi, k., paganetti, m., mossi, w., guainazzi, p., & calderari, g. aspects of melatonin manufacturing and requirements for a reliable active component. neurosignals 1999;8(1-2):143-146. http://www.ajecr.org/ 304 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(4):304-309 original article compounding efficiency of snap-n-go vials compared to traditional sterile compounding techniques for vancomycin 1.5g and 2.0g dave shannon*, jacqueline tualla, raoof abdellatif department of pharmacy, northwestern memorial hospital, east huron street, chicago, illinois, usa abstract. the current procedure for compounding vancomycin is an inefficient, time-consuming process that has been shown to result in more human error and leads to an overabundance of waste due to its short half-life after compounding. in an attempt to mitigate these inefficiencies, pentec health has developed a new medication formulation called snap-n-go™ to eliminate many of the unnecessary steps utilized by the traditional method. their product may help to eliminate drug waste due to its longer half-life and may improve safety because the vial contains all the drug information on its label. this will assist the pharmacist in verifying exactly what the pharmacy technician used to compound the product in the cleanroom and potentially reduce administration errors at the patient’s bedside. this study will primarily determine how much time can be saved by using snap-n-go™ versus traditional methods and compare the cost differences between the products used in each process. keywords: snap-n-go™, vancomycin, compounding, stability, cleanroom introduction compounding sterile products is a key responsibility of hospital pharmacies in providing a high volume of patients with intravenous (iv) medications. the current procedure for compounding vancomycin is a lengthy process that involves reconstituting lyophilized powder with sterile water, then drawing this solution into a syringe to be added to an iv fluid bag of normal saline. because of the multitude of steps in this process, there have been numerous reports of medication errors due to inaccuracies and impurities despite additional regulations and guidelines to improve compounding practices [1]. according to an observational study done at five u.s. hospitals, the error rate for compounding iv admixtures is 9% [2]. errors that occur throughout the compounding procedure include choosing incorrect ingredients, physical and chemical contaminants, and inappropriate compounding methods.3 because of these ongoing issues, the need for further enhancement and optimization of sterile compounding processes at institutions is imperative to ensure patient safety. at northwestern memorial hospital, 160 doses of vancomycin 1.5 g and 50 doses of vancomycin 2.0 g are batched each week. in addition to this process being lengthy and error-prone, the final product of vancomycin has limited variability in strength and a stability of only 48 hours at room temperature or 14 days refrigerated between 2-8℃. the amount of vancomycin that is wasted as a result of this stability is an average of 24 units of 1.5 g vancomycin and 42 units of vancomycin 2.0 g per quarter. pentec health has developed a new medication formulation called snap-n-go™ in an attempt to resolve some of the deficiencies in the current sterile compounding procedure. this innovative product consists of a glass vial filled with dissolved medication and a cap that is compatible with multiple iv bag adapters including vialmate™ and mini-bag plus containers™. to compound using this product, the vial is attached to an iv bag of normal saline without the need for additional reconstitution and is mixed into the normal saline solution at the time of administration. since the initial solution is prepared in a 503b compliant facility, depending on the vial docking device used following the manufacturers recommendation on bud’s the vancomycin, for example, is 30 days at refrigerated temperature after docking in the appropriate iso environment. pentec health claims it will also improve workflow efficiency, reduce waste, and decrease human errors. this study will attempt to determine the validity of these claims by comparing the traditional method of compounding vancomycin to compounding using the snap-n-go™ vials. the purpose of this study is to determine the safety and ___________________________________________________________ * corresponding author: dave shannon, pharmd (dshannon02@mail.roosevelt.edu). http://www.ajecr.org/ mailto:dshannon02@mail.roosevelt.edu 305 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org figure 1 traditional vancomycin 1.5 g compounding procedure. outline of the staging, compounding, and verification steps taken to compound vancomycin 1.5 g using the traditional method of sterile compounding at northwestern memorial hospital. nss, normal saline solution; swfi, sterile water for injection; ipa, isopropyl alcohol. figure 2 traditional vancomycin 2 g compounding procedure. outline of the staging, compounding, and verification steps taken to compound vancomycin 2 g using the traditional method of sterile compounding at northwestern memorial hospital. nss, normal saline solution; swfi, sterile water for injection; ipa, isopropyl alcohol. http://www.ajecr.org/ 306 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org figure 3 snap-n-go™ compounding procedure. outline of the staging, compounding, and verification steps taken to compound vancomycin 1.5 g and vancomycin 2 g using the snap-n-go™ method of sterile compounding introduced by pentec health. sng, snap-n-go; nss, normal saline solution; swfi, sterile water for injection; ipa, isopropyl alcohol. efficiency of the snap-n-go™ compounding procedure compared to traditional compounding techniques. materials and methods we conducted this prospective study over the course of five days from april 2, 2018 to april 6, 2018 in the central pharmacy of northwestern memorial hospital. all compounding was performed in an iso class 5 laminar airflow workbench located within an iso class 7 cleanroom. on day one of the study, pharmacy technicians were observed in order to analyze their current procedure for compounding vancomycin. a workflow diagram was developed to map each step taken in the process and divided into three main categories: staging, compounding, and verification. each step in the traditional procedure was timed on days two and three. an observer from pentec health recorded the times to compound 30 doses of vancomycin 1.5 g and 15 doses of vancomycin 2.0 g using vials of lyophilized powder. three pharmacy technicians were timed during the compounding stage. each technician compounded ten doses of vancomycin 1.5 g and five doses of vancomycin 2.0 g. vancomycin 1.5 g iv bags were made by reconstituting 1.0 g vials of vancomycin and adding the contents of one-and-a-half vials to a 500 ml fluid bag of normal saline. vancomycin 2.0 g iv bags were made by reconstituting a 10 g vial of vancomycin and distributing the contents over five 500 ml fluid bags of normal saline. the 1.0 g vials of vancomycin were used to better illustrate the usual practice of compounding individualized doses at community hospitals, while the 10 g vials represented the routine compounding procedure of larger hospitals that batch multiple doses of vancomycin at a time. the vancomycin lyophilized powder was reconstituted using sterile water for infusion. the details of each procedure is seen on the workflow diagrams, figure 1 and figure 2. on days four and five, the observer timed the technicians and pharmacist following the same procedure as days two and three, but utilizing the snap-n-go™ reconstituted vials of vancomycin instead. each technician compounded 10 doses of vancomycin 1.5 g and 5 doses of vancomycin 2.0 g by attaching the snap-n-go™ vials to 500 ml normal saline iv bags with vial-mate™ adapters. the primary endpoint was the time to compound vancomycin using lyophilized powder vials compared with snap-n-go™ vials to compare the efficiency of each process. secondary endpoints included technician and patient safety, amount of waste produced, and dissolution times of medication. statistical analysis the primary analysis was designed to show whether the snap-n-go™ method was significantly faster than the standard approach for compounding vancomycin. the sum of reconstitution and compounding times using lyophilized powder were compared with the entire time duration of compounding the snap-n-go™ vials. one-way anova was performed to test the null hypothesis between multiple independent variables (1.5 g traditional, 2.0 g traditional, 1.5 g sng, and 2.0 g sng) and a continuous dependent variable of time to compound. the null hypothesis was that no significant difference exists in the time it takes to compound using traditional vials vs sng vials. a student t-test was then used to compare the continuous outcomes http://www.ajecr.org/ 307 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org figure 4: average time to reconstitute and compound. figure 5: distribution of compounding time. between the two 1.5 g vials (traditional vs sng) and also between the two 2.0 g vials (traditional vs sng). this test was two-sided with a p value of 0.05 set to determine significance. additionally, a 95% confidence interval was calculated to analyze the difference of means between both groups. data analysis was conducted using excel 2016.was two-sided with a p value of 0.05 set to determine significance. additionally, a 95% confidence interval was calculated to analyze the difference of means between both groups. data analysis was conducted using excel 2016. results one-hundred-and-twenty doses of vancomycin 1.5 g and 60 doses of vancomycin 2.0 g were compounded. all doses were evenly distributed between the traditional compounding technique and the snap-n-go™ method. two of the three pharmacy technicians remained active for the full duration of the study. the same pharmacist verified all doses of vancomycin and the observer from pentec health recorded all times. the shortest time to reconstitute and compound 1.5 g vancomycin (n = 60) using the table 1 one-way anova summary table 2 anova data lyophilized powder vial was 84 seconds and the longest time was 257 seconds, with the average being 165 seconds (sd = 54.6). for the 2.0 g dose of vancomycin (n = 30), the shortest time to reconstitute and compound each dose was 67 seconds and the longest was 102 seconds, with an average of 82 seconds (sd = 13.4). the fastest time to compound snap-n-go™ was 17 seconds for vancomycin 1.5 g vials (n = 60) and 15 seconds for the vancomycin 2.0 g vials (n = 30). the longest compounding times for snapn-go™ were 37 seconds for vancomycin 1.5 g vials (n = 60) and 34 seconds for vancomycin 2.0 g vials (n = 30). twenty-six seconds was the average time to compound both the snap-n-go™ vancomycin 1.5 g (sd = 6) and 2.0 g vials (sd = 4.9). the average time for the lyophilized powder vials to completely dissolve in solution was 282 seconds (sd = 70.1) for the 1.0 g vials and 530 seconds (sd = 285) for the 10 g vials. both snap-n-go™ doses, 1.5 g and 2.0 g, needed one snap-n-go™ vial, one vialmate™ adapter, and one 500 ml ns bag. compounding vancomycin 1.5 g with the lyophilized powder vials required an average of 2 needles, 4 alcohol swabs, and 1 syringe per dose. compounding vancomycin 2.0 g with the lyophilized powder vials required an average of 1 needle, 3 alcohol swabs, 2 syringes, and 1 vented spike per dose. the one-way anova found an f-observed value (223.58) that was larger than the f-crit value (2.66) so we rejected the null hypothesis and determined that a significant difference in mean compounding time existed between our independent variables. an independentsamples t-test was then conducted to compare overall process times between the snap-n-go™ and original compounding methods based on which strength was compounded. a statistically significant difference in the vancomycin 1.5 g procedure time was found between the original lyophilized powder vials and snap-n-go™ vials, t (118) = 19.68, p = 6.21 x10-28. there was also a significant difference in the process times for vancomycin 2.0 g snapn-go™ vials and original lyophilized powder vials, t(58) = 21.61, p = 2.11 x10-22. the vancomycin 1.5 g snap-ngo™ vials averaged 139 seconds faster to compound than the original lyophilized powder vials, 95% ci [129,150]. the average compounding time of the vancomycin 2.0 g snap-n-go™ vials was 56 seconds faster than the groups count sum average variance column 1 60 9894 164.9 2977.074576 column 2 30 2449 81.63333333 180.516092 column 3 60 1531 25.51666667 32.79632768 column 4 30 766 25.53333333 23.70574713 source of variation ss df ms f p-value f crit between groups 699345.1833 3 233115.0611 223.5813288 8.70e-60 2.655938877 within groups 183504.8167 176 1042.641004 total 882850 179 http://www.ajecr.org/ 308 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org lyophilized powder vials, 95% ci [54, 59]. the mean number of seconds taken to reconstitute and compound using the traditional sterile compounding technique and the snap-n-go method. dissolution times were omitted from this data because they did not affect the overall time required to compound (technicians were able to compound additional doses while waiting for complete dissolution of medication). compounding times differed between the vancomycin 1.5 g, vancomycin 2.0 g, and sng vials, but were similar between sng vials of differing strengths. vancomycin 2.0 g took less time to compound than vancomycin 1.5 g because it was compounded as a batch dose rather than individual doses. the distribution follows a standard bell curve for each method used to compound vancomycin. a wider distribution is observed using the traditional method due to the skill level required and differences between technicians performing the compounding. the narrow distribution observed for the sng vials indicates that skill level is less of a factor in the speed of compounding vancomycin and all technicians were able to compound using this technique significantly faster than the traditional method. f (observed) > f-crit so we rejected the null hypothesis that there is no significant difference between the time it takes to compound vancomycin 1.5 g and 2.0 g using the traditional sterile compounding method and the sng method. discussion throughout the course of this research we found that the time to compound snap-n-go was significantly faster than the lyophilized powder. the difference in compounding times between the two groups would be even more significant if dissolution times were included, but they were reported separately because it was observed that pharmacy technicians typically complete other tasks while waiting for the contents to dissolve. however, this increases the risk for human error when vials are left in the hood unsupervised. one limitation of this study are the differences between technicians who performed the compounding. technicians were selected to participate based on when they were scheduled to work in the cleanroom throughout the study week. this lack of consistency may have altered compounding times due to differences in their skill level and compounding methods, thus weakening the internal validity. another limitation is that the study participants were not blinded to the fact that they were being timed. this could have altered the speed at which they compounded vancomycin compared to a regular work day. the longest times of the doses made with lyophilized powder were delayed due to coring of the vials in which additional vials were required from outside of the cleanroom. we observed an inherent safety feature of the snap-ngo™ product in that it allowed the pharmacist to verify exactly what was compounded together. when verifying doses made with lyophilized powder, the accuracy in strength and ingredient used relies solely on the technician preparing the product. premanufactured vials removes this potential error and allows for accuracy even before the final verification by the pharmacist. the iv solution of vancomycin has a short stability of 14 days when refrigerated and two days at room temperature, causing many doses to go unused before expiring and creating more waste. the snap-n-go™ product provides an additional benefit with its prolonged stability of one month, by potentially decreasing the amount of product wasted. the main disadvantage of snap-n-go™ is its cost. a secondary cost analysis was run, although the pricing data varies and is exclusive to each institution. nonetheless, snap-n-go™ appeared to be more expensive as a cost per dose in this particular study. although the price of snap-ngo™ may vary with different contracts and medications, the overall cost still seems to be higher with supplies and labor factored in as compared to high volume batching. another inconvenience is the inability to batch a large volume of single doses using one multi-dose vial. using a multi-dose vial, such as the 10 g lyophilized powder vial, can potentially reduce waste when a large number of doses are compounded. however, we found that an equivalent number of doses is compounded more quickly using the snap-n-go™ vials versus the 10 g multi-dose vials. snapn-go™ comes in six strengths including 0.75 g, 1.0 g, 1.25 g, 1.5 g, 1.75 g, and 2.0 g for ease of use in compounding, which is especially beneficial for smaller hospitals requiring individual doses as needed. future studies for snap-n-go vials should be conducted using a larger sample size of product and uniformity between the pharmacy technicians performing the compounding. other studies should also be conducted to determine the most efficient adapter to be used with snap-n-go™ vials, whether it be vial-mate™ or minibag plus containers™. the ease of use during administration has yet to be determined and could also be included in future studies. conclusion snap-n-go™ vials have been shown to significantly increase efficiency while also enhancing the stability and safety of compounding vancomycin. snap-n-go™ comes at a greater initial monetary cost, but may be of more value to certain institutions based on their needs. some of this cost is recovered by utilizing fewer materials to compound and allowing more time for pharmacy technicians to accomplish additional tasks. the larger cost appears to be most beneficial for smaller institutions, as most do not have the patient capacity necessary for batching large doses of vancomycin. furthermore, smaller institutions have less personnel and time to complete the lengthy standard compounding process required to make individual doses. conflict of interest the authors declare no conflicts of interest. acknowledgement this study was an unbiased study funded by northwestern memorial hospital and pentec health. http://www.ajecr.org/ 309 am j exp clin res, vol. 5, no. 4, 2018 http://www.ajecr.org the authors would like to acknowledge john lee, pharmd, gail santucci, pharmd, basil hussein cpht, tia williams cpht, erica sanchez, and shakela goss for their assistance associated with this manuscript. references 1. american society of health-systems pharmacists. ashp guidelines on compounding sterile preparations. am j health syst pharm 71:145-166, 2014. 2. flynn ea, pearson re, barker kn. observational study of accuracy in compounding i.v. admixtures at five hospitals. am j health syst pharm 54:904-912, 1997. 3. westbrook ji, rob mi, woods a, et al. errors in the administration of intravenous medications in hospital and the role of correct procedures and nurse experience. bmj qual saf 20:1027-1034, 2011. http://www.ajecr.org/ 102 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(2):102-104 original article enhancing turn-around-time (tat) on stroke protocol head ct reports via continuous quality improvement (cqi) methodology in a busy teleradiology practice eigles sb*, panughpath sg, kalyanpur a teleradiology solutions, inc., bangalore, india abstract. through strict adherence to continuous quality improvement (cqi) methodology, over a 3 year period our teleradiology practice was able to achieve ongoing 100% compliance with our turn-around-time (tat) goal for interpretation of stroke protocol head cts. during the 3 years prior to implementing cqi, our efforts to reduce tat through traditional less systematic methods had been ineffective, while through the iterative cqi method of defining metrics, measuring, planning, implementation, and re-evaluation, we progressively achieved our goal of interpreting all stroke head cts in less than 15 minutes. keywords: head ct, stroke protocol, tat, cqi, teleradiology introduction in recent years, there has been increasing recognition of the importance of rapid triage and treatment of nonhemorrhagic stroke, with thrombolysis predicated on first excluding intracranial hemorrhage [1, 2]. to this end, beginning in 2006, our teleradiology company began to prioritize reports for non-contrast head cts obtained to rule out stroke (stroke protocol). we internally set a goal of 15 minute tat, a requirement subsequently adopted by one client hospital in 2009, and later by all of our clients. in 2010, the american heart association publicized a 1hour door-to-needle goal, including up to 20 minutes for radiology reporting (aha/asa 2010 stroke campaign manual) [3]. after three years of monitoring stroke protocol tat, as of late 2009, administrative efforts to reduce tat had been relatively ineffective. between 2006 and 2009, average stroke head ct tat was only 16.0 minutes, but 40% of cases exceeded 15 minutes (fig. 1). we then began a long-term systematic cqi initiative, which we continue to the present time. this project has resulted in a dramatic sustained decrease in the number of delayed cases, achieving our goal 15 minute tat goal as well as reducing average tat. materials and methods we first selected our metric and in stages more accurately defined it. we replaced our focus on average tat in favor of percentage of cases <15 minutes, a metric which more accurately reflected client concerns. we defined tat as starting when all images and the order are received by our company, and ending at the commencement of a phone call communicating the results. we required all parties handling each delayed case to comment on reasons for delay (rfd), which previously had been largely unknown to our administration. the most common rfd were: complex protocol (brain cta, perfusion, etc.), complex report, order changed to stroke, tat not adjusted per metric, and long hold time. in addition to the self-reported rfd, we examined the correlation between tat and a spectrum of potentially unreported factors, including individual radiologist, hospital, report type (positive versus negative), radiologist location, weekday versus weekend, time of day, patient age and gender, multiple simultaneous exams, number of images, availability of prior exams, completeness of images, and whether the report was called by the radiologist or a clerk. we found particularly positive correlations between the number of delayed cases and (a) individual radiologists, (b) specific hospitals, (c) positive reports, (d) the transmission of multiple simultaneous exams on the same patient, and (e) calls made by the radiologists (see example, table 1). with regard to the radiologists, there was no correlation between delayed cases and specialty training or years of experience. on a monthly basis, we implemented specific workflow changes to address the most significant issues. examples of such changes include: 1. coordinator immediately notifies radiologists of ___________________________________________________________ * corresponding author: stephen b eigles, md (stephen.eigles@gmail.com). http://www.ajecr.org/ mailto:stephen.eigles@gmail.com 103 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org 0 10 20 30 40 50 60 70 aug-06 feb-07 aug-07 feb-08 aug-08 feb-09 aug-09 % delayed ave tat stroke head ct figure 1 average stroke head ct tat between 2006 and 2009 was only 16.0 minutes but 40% of cases exceeded 15 minutes. 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 c a se v o lu m e s m in u te s % delayed average tat case volumes implementation of cqi initiative figure 2 cqi step 6: goal achieved. stroke exams. 2. cases are assigned to the fastest radiologist. 3. radiologist informs if busy, and the stroke case is reassigned. 4. if a probable stroke exam is ordered incorrectly, it is treated as a stroke while confirming. 5. tat times are calculated as per metric definition. 6. radiologist calls results before entering the report or receiving prior images/report. 7. all teams (order entry, radiologist, proofer, call center) are trained re importance of stroke exams and protocols. 8. mandatory rfd on delayed cases 9. stroke tats are discussed in every monthly radiologist conference. 10. work with hospitals to: a. unbundle multiple exams b. specify stroke exams c. facilitate radiologist calls to providers we subsequently evaluated whether the changes were having the desired effect. summarized data was shared with staff and radiologists, and we determined which rfd had resolved, and which still needed action. through group discussion of the findings, brainstorming led to suggested workflow changes that achieved the desired outcome. we used control charts to assess our performance on an ongoing basis, such as in this sample (table 2). results over a period of several years, by iteratively performing the cqi process on a monthly basis, we gradually reduced the number of delayed cases, to the extent that in the ensuing years, despite increasing case volumes, we have had no more than 1 delayed case each month. in 2006, we labeled just 54 cases as stroke protocol, but only 57% had a tat<15 minutes. by 2012, we labeled 2,454 cases as stroke protocol and yet achieved http://www.ajecr.org/ 104 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org table 1 calls made by radiologists all rads rad a mean tat (min) 10.6 16.2 no. of observations 113 8 table 2 sample month rfd analysis 2010 aug & sept oct reason for delay # cases # cases unknown 4 2 order needs corrections 1 images arrived late 1 delated order entry 1 6 prior exam 1 on hold with hospital 3 3 busy 1 report not noticed, resent 1 additional history needed 1 2nd opinion needed 1 doc would only speak ww rad 1 complex 2 radiologist busy 4 ris/pacs problem 2 delayed cases covered by prior policies delayed cases covered by new policies >99% tat<15 minutes, as well as cut the average tat in half (fig. 2). conclusion cqi has been a highly effective method of treating the complex problem of report delays, allowing us to achieve our tat goals through incremental workflow changes. acknowledgement special thanks to dipika bedi, operation head, akran pervez, operation manager and the teleradiology solutions staff and radiologists for making this cqi project successful. conflict of interest the authors declare no conflicts of interest. references 1. zuckerman s, magarik j, espaillat k, bhatia r, dewan m, morone p, mocco j. o-031 implementation of an institutional-wide acute stroke algorithm: improving stroke quality metrics. j neurointerv surg 1:a16, 2014. 2. ford, al, williams ja, spencer m, mccammon c, khoury n, sampson tr, panagos p, lee jm. reducing door-to-needle times using toyota's lean manufacturing principles and value stream analysis. stroke 12:3395-3398, 2012. 3. aha/asa 2010 stroke campaign manual: http://www.strokeassociation.org/idc/groups/heart-public/ @wcm/@hcm/@gwtg/documents/downloadable/ucm_308 277.pdf. http://www.ajecr.org/ 141 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(1):141-145 original article types of dizziness and its relationship with psychological symptoms in patients with chronic dizziness chitsaz a 1 , khourvash f 1 , tolou-ghamari z 2 *, gholamrezaei a 3 , noormohamadi a 3 1 department of neurology, isfahan neurosciences research center (inrc), faculty of medicine, isfahan university of medical sciences, isfahan, iran 2 department of urology, isfahan urology and renal transplantation research center, al-zahra research centers, isfahan university of medical sciences, isfahan, iran 3 student research center, faculty of medicine, isfahan university of medical sciences, isfahan, iran abstract. dizziness could be categorized as one of the most common medical complaints of patients referred to the neurology clinics. the aim of this study was to evaluate the types of dizziness and its relationship with psychological disorders in patients with chronic complaints. we studied 179 patients ranging in age from 18 to 65 years old. patients were asked to complete a revised questionnaire form that contained 90 questions related to the signs based on severity scale vertigo tool. subsequently, to detect organic or non-organic vertigo, the patients were divided into two groups. for psychiatric disorders, screening questions of international standard for testing scl-90-r were used. vertigo severity scale was used for the evaluation of dizziness severity. to compare quantitative variables between the two groups independent t-test was used and p value of ≤0.05 was considered as significant. of the total patient population, 70.9% were females and 74% of patients had dizziness due to organic causes while 26% had dizziness due to non-organic causes. dimension scores related to somatic complaints, obsessive-compulsive, depression, anxiety, paranoid ideation and global severity index in individuals with nonorganic vertigo was significantly higher than other groups. there was a significant direct relationship between the overall score related to intensity of dizziness and all of the questionnaire’s dimensions. there was significant correlation between the extent of physical complaints and the fear for morbid (p<0.001). the score of extent related to dizziness/balance in patient with vertigo due to organic causes and score of extent related to autonomic/anxiety in patient with vertigo due to non-organic causes was significantly higher than in other groups. keywords: dizziness, psychological symptoms, somatoform, disorders introduction dizziness is the most common medical complaints of patients referred to the neurology clinic. in therapeutic practice dizziness often takes a prolonged sequence and can indulge the quality and quantity of life. psychiatric comorbidity is common in vertiginous patients [1-6]. there is high variability related to underlying causes of dizziness. these involve as organic causes with: 1) central and peripheral vestibular disorders, 2) non-vestibular and nonorganic causes including 1) somatoform disorders 2) psychiatric origin [7, 8]. dizziness or vertigo could be related with both vestibular-balance and psychological features. a common valuation tool is the vertigo symptom scale (vss) -short form, which has two subscales: vestibular-balance and autonomic-anxiety [9]. european evaluation of vertigo scale (eev) is a physician-administered questionnaire that only assesses symptoms of the vestibular syndrome: illusion of movement, duration of illusion, motion intolerance, neurovegetative signs, and instability [10]. it looks that recently particular attentions have been made to the prevalence of psychological disorders and associated complications in patients with dizziness. approximately 20-50% of all cases with vertigo disorders, psychological disorders have significant impact on the course of disease. according to previous study, anxiety is the most common disorders in these population which could increase their mortality and results to a worsen prognosis [11]. there are mutual relationships between anxiety and dizziness. it means that patients with unpredictable attacks of vertigo may be more prone to anxiety and develop phobic avoidance behavior subsequently. also, patients who suffer from anxiety and severe fear during attacks of vertigo could experience more physical symptoms (dizziness) [6, 12]. in connection with high cases of dizziness associated with psychological disorders two ___________________________________________________________ * corresponding author: dr zahra tolou ghamari (toloeghamari@pharm.mui.ac,.ir). mailto:toloeghamari@pharm.mui.ac,.ir 142 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org models have been proposed: 1) psychosomatic model in which patients' dizziness could be caused without any primary organic origin (somatoform original). the most common underlying causes of this kind of dizziness are anxiety disorders and phobias [13-15]. the attacks of vertigo could be as a demonstration of panic disorder [16]. 2) somatopsychic model in which pathological effects of organic disorder is on the patient's mind, therefore it could leads to such symptoms such as dizziness, despite the determination of the original organic disorder [17]. with not any clear diagnosed cause, patients with acute vestibular disease seem to be at risk for prolonged dizziness and disability in daily life (secondary somatoform vertigo). vestibular assessments in its' own could not determine the actual degree of disability rate in these patients [18-21]. the frequency of acute vestibular disease, the risk of psychiatric disorders such as anxiety, somatoform disorders and depression have been reported as 37.5, 14, 15 and 9 % respectively [22]. study performed by tschan et al., in 2010 confirmed that in patients with vestibular disorders psychiatric history are important factors linked to secondary risk of somatoform vertigo [23]. another study on patients with organic vestibular vertigo revealed that the vestibular deficit and it's dysfunction over time have no effect on creation of the secondary somatoform vertigo [24]. it has been confirmed that organic vestibular disorder alone could not be the cause of anxiety disorders and depression in these patients [25]. half of patients with organic vertigo syndromes suffer from secondary somatoform vertigo. differentiation between vertigo with an organic origin underlying psychological disorders seems to be difficult. prevalence of anxiety and fear disorders in patients with primary somatoform dizziness (non-organic) has been reported with an incidence of 45% that are comparable with a prevalence of 41% in patients with different organic vertigo syndromes. in most patients a psychological assessment (psychometric) does not perform, because the compliant is dizziness. as a result in the differential diagnosis non-organic vertigo disorders are not considered or reached to mind very late, that resulting in chronic illness and reduced quality of life. it could cause large costs to the health system [26, 27]. considering the dizziness accompanied by psychological disorders and its impact on quality of life, the aim of this study was to determine the type of vertigo and its association with psychological disorders in patients complaining of chronic dizziness. materials and methods this cross-sectional study was carried out in patients suffered with chronic dizziness and referred to neurology clinics between the years 2011 to 2013. inclusion criteria were: 1) age between 18 to 65 years old, 2) chronic dizziness at least for three months, 3) absence of malignancy or other known central nervous system disease, 4) absence of cognitive disorders due to recent brain disease or psychotic illness, and 5) lack of recent psychotherapy and psychiatric drugs. all participated patients were informed and signed the consent form. table 1 pateints demographic charachteristica and severity of dizziness based on the type of vertigo o rgan i c non -o rgan i c n =133 n =46 age (year) 44.5±13.5 (1.167) 39.6±12.2 (1.791) 0.029 female/male 91/42 36/10 0.14 severe dizziness (vss) 18.8±10.3 (0.900) 19.3±9.5 (1.411) 0.786 dizziness/balance subscale (vss-v) 10.7±6.9 (0.608) 5.8±4.4 (0.657) < 0.001 aut onomic/anxiet y subscale (vss-a) 8.6±5.9 (0.536) 14±7.0 (1.054) < 0.001 vari abl e s p val u e table 2 comparison of the two groups of pateints with organic and non-organic vertigo organic non-organic n=133 n=46 somatization 14.0±8.4 (0.726) 17.57±9.744 (1.421) 0.018 obsessivecompulsive 9.6±7.45 (0.643) 12.9±7.4 (1.087) 0.005 interpersonal sensitivity 7.9±6.5 (0.563) 8.5±5.61 (0.827) 0.366 depression 13.8±10.1 (0.875） 17.4±10.1 （1.477） 0.036 anxiety 10.1±7.2 （0.623） 12.8±7.4 （1.084） 0.027 aggression 4.89±4.468 (0.386) 5.1±3.43 （0.506） 0.272 fear 4.6±4.7 (0.407) 4.5±4.6 (0.686) 0.911 paranoid thoughts 5.7±4.6 [0.397] 7.8±5.1 (0.751) 0.012 psychosis 7.3±6. 4 (0.559) 6.8±4.6 (0.680) 0.853 additional questions 7.4±5.3 (0.461) 8.9±5.6 (0.816) 0.112 general severity index (gsi) 0.95±0.61 (0.053) 1.15±.54 (0.081) 0.029 variables p value sampling was based on the repeated procedure and a sample size of 179 was considered for this study. all patients were assessed by a neurologist for neurovestibular examinations included vestibular testing, equalometor performance and psychometric evaluation. whenever it was necessary, brain mri was requested to evaluate organic causes. for psychometric evaluation, patients were asked to complete the given questionnaire form. for psychiatric disorders screening questions of international standard for testing scl-90-r were used that standardized and translated in islamic republic of iran [30]. this test consists of 90 questions, for evaluation of psychiatric symptoms that is answered and reported by the subjects. all provided answers to each of the tests' material on a five degrees scale were determined. the rate of discomfort was reported from none to severe. the 90 questions of this test included 9 different psychiatric dimensions related to somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobia, paranoid ideation, and psychosis. general score index or gsi for this indicator is the average score of all test questions (the scores range from zero to four can be varied). also for the evaluation of dizziness severity in patients vertigo severity scale (vss-sf) was used. it was included 15 questions linked to the two subscales of vertigo/balance (vss-v) 143 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org and autonomic/ anxiety (vss-a). on a scale of 5 degrees questions were answered from 0 to 4. the final questionnaire score is the summing of the scores from 0 to 60 score that greater score indicating greater symptom severity. a score equal or more than 12 were defined as severe dizziness. data were recorded in d-base and statistical analysis was performed using spss for windows (version 16). statistical analysis mean ± sd and percentage for all variables were used. to compare quantitative variables between the two groups independent t-test (in case of non-normal distribution of test data and mann-whitney) was used. analyzes at the level of less than 0.05 was considered statistically significant. results from 184 subjects who entered the study, the scl-90r questionnaires form 5 subjects were incomplete which were excluded from the study. thus the data related to 179 patients were evaluated. the mean age of all patients was 43.2±13.2 years old and 70.9% of the patients were females. there were 133 (74%) patients who had organic dizziness and 46 (26%) who had non-organic dizziness. recognition of the frequency related to causes of organicdizziness was as: benign paroxysmal positional vertigo (35.6), meniere's disease (18.2), acute vestibulitis (16.7), vestibulopathy (9.8), migraine (7.6), vertiginous migraine (3.0), labyrintitis (2.3), multiple sclerosis (2.3), vertical blanking interval (2.3), vertiginous seizures (1.5), and chronic inflammatory demyelinating polyneuropathy (0.8). figure 1 shows the relationship between the overall index of illness and severity of vertigo. comparison of demographic and clinical characteristics of patients with organic vertigo is shown in table 1. however, the patients with organic vertigo were older than patients with nonorganic vertigo but there was not any significant differences related to gender. between the two groups vertigo intensity scores were not different. scores of dizziness/balance in subjects with organic-dizziness and figure 1 the relationship between the overall index of illness and severity of vertigo. scores of autonomic/anxiety in patients with non-organic vertigo was significantly higher than the other group. table 2 shows the comparison of the two groups of patients with organic and non-organic dizziness based on scl-90-r questionnaire analysis. thus, the dimensions of physical complaints, obsessive-compulsive, depression, anxiety, paranoid thought and general severity index score (gsi) in patients with non-organic vertigo was significantly higher than other groups. there was a positive correlation between the total score of severity of vertigo and all aspects of scl-90-r questionnaire analyzed data. pearson correlation coefficient from somatization to phobia was significant (p<0.018). also, there was a significant positive correlation between the scores of dizziness/balance and all aspects of scl-90-r questionnaire analyzed data (pearson correlation coefficient of 0.398 for physical complaints to 0.206 for the paranoid ideas, p<0.001). between the scores of autonomic/anxiety and all aspects of scl-90-r questionnaire analyzed data, significant positive correlation was found (pearson correlation coefficient of r= 0.473 for general indicator of imperilments to t=0.279 for phobic disorders, p<0.001). discussion with the aim related to the assessment of dizziness and its' association to psychological disorders in patients with chronic dizziness, it seems that psychological symptoms have a significant prevalence. there was a correlation between psychological symptoms and severity of vertigo, as psychological disorders tightened dizziness. patients with non-organic vertigo suffer from sever dizziness. accompanying of psychological symptoms with dizziness that has a negative impact on quality of life, could be mentioned as an important issue. this also could increase the health cost. it could be helpful for better understanding related to the pathophysiology and etiology of this association. therefore in treatment of patients with dizziness physicians should have adequate attention to psychological symptoms and subsequently offer appropriate treatment. the result of this study is in agreement with previous publication, because benign positional vertigo was the most common diagnosed among the organic causes [3, 17, 22]. the scores related to the physical complaints, obsessive-compulsive, depression, anxiety, paranoid ideation and general severity index score (gsi) was higher in patients with non-organic vertigo than organic group, which was significantly more predictive of severe disability and emotional distress in this group [8, 26]. in another study with a sample size of 202 patients complaining of chronic dizziness, which was assessed with scl-90-r, the highest score were related to anxiety, fear and depression [3]. however, in our study the highest score were linked to somatization, depression and anxiety. this could be due to the differences in the population studied, or might be considered as non-significant in previous studies. the importance of considering the differential diagnosis of depression and somatization disorder related to chronic dizziness has been confirmed by this investigation. 144 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org relatively high scores of somatization, depression, anxiety and obsessive-compulsive within organic groups, indicating the need for the simultaneous use of diagnostic procedures based on organic and psychometric methods in patients with chronic dizziness. it could be suggested that this group is highly capable of presenting secondary somatoform vertigo [5, 11]. previous publications confirmed that simultaneous presentation of organic vestibular disorders and anxiety disorders indicate neuroanatomical communication between vestibular system and emotional response. that could have consequences as: 1) monoaminergic input to vestibular system that could makes interface associated to the effects of stress, 2) parabrachial nucleus, that is a place for the information received from vestibular system, somatic and visceral receptors that through its' association with the breathing controller region in the brainstem and autonomic nervous system involved in the incidence of presentation related to physical anxiety. also it is connected with the central nucleus of the amygdale and infralimbic cortex, which is a structure, related to the fear and avoidance behaviors. patients older than 65 years were excluded from the study, while the highest organic vertigo has been reported in this group [26-33]. finally, the high prevalence of psychological symptoms in this study identifies the necessity to find for scientific and practical strategies that could be able to deal effectively with these disorders. diagnosis and effective treatment related to these disorders could improve prognosis, quality of life and patients' survival. in addition to patients' training related to methods of control and prevention of depression and anxiety, further studies related to the methods for early detection and screening of these disorders is also recommended. acknowledgement this study was supported by isfahan university of medical sciences. conflict of interest the authors declare no conflicts of interest. references 1. weidt s, bruehl ab, straumann d, hegemann sc, krautstrunk g, rufer m. health-related quality of life and emotional distress in patients with dizziness: a crosssectional approach to disentangle their relationship. bmc health serv res 14:317-325, 2014. 2. ketola s, havia m, appelberg b, kentala e. psychiatric symptoms in vertiginous patients. nord j psychiatry 69:287-291, 2015. 3. eckhardt-henn a, breuer p, thomalske c, hoffmann so, hopf hc. anxiety disorders and other psychiatric subgroups in patients complaining of dizziness. j anxiety disord 17:369-388, 2003. 4. best c, eckhardt-henn a, tschan r, dieterich m. why do subjective vertigo and dizziness persist over one year after a vestibular vertigo syndrome? ann n y acad sci 334-337, 2009. 5. dieterich m, eckhardt-henn a. neurological and somatoform vertigo syndromes. nervenarzt 75:281-302, 2004. 6. staab jp. chronic dizziness: the interface between psychiatry and neuro-otology. curr opin neurol 19: 41-48, 2006. 7. andersson g, hagman j, talianzadeh r, svedberg a, larsen hc. dual-task study of cognitive and postural interference in patients with vestibular disorders. otol neurotol 24:289-293, 2003. 8. dieterich ae-ham. psychiatric disorders in otoneurology patients. . neurol clin 23:731-749, 2005. 9. kondo m, kiyomizu k, goto f, kitahara t, imai t, hashimoto m, shimogori h, ikezono t, nakayama m, watanabe n, akechi t. analysis of vestibular-balance symptoms according to symptom duration: dimensionality of the vertigo symptom scale-short form. health qual life outcomes 3:4, 2015. 10. mègnigbêto ca, sauvage jp, launois r. the european evaluation of vertigo (eev) scale: a clinical validation study. rev laryngol otol rhinol (bord) 22:95102, 2001. 11. eckhardt-henn a, breuer p, thomalske c, hoffmann so, hopf hc. anxiety disorders and other psychiatric subgroups in patients complaining of dizziness. j anxiety disord. 17:369-388, 2003. 12. paillard ac, quarck g, paolino f, denise p, paolino m, golding jf, ghulyan-bedikian v. motion sickness susceptibility in healthy subjects and vestibular patients: effects of gender, age and trait-anxiety. j vestib res 23:203-209, 2013. 13. schniepp r, brandt t, huth s, pradhan c, ja hn k, wuehr m. gait characteristics of patients with phobic postural vertigo: effects of fear of falling, attention, and visual input. j neurol 261:738-746, 2014. 14. schmid g, dieterich m, henningsen p, lahmann c, sattel h,. psychotherapy in dizziness: a systematic review. j neurol neurosurg psychiatry 82:601-606, 2011. 15. zur o, carmeli e. the university of california los angeles dizziness questionnaire: advantages and disadvantages. j vestib res 23:279-283, 2013. 16. schniepp r, brandt t, jahn k, krafczyk s, novozhilov s, pradhan c, wuehr m. nonlinear variability of body sway in patients with phobic postural vertigo. front neurol 4:115, 2013. 17. jacob rg rm, furman jm. optic flow-induces sway in anxiety disorders associated with space and motion discomfort. j anxiety disord 9:411-425, 1997. 18. erin g. piker gpj, devin l. mccaslin,sarah l. grantham. psychological comorbidities and their relationship to self-reported handicap in samples of dizzy patients. j am acad audiol 19:337-347, 2008. 19. wada m, takeshima t, nakamura y, nagasaka s, kamesaki t, oki h, kajii e. incidence of dizziness and vertigo in japanese primary care clinic patients with lifestyle-related diseases: an observational study. int j gen med 8:149-154, 2015. 20. zalewski ck, chien ww, king ka, muskett ja, baron re, butman ja, griffith aj, brewer cc. vestibular dysfunction in patients with enlarged vestibular aqueduct. otolaryngol head neck surg 153:257-262, 2015. 145 am j exp clin res, vol. 3, no. 1, 2016 http://www.ajecr.org 21. muller i, kirby s, yardley l. understanding patient experiences of self-managing chronic dizziness: a qualitative study of booklet-based vestibular rehabilitation, with or without remote support. bmj open 5:e007680, 2015. 22. jacob rg, whitney sl, detweiler-shostak g, furman jm. vestibular rehabilitation for patients with agoraphobia and vestibular dysfunction: a pilot study. j anxiety disord 15:131-146, 2001. 23. best c, eckhardt-henn a, tschan r, dieterich m. psychiatric morbidity and comorbidity in different vestibular vertigo syndromes. results of a prospective longitudinal study over one year. j neurol 256:58-65, 2009. 24. tschan r, wiltink j, best c, beutel m, dieterich m, eckhardt-henn a. validation of the german version of the vertigo handicap questionnaire (vhq) in patients with vestibular vertigo syndromes or somatoform vertigo and dizziness. psychother psychosom med psychol 60:e1-12, 2010. 25. best c, tschan r, eckhardt-henn a, dieterich m. who is at risk for ongoing dizziness and psychological strain after a vestibular disorder? neuroscience 164:15791587, 2009. 26. best c, eckhardt-henn a, diener g, bense s, breuer p, dieterich m. interaction of somatoform and vestibular disorders. j neurol neurosurg psychiatry 77:658-664, 2006. 27. eckhardt-henn a, tschan r, best c, dieterich m. somatoform vertigo syndrome. nervenarzt 80:909-917, 2009. 28. schmid g, henningsen p, dieterich m, sattel h, lahmann c. psychotherapy in dizziness: a systematic review. j neurol neurosurg psychiatry 82:601-606, 2011. 29. monzani d, casolari l, guidetti g, rigatelli m. psychological distress and disability in patients with vertigo. j psychosom res 50:319-323, 2001. 30. wiltink j, tschan r, michal m, subic-wrana c, eckhardt-henn a, dieterich m, beutel me. dizziness: anxiety, health care utilization and health behavior--results from a representative german community survey. j psychosom res 66:417-424, 2009. 31. r. m. standardizing and validating of scl-90-r in iran [msc thesis]. tehran university of medical sciences. 32. balaban cd, jacob jr, and furman jm. neurologic bases for comorbidity of balance disorders, anxiety disorders and migraine: neurotherapeutic implications. expert review of neurotherapeutics 11:379-394, 2011. 33. ettigi p, meyerhoff as, chirban jt, jacobs jr, wilsonrr. the quality of life and employment in panic disorder. j nerv ment dis 185:368-372, 1997. 83 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(1):83-86 original article effect of 17β estradiol on hippocampus region of aging female rat brain: ultrastructural study rashmi jha 1, 2 , ranjeet kumar 1 , priyanka mishra 1 , abbas ali mahdi 2 , shivani pandey 2 , najma zaheer baquer 1 , sudha mahajan cowsik 1 * 1 school of life sciences, jawaharlal nehru university, new delhi-110067, india 2 department of biochemistry, king george’s medical university, lucknow, up -226021. india abstract. estradiol has direct membrane-mediated effects on neurons and its effects are both neuroprotective and neurotrophic. this hormone modulates brain development and aging and affects neurochemical systems which are affected in age-related cognitive decline, ad and other neuropsychiatric disorders. the aim of the present study was to determine the effect of 17β estradiol (e2) in hippocampus region of different age groups of rats. the changes in the hippocampus region of female rat brain of different age groups with and without e2 treatment were observed by transmission electron microscopy. age dependent changes in myelin sheath, axon and cytoplasm membrane were observed with aging in control group rat brain but the e2 treated rats showed significantly stable myelin sheath, myelin axon and cytoplasm structure. our results showed that e2 treatment significantly effects hippocampus brain region of aging rats. these analyses revealed that fundamental age-related changes in brain and estrogen have important implications when estrogen levels and hippocampus dependent functions decline. keywords: brain aging, estradiol, estrogen receptor, hippocampus introduction aging is associated with a decline in metabolic function, affects the endocrine system by altering endocrine cells, the hormones produced by these cells, hormone receptors or post receptor processes in the target cells [1, 2, 3]. the process of aging presents itself with various alterations in physiological events. advanced age is commonly associated with many of the serious neurological disorders and among them alzheimer’s diseases (ad) is the most important age related neurological disorder in elderly population and there lies a difficulty in diagnosing and treating ad. age-related impairment of functionality of the central nervous system (cns) is associated with increased susceptibility to develop many neurodegenerative diseases. increased oxidative stress in the cns of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deficits. the ovarian steroid hormone 17β estradiol (e2) is an essential hormone which protects neurons against aβ toxicity, oxidative stress and excitotoxicity [4-7]. hormone replacement therapy (hrt) has effects on the brain at the functional, metabolic and neurotransmitter levels. estrogens significantly affect the microstructure of brain regions, which are crucial to higher cognitive function and implicated in ad. intracellular estrogen receptors (er) are widespread and are found in the hippocampus, cerebral cortex, midbrain, brain stem, hypothalamus and pituitary gland. the distribution of er is well established, being present in a high concentration in the hippocampus, hypothalamus, pituitary and amygdala. the action of estrogen has been shown to bind to cell membrane receptors, and affect the same second messenger systems used by growth factors and neurotransmitters [8]. however, estrogens also affect synaptic communication in brain regions involved in cognitive processing, such as the hippocampus [9], and these effects may be of particular importance in the context of aging, when both circulating estrogen levels change and hippocampus dependent functions decline [10]. the population of healthy neurons might be reduced, particularly in the hippocampus, leaving the brain with impaired ability to tolerate the neurodegenerative processes of aging and alzheimer's disease. males have higher brain levels of e2 than females, and this has been demonstrated for the hypothalamus, the major brain region undergoing sexual differentiation [11]. e2 is also synthesized de novo in the developing hippocampus and cortex. the amount of e2 measured in neonatal hippocampus and cortex is the same in males and females, and this may be the result of de novo local e2 synthesis [11]. the expression of er-α and er-β in the brain, in particular in the hippocampal formation, provides ___________________________________________________________ * corresponding author: sudha cowsik, phd (scowsik@yahoo.com). http://www.ajecr.org/ mailto:scowsik@yahoo.com 84 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org a potential target and mechanism for effects of estrogen in brain regions and circuits mediating cognitive processes such as memory [12-20]. the potential for e2 as a therapeutic neuroprotective agent in the aging brain has been of intense interest for over a decade. the aim of the present study was to investigate the age related changes in the hippocampal region of female rat brain of different age groups in the presence of e2 by means of the transmission electron microscopy (tem). materials and methods animals the present study was conducted on female albino rats of the wistar strain in different age groups (3, 12 and 24 months). animals were maintained in the animal house facility of jawaharlal nehru university (jnu), new delhi, india at a constant temperature of 25°c, humidity 55% and 12 h dark and light cycle. the animals were fed standard chow rat feed (hindustan leaver ltd., india) and given tap water until the time of sacrifice. the institutional animal ethics committee (iaec) of jnu approved all the animal experiments; all institutional guidelines for care of animals were followed. hormone administration subcutaneous injections of e2 (0.1 μg/g body weight) were given daily for one month, to the aged rats (12 and 24 months old; n = 8 for each group). e2 was dissolved in propylene glycol in appropriate concentrations [21]. control animals received an equal volume of vehicle. there was no treatment on the day of the sacrifice. animals of all the groups were sacrificed and brains were isolated for further study. rat perfusion for rat perfusion 4% paraformaldehyde2.5 % glutaraldehyde [22] has been used. we dissolved 4g paraformaldehyde in 100ml of double distilled water at 60ºc with contunious stirring in a covered beaker to avoid evaporation. 1n naoh was added drop wise with stirring until the solution become clear. to 100ml solution, 2.5% glutaraldehyde was added. thereafter, the volume was made to 100ml with buffer solution. the ph of solution was adjusted to 7.4. paraformaldehydeglutaraldehyde was used to fix brain tissue after the rat perfusion experiments. in this experiment the rat was deeply anesthetized, and then perfused intracardially with a paraformaldehydeglutaraldehyde solution. the brain was then removed from the rat, placed into a vial containing paraformaldehyde-glutaraldehyde and incubated at least 20 hours. the brain was then removed for further anatomical examination and processed for tem examination.. results anatomical changes in control and e2 treated female rats of ages 3, 12 and 24 months were assessed in the hippocampus region of female rat brain by tem. in 3 month old rats, we observed the stable myelin sheath, axon and cytoplasm membranous structure (fig. 1a). figure 1 (a-e) transmission electron micrographs of the rat hippocampus region showing an increase in myelin sheath after e2 treatment (arrow) (bar = 1m) in 12 month old rats we found the age dependent decrease in myelin sheath and changes in axon and cytoplasm membrane (fig. 1b). however, e2 treated 12 3m 12m 12m e2 24m 24m e2 http://www.ajecr.org/ 85 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org months rat brain show significantly stable myelin sheath, axon and membrane as compared to 12 month control rats (fig. 1c). similarly the treatment with e2 in 24 month group showed increase in myelin sheath, changes in axon and stable membranous structure as compared to the 24 month age. in this study, after the treatment with e2 to aging animals, an increase in myelin sheath, axon and cytoplasm membranous stability in hippocampus region was found as compared with 12 and 24 month control rats (figs. 1 a-e). discussion e2 is synthesized in hippocampus and being present in a high concentration in the hippocampus, hypothalamus, pituitary and amygdale [11]. the aged synapses fundamentally different from the young synapse in its capacity for plasticity, particularly in response to estrogen [23]. in the present study, a quantitative anatomical study was carried out in hippocampus region of female rat brain of different age group, with and without e2 treatment. tem was used to verify the ultrastructural level. accurate electron microscope analysis has proved to be very tedious and provides a relatively small sample from which to generalize the result [24, 25]. the anatomical structure study of hippocampus brain region was done by tem. in this study, progressively with age, decrease in myelin sheath and less stable axon and cytoplasm structure were found. this observation supported by earlier report of bertoni-freddari et al.,1993, 2006 and solmi et al., 1994) [26-28]. earlier results also showed the changes in the fluidity of membrane and myelin sheath are known to occur during aging [29-31]. further we studied the anatomical structure of hippocampus region of aging rats in e2 treated rats and found significant increase in myelin sheath and stable cytoplasm membrane in 12 month and 24 months aging rats. changes were seen in cytoplasm, plasma membrane, myelin axon as well as mitochondrial membrane. from this result we can say that estrogens significantly affect the microstructure of brain regions and state that e2 maintains cytoplasm membrane and myelin axon of aged rats. this study suggests that significant morphological variations may exist among different neurotransmitterspecific nerve terminals in the brain. e2 interact with neuronal networks at many different levels and affect brain development and aging. however, it is possible that various effects of homogenization may produce some of the morphologic differences. these analyses revealed that fundamental age-related changes in brain and estrogen have important implications when estrogen levels and hippocampal dependent functions decline. acknowledgement financial grant from university grant commission, new delhi, india in the form of project is gratefully acknowledged. conflict of interest the authors declare no conflicts of interest. references 1. harman d. free radical theory of aging. mutat res 275:257-266, 1992. 2. veiga, s., melcangi, r.c., doncarlos, l.l., garciasegura l.m., azcoitia, i. sex hormones and brain aging. exp gerontol 39:1623-1631, 2004. 3. baquer nz, taha a, kumar p, mclean p, cowsik sm, kale rk, singh r, sharma d. a metabolic and functional overview of brain aging linked to neurological disorders. biogerontology 10, 377-413, 2009. 4. brann dw, krishnan d, chandramohan w, virendra bm, mohammad mk. neurotrophic neuroprotective actions of estrogen: basic mechanisms and clinical implications. steroids 72:381-405, 2007. 5. jha r, mahdi aa, pandey s, baquer nz, cowsik sm. effects of tachykinin neuropeptide nkb and aβ (2535) on antioxidant enzymes status in 17β estradiol treated aging female rats. adv aging res 2:137-143, 2013. 6. jha r, mahdi aa, pandey s, baquer nz, cowsik sm. neuroprotective role of 17β estradiol with tachykinin neuropeptide nkb and aβ (25 35) in aging female rat brain. adv aging res 2:130-136, 2013. 7. meyers b, agostino da, walker j, kritzer mf. gonadectomy and hormone replacement exert region-and enzyme isoform-specific effects on monoamine oxidase and catechol-o-methyltransferase activity in prefrontal cortex and neostriatum of adult male rats. neuroscience 165:850-862, 2010. 8. mcewen bs. clinical review 108: the molecular and neuroanatomical basis for estrogen effects in the central nervous system. j clin endocrinol metab 84:1790-1797, 1999. 9. woolley cs, schwartzkroin pa. hormonal effects on the brain. epilepsia 39 (suppl) 8:s2-s8, 1998. 10. sherwin bb. oestrogen and cognitive function throughout the female lifespan. novartis found symp 230:188-196, 2000. 11. amateau sk, alt jj, stamps cl, mccarthy mm. brain estradiol content in newborn rats: sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. endocrinology 145:2906–17, 2004. 12. li x, schwartz pe, rissman ef. distribution of estrogen receptorβ like immunoreactivity in rat forebrain. neuroendocrinology 66:63-67, 1997. 13. shughrue pj, lane mv, merchenthaler. comparative distribution of estrogen receptor-α and -β mrna in the rat central nervous system. j comp neurol 388:507-525, 1997. 14. shughrue pj, scrimo pj, merchenthaler i. evidence for the colocalization of estrogen receptor-β mrna and estrogen receptor-α immunoreactivity in neurons of the rat forebrain. endocrinology 139:5267-5270, 1998. 15. weiland ng, orikasa c, hayashi s, mcewen bs. distribution and hormone regulation of estrogen receptor immunoreactive cells in the hippocampus of male and female rats. j comp neurol 388:603-612, 1997. 16. pau cy, pau k-yf, spies hg. putative estrogen receptor β and α mrna expression in male and female http://www.ajecr.org/ 86 am j exp clin res, vol. 2, no. 1, 2015 http://www.ajecr.org rhesus macaques. mol cell endocrinol 146:59-68, 1998. 17. petersen dn, tkalcevic gt, koza-taylor ph, turi tg, brown ta. identification of estrogen receptor β2, a functional variant of estrogen receptor β expressed in normal rat tissues. endocrinology 139:1082-1092, 1998. 18. register tc, shively ca, lewis ce. expression of estrogen receptor α and β transcripts in female monkey hippocampus and hypothalamus. brain res 788:320-322, 1998. 19. shughrue pj, merchenthaler i. evidence for novel estrogen binding sites in the rat hippocampus. neuroscience 99:605-612, 2000. 20. brinton rd. cellular and molecular mechanisms of estrogen regulation of memory function and neuroprotection against alzheimer’s disease: recent insights and remaining challenges. learn mem 8:121-133, 2001. 21. moorthy k, yadav ucs, siddiqui mr, basir sf, sharma d, baquer, nz. effect of estradiol and progesterone treatment on carbohydrate metabolizing enzymes in tissues of aging female rats. biogerontology 5: 249-259, 2004. 22. karnovsky mj. 1965. a formaldehydeglutaraldehyde fixative of high osmolarity for use in electron microscopy. j cell biol 27:137a, 1965. 23. adams mm, fink se, shah ra, janseen wgm, hayashi s, milner ta, mcewen bs, morrison jh. estrogen and aging affect the subcellular distribution of estrogen receptor-α in the hippocampus of female rats. the journal of neuroscience 22:3608-3614, 2002 24. harris km, jensen fe, tsao b. development of hippocampal synapses, spines, and ltp. sot neurosci abstr 13:1429, 1987. 25. harris km, jensen fe, tsao b. ultrastructure, development, and plasticity of dendritic spine synapses in area ca1 of the rat hippocampus: extending our vision with serial electron microscopy and three-dimensional analyses. in: the hippocampus-new vistas, neuroloav and neurobioloav. vol 52 (ghan-palav v. kohler c. eds). pp 33-g. new york: list, 1989. 26. bertoni-freddari c, fattoretti p, casoli t, spagna c, meier-ruge w, ulrich j. morphological plasticity of synaptic mitochondria during aging brain res 628:193200, 1993. 27. bertoni-freddari c, mocchegiani e, malavolta m, casolit, di stefano g, fattoretti p. synaptic and mito chondrial physiopathologic changes in the aging nervous system and the role of zinc ion homeostasis. mech ageing dev 127:590-596, 2006. 28. solmi r, pallotti f, rugolo m, genova ml, estornell e, ghetti p, pugnaloni a, biagini g, rizzoli c, lenaz g. lack of major mitochondrial bioenergetic changes in cultured skin fibroblasts from aged individuals. biochem mol biol int 33:477-484, 1994. 29. choi jh, yu bp. brain synaptosomal aging: free radicals and membrane fluidity. free radic. biol. med. 18, 133-139, 1995. 30. mantha, ak, moorthy k, cowsik sm, baquer nz. membrane associated functions of neurokinin b (nkb) on amyloid-beta (25–35) induced toxicity in aging rat brain synaptosomes. biogerontology 7:19-33, 2006. 31. jha r, mahdi aa, pandey s, baquer nz, cowsik sm. age-related changes in membrane fluidity and fluorescence intensity by tachykinin neuropeptide nkb and aβ (25-35) with 17β estradiol in female rat brain. am j exp clin res 1 (2), 25-30, 2014. http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/pubmed?term=lenaz%20g%5bauthor%5d&cauthor=true&cauthor_uid=7951066 299 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(3):299-303 original article theophylline and silymarin are potent inhibitors of il-4 secretion from activated human basophils hamidreza jahanbani-ardakani 1 , ghazale bordbari 2 , nahid eskandari 2 *, ali mohammad sabzghabaee 3 , alireza andalib 2 1isfehan medical students resaerch center (imsrc), isfahan university of medical sciences, isfahan, iran 2departmen of immunology, school of medicine, isfahan university of medical sciences, isfahan, iran 3isfahan clinical toxicology research center, isfahan university of medical sciences, isfahan, iran abstract. basophils are the most important cells in allergic reactions and secret interleukin 4 (il-4) and tumor necrosis factor α (tnfα) mediators. theophylline as a phosphodiesterase 4 inhibitor and silymarin with its anti-inflammatory effects seem to be useful drugs in treatment of allergic reactions. the aim of the present study was comparing effects of theophylline and silymarin on il-4 and tnfα secretion by human basophils. after isolation of peripheral blood mononuclear cells, three cultures were designed; theophylline, silymarin and control cultures. cultures were incubated for 15 minutes with theophylline, silymarin and buffer before challenging with bacterial lipopolysaccharide and anti-ige antibody. il-4 and tnfα mediators were assessed by elisa assay and values of each culture were compared with control group. silymarin and theophylline as potent inhibitors decreased secretion of il-4 from human basophils significantly (p<0.01 and p<0.01, respectively). however, neither theophylline nor silymarin had significant effect on tnfα secretion (p=0.14 and p=0.08, respectively). these data suggest that silymarin and theophylline could regulate the generation of il-4 from human basophils. keywords: theophylline, silymarin, interleukin-4, tumor necrosis factor α, basophil introduction basophils are well known for their pivotal role in allergic reactions. these cells are at least abundant leukocytes in peripheral blood (less than 1 percent). basophils like mast cells have high-affinity ige receptors on cell surface and also contain granules in their cytoplasmic space that will be exocytose when crosslinked antigen-specific ige occurred. granules consists of histamine, leukotrienes, cytokines, chemokines and other mediators that are responsible for early and late allergic reactions [1]. in allergic disorders, basophils promotes t helper2 (th2) differentiation by il-4 production and other co-stimulatory molecules such as cd40, cd80 and cd86 [2, 3]. basophils would consider as a key role in allergic reaction for major source of cytokines such as il-4 and tissue necrosis factor alpha (tnfα) after activation through ige receptors [4-6]. production of these cytokines could be induced either by ige-dependent mechanism or non-ige dependent mechanism. silymarin is a flavonoid derived from milk thistle, silybum marianum l. it is an antioxidant agent with protective effects on liver organ and prescribed clinically in liver diseases [7, 8]. in addition, silymarin has been described to represent antioxidant, immunomodulatory, anti-proliferative, anti-fibrotic, and antiviral activities. the mechanisms of silymarin is elusive and its clinical efficacy is currently uncertain. several studies, have reported immunomodulatory actions of silymarin in vitro. in addition, silymarin could modulate immune system and stabilizes cell membrane. previously, it was shown that silymarin could reduce histamine release from basophils and also promotes of th2 cytokines and suppresses of th1 cytokines [9, 10]. however, data on its effects on th1/th2 balance is in controversy. cyclic monophosphate phosphodiesterases (pde) are an enzyme family that degrade cyclic adenosine monophosphate (camp) and guanosine monophosphate. pde-4 enzyme, is a member of pde enzyme that exist mostly in immune cells including neutrophils, monocytes, eosinophils and lymphocytes. also, pde-4 express highly in epithelial cells of lung and play a crucial role in pathogenesis of inflammatory lung diseases such as chronic obstructive pulmonary disease (copd). pde inhibitors prevent degradation of cyclic adenosine monophosphate (camp) and guanosine monophosphate. moreover, pde-4 inhibition results to accumulation of ___________________________________________________________ * corresponding author: nahid eskandari, md, phd (neskandari@med.mui.ac.ir) http://www.ajecr.org/ 300 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org figure 1 effects of theophylline and silymarin on il-4 secretion. cells were incubated for 15 minutes before challenging with anti-ige and lps for 4 hours (n=5). mean value of il-4 was 60.06±10.73 and 31.83±12.43 in cultures treated with theophylline and silymarin respectively. camp and consequently leads to suppressing expression of pre-inflammatory cytokines and chemokines [11, 12]. theophylline (1, 3 dimethylxanthine) is a non-selective pde inhibitor with anti-inflammatory and bronchodilator effects. previously, it has been demonstrated that theophylline administration could leads to statistically improvement of forced expiratory volume in 1 second, forced vital capacity, and fev1/fvc [13]. also, long term administration of theophylline leads to reduction of interleukin 8 and tissue necrosis factor alpha in patients with chronic obstructive pulmonary disease [14]. furthermore, theophylline administration in asymptomatic atopic asthmatic patients could reduce the serum level of il-4 and also interleukin 5 [15]. the aim of the present study was to determine whether silymarin and theophylline regulates the generation of the cytokines, il-4 and tnfα, from activated basophils. materials and methods preparation of inhibitors and stimuli silymarin (sigma, poole, u.k.) was dissolved in 100% dimethyl sulfoxide (sigma, poole, u.k.) to achieve a 100mm stock solution. we used 10-2 concentration of silymarin stock solution. theophylline (sigma, poole, uk) was prepared daily as stock solutions (2 mm) in the buffer. escherichia coli bacterial lipopolysaccharide (lps) (enzo life sciences, exeter, uk) was diluted with sterile hepes buffer (2000ng/ml). neat polyclonal goat antihuman ige antibody (sigma, poole, uk) was made up in distilled water and stored at 4ºc. all drugs were diluted to desired concentration in the buffer just before use. isolation of human basophils human peripheral blood mononuclear cells (pbmcs) were collected by vein puncture in heparinized tubes from whole blood obtained from fifteen healthy volunteers, who gave informed written consent. the study protocol was approved by ethics committee of isfahan university of medical sciences. our exclusion criteria were as follows: any acute or chronic infectious disease, any neurological disorder, addiction and current smokers. the cells were isolated by using standard ficoll density-gradient centrifugation. shortly, heparinized blood (50ml) was mixed with phosphate-buffered saline (pbs) (50ml) and after coating samples with ficoll, the centrifugation (2800 rpm for 20 min) was performed and the interface layer of pbmcs was harvested and washed twice with pbs. cell viability was assessed by trypan blue dye exclusion (0.4% trypan blue in pbs). cells with viability of 95% and more were selected for further experiments. cell culture and mediator release the cells were categorized in three cultures; control, theophylline and silymarin cultures. the cultures contained rpmi 1640 buffer (gibco brl, dundee, u.k.) supplemented with bsa (sigma, poole, u.k.) (1 mg/ 1ml), gentamicin (gibco brl, dundee, u.k.) (10 mg /ml) and calcium chloride (made up to 1 mm). cultures were incubated with theophylline, silymarin and buffer for duration of 15 minutes before challenging with lps and anti-ige antibody. cells incubated in buffer alone were considered as values of spontaneous mediator release, and all values cited for other cultures were corrected by subtracting values of spontaneous mediator release. in experiments monitoring mediator generation, basophils were activated for 4 hours to assess il-4 generation and 24 hours to assess tnfα generation. these conditions for optimal generation of il-4 and tnfα generation have been reported by others [16]. after activation, the cells were centrifuged at 450 g for 4 min and the supernatants saved and analyzed for mediator release. the supernatant was assayed for il-4 and tnfα mediators by available commercial enzyme linked immunosorbent assay (elisa) kit (biolog life science institute, bremen, germany). the od of samples was measured at 450 nm using a dynatech plate reader. statistical analysis wilcoxon test were applied for comparing data between study groups. data are expressed as means ± standard deviation. a p value of less than 0.05 was considered statistically significant. all of the analysis was performed by using spss software (spss inc., chicago, il, usa). results effects of theophylline and silymarin on il-4 secretion the means ± sem generation of il-4 concentration inhibited by theophylline and silymarin were 5.68±1.53 ng/ml and 9.69±1.77ng/ml respectively. both theophylline and silymarin had significant effects on reduction of il-4 secretion (p<0.01 and p<0.01 respectively) (figs. 1 and 2). as figures 1 depicts, theophylline is more effective inhibitor than silymarin. http://www.ajecr.org/ 301 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org figure 2 effects of theophylline and silymarin on tnfα secretion. cells were incubated for 15 minutes before challenging with antiige and lps for 24 h (n=5). mean value of tnfα was 18.08±2.61 and 22.93±1.94 in cultures treated with theophylline and silymarin, respectively. effects of theophylline and silymarin on tnfα secretion the mean value of tnfα inhibition in silymarin and theophylline cell cultures were 1.53±0.04 ng/ml and 1.62±0.52 ng/ml respectively. neither silymarin nor theophylline had significant effects on reduction of tnfα secretion (p=0.08 and p=0.14 respectively) (fig. 2). discussion in the present study, we have investigated the effects of silymarin and theophylline on il-4 and tnfα generation from human basophils. a recent study showed topical administration of silymarin in nc/nga mice with atopic dermatitis showed significant decrement of serum level of il-4 [16]. bakhshaee et al. have shown that prescription of silymarin with anti-histamine had significantly better improvement in clinical symptom severity of allergic disorders and the level of il-4 decreased numerically in the sera of patients [17]. in the present study, it was shown that silymarin is effective inhibitor of the il-4 generation from basophils, confirming observations made by others [16]. previously inhibition of histamine releasing by theophylline from basophils has been reported [18]. our data demonstrate the inhibitory effects of theophylline on il-4, as reported by others [19, 20]. further studies were performed to determine silymarin and theophylline effects that regulate cytokine generation from human basophils. both compounds inhibited the igemediated generation of tnfα. earlier, it has been shown that long term treatment with theophylline in copd patients lead to significant reduction of tnfα in sputum and could be associated with fev1 increase [21]. in addition, theophylline could enhance basophil apoptosis and maybe this effect is one of the explanations of its affectivity in allergic diseases [9]. the mechanism of theophylline on decrement of pro inflammatory cytokines generation is inhibition of nfkappab activation via protection of ikappabalpha protein [19]. many properties of silymarin has been reported; some of them are anti-oxidant activity, anti-inflammatory activity, immuno-suppressive and immunomodulatory effects [22, 23]. the large body of data showed that silymarin increases lymphocyte proliferation, interferon gamma (ifn-γ), il-4 and il-10 secretion by activated lymphocytes, in a dose-dependent manner [24]. previously han and colleagues reported that silymarin inhibits chemical-induced irritant contact dermatitis in mice [25]. in individual studies, administration of silymarin in patients with peritoneal dialysis leads to decreasing in serum level of tnfα. in another study in patients with βthalassemia major it was shown that silymarin could significantly decrease tnfα in serum of patients [26, 27]. inhibition of tnfα expression by silymarin had been reported by han et al. and zi and colleagues [25, 28]. the recent meta-analysis showed that administration of silymarin in patients with hepatitis b can decrease tnfα, tgf-β1 and il-6 [29]. our results showed numerically but not significantly decrement of tnfα secretion by human basophils. this data could suggest that silymarin affects other sources of tnfα secretors except basophils; however more studies are suggested in this regard. silymarin exerts its anti-inflammatory effects via phosphorylation of iκba and consequently it leads to inhibition of nf-κb. nfκb influence production of cytokines, chemokines and other mediators related to inflammation [30, 31]. therefore it could be concluded that pde-4 inhibitors exerts their effect via inhibition on nfκb and due to wide range of activity of nfκb. it seems that more inflammatory mediators will be affected by pde-4 inhibitors treatment. our study limitations were low number of cell cultures due to financial problems and lack of assessment of other involved cytokines in allergic reactions such as interleukin13 and interleukin 5. we recommend future studies to investigate inhibitory effects of silymarin and theophylline on il-6, il-13, il-25 and other involved biomarkers in allergic reactions secreted from human basophils. conclusion in conclusion, the present study demonstrates significant effects of silymarin on mediator releasing from human basophils, and it seems that theophylline and silymarin could be useful drugs in allergic diseases. acknowledgment this project was supported by isfahan university of medical sciences (grant no: 393412). also, the authors would like to greatly appreciate dr. abolfazl fallah for his kind help. conflicts of interest the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. http://www.ajecr.org/ 302 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org references 1. siracusa mc, kim bs, spergel jm, artis d. basophils and allergic inflammation. j allergy clin immunol 132:789-801, 2013. 2. sokol cl, medzhitov r. role of basophils in the initiation of th2 responses. cur opin immunol 22:73-77, 2010. 3. sokol cl, chu n-q, yu s, nish sa, laufer tm, medzhitov r. basophils function as antigen-presenting cells for an allergen-induced t helper type 2 response. nature immunol 10:713-720, 2009. 4. schneider e, thieblemont n, de moraes ml, dy m. basophils: new players in the cytokine network. eur cytokine network 21:142-153, 2010. 5. steiner m, huber s, harrer a, himly m. the evolution of human basophil biology from neglect towards understanding of their immune functions. biomed res int 8232830, 2016. 6. gibbs bf. basophils as key regulators of allergic inflammation and th2-type immunity. world allerg org j 1:123-128, 2008. 7. bannwart cf, peracoli jc, nakaira-takahagi e, peracoli mt. inhibitory effect of silibinin on tumour necrosis factor-alpha and hydrogen peroxide production by human monocytes. natural prod res 24:1747-1757, 2010. 8. dixit n, baboota s, kohli k, ahmad s, ali j. silymarin: a review of pharmacological aspects and bioavailability enhancement approaches. indian j pharmacol 39:172-179, 2007. 9. kawakami a, suzukawa m, koketsu r, komiya a, ohta k, yamamoto k, et al., editors. enhancement of basophil apoptosis by olopatadine and theophylline. allergy and asthma proceedings. oceanside publications, inc., 2008. 10. choi yh, yan gh. silibinin attenuates mast cellmediated anaphylaxis-like reactions. biolog pharmaceut bulletin 32:868-875, 2009. 11. serezani ch, ballinger mn, aronoff dm, petersgolden m. cyclic amp: master regulator of innate immune cell function. am j resp cell mol biol 39:127132, 2008. 12. page cp, spina d. selective pde inhibitors as novel treatments for respiratory diseases. cur opin pharmacol 12:275-286, 2012. 13. margay sm, farhat s, kaur s, teli ha. to study the efficacy and safety of doxophylline and theophylline in bronchial asthma. j clin diag res 9:fc05-08, 2015. 14. iiboshi h, ashitani j, katoh s, sano a, matsumoto n, mukae h, et al. long-term treatment with theophylline reduces neutrophils, interleukin-8 and tumor necrosis factor-alpha in the sputum of patients with chronic obstructive pulmonary disease. pulm pharmacol ther 20:46-51, 2007. 15. kosmas e, michaelides s, polychronaki a, roussou t, toukmatzi s, polychronopoulos v, et al. theophylline induces a reduction in circulating interleukin‐4 and interleukin‐5 in atopic asthmatics. eur resp j 13:53-58, 1999. 16. kang js, yoon wk, han mh, lee h, lee cw, lee kh, et al. inhibition of atopic dermatitis by topical application of silymarin in nc/nga mice. int immunopharmacol 8:1475-1480, 2008. 17. bakhshaee m, jabbari f, hoseini s, farid r, sadeghian mh, rajati m, et al. effect of silymarin in the treatment of allergic rhinitis. otolaryng head neck surg 145:904-909, 2011. 18. nakano j, yano t, yamamura k, yoshihara h, ohbayashi o, yamashita n, et al. aminophilline suppress the release of chemical mediators in treatment of acute asthma. respir med 100:542-550, 2006. 19. chen zc, ding jj, qian hy, lin xh, feng kq, wang j. clinical efficacies of inhaled corticosteroids plus theophylline in the treatment of bronchial asthma]. zhonghua yi xue za zhi 93:1076-1079, 2013. 20. eskandari n, wickramasinghe t, peachell pt. effects of phosphodiesterase inhibitors on interleukin‐4 and interleukin‐13 generation from human basophils. brit j pharmacol 142:1265-1272, 2004. 21. iiboshi h, ashitani j-i, katoh s, sano a, matsumoto n, mukae h, et al. long-term treatment with theophylline reduces neutrophils, interleukin-8 and tumor necrosis factor-α in the sputum of patients with chronic obstructive pulmonary disease. pulm pharm therap 20:4651, 2007. 22. min k, yoon w-k, kim sk, kim b-h. immunosuppressive effect of silibinin in experimental autoimmune encephalomyelitis. arch pharm res 30:12651272, 2007. 23. kang js, park s-k, yang k-h, kim hm. silymarin inhibits tnf‐α‐induced expression of adhesion molecules in human umbilical vein endothelial cells. febs letters 550:89-93, 2003. 24. alidoost f, gharagozloo m, bagherpour b, jafarian a, sajjadi se, hourfar h, et al. effects of silymarin on the proliferation and glutathione levels of peripheral blood mononuclear cells from beta-thalassemia major patients. int immunopharmacol 6:1305-1310, 2006. 25. han mh, yoon wk, lee h, han s-b, lee k, park s-k, et al. topical application of silymarin reduces chemical-induced irritant contact dermatitis in balb/c mice. int immunopharmacol 7:1651-1658, 2007. 26. gharagozloo m, karimi m, amirghofran z. immunomodulatory effects of silymarin in patients with βthalassemia major. int immunopharmacol 16:243-247, 2013. 27. nazemian f, karimi g, moatamedi m, charkazi s, shamsara j, mohammadpour ah. effect of silymarin administration on tnf‐α serum concentration in peritoneal dialysis patients. phytother res 24:1654-1657, 2010. 28. esmaeil n, anaraki sb, gharagozloo m, moayedi b. silymarin impacts on immune system as an immunomodulator: one key for many locks. int immunopharmacol 50:194-201, 2017. 29. wei q, xu x, ling q, zhou b, zheng s-s. perioperative antiviral therapy for chronic hepatitis brelated hepatocellular carcinoma. hepatobil pancreat dis int 12:251-255, 2013. 30. kim b-r, seo h-s, ku j-m, kim g-j, jeon cy, park jh, et al. silibinin inhibits the production of prohttp://www.ajecr.org/ 303 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org inflammatory cytokines through inhibition of nf-κb signaling pathway in hmc-1 human mast cells. inflamm res 62:941-950, 2013. 31. striz i, brabcova e, kolesar l, liu x, barcova i, sekerkova a, et al. epithelial cells modulate genes associated with nf kappa b activation in co-cultured human macrophages. immunobiology 216:1110-1116, 2011. http://www.ajecr.org/ 133 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(4):133-135 original article plasma exchange in progressive systemic sclerosis mohammad bagher owlia* department of medicine, shahid sadoughi university of medical sciences, yazd, iran abstract. systemic sclerosis (ssc) is an autoimmune systemic disease of unknown etiology. present treatment modalities have limited impact on clinical/ laboratory outcomes. for the first time in our center, we used plasma exchange (pex) in a rather young woman with recent onset but progressive ssc. she is a 39-year-old woman with a recent history of skin stiffness, raynaud’s phenomenon, nail fold capillary changes and newly diagnosis of ssc presented to us due to worsening her clinical symptoms even after initiation of routine remedies such as low dose oral prednisolone, ca-channel blockers, azathioprine and pentoxyfylline. after obtaining written consent, interdisciplinary discussion with experts in this field and agreement we started a series of plasma exchange with ffp replacement for her. a dramatic clinical response was observed in respect to raynaud’s phenomenon, skin stiffness, tendon rub after three sessions of pex. her modified rodnan skin score (mrss) dropped from 36 (before commencement of therapy) to 28 in day 4 and 18 in day 20 after 15 sessions of pex. in conclusion pex could significantly modify the course of ssc as observed in our case study. elimination of culprit immune mediators/cytokines/autoantibodies could be the possible mechanism of action of pex. keywords: systemic sclerosis, plasma exchange, therapy, plasmapheresis, treatment introduction systemic sclerosis (ssc) is one of the most devastating and chronic rheumatic diseases with disappointing results despite explosive discoveries of new treatment options in recent era. progressive organ fibrosis and obliterative vasculopathy are two major pillars of ssc pathophysiology. skin, lungs and kidneys are the major organs involved in the process of ssc. but other organs like gastrointestinal and conductive system of heart may also be damaged during disease pathology [1]. ssc usually attacks young women of reproductive age with many physical, social and emotional consequences. besides, current therapies are of very limited value in acceptable control or quenching disease. actually most of observed dramatic improvements in ssc could be due to spontaneous regression rather than therapeutic intervention per se. innate and adaptive immunity also cellular and humoral mediators are involved in the pathogenesis of ssc resulting auto-antibody formation and cell-medicated tissue injury [2]. genetic susceptibility along with environmental factors is shown to be important [3]. circulatory autoantibodies or immune mediators which are more than simple epiphenomena are shown to play an effective role in disease activity in ssc [4]. some antibodies are associated with specific organ involvement and others like autoantibodies against angiotensin ii receptor and antiendothelin1 receptor are associated with more severe disease [5, 6]. other kinds of autoantibodies such as antirna polymerase iii antibody are associated with underlying malignancies [7]. current pharmacologic therapies are based on immunomodulation, vasodilators, anti-platelet agents. tyrosine kinase inhibitors (imatinib) are rather new agents used in ssc in order to regulate growth, differentiation and apoptosis of aberrant cell function. considering the abundant circulatory factors (known and unknown) in sera of patients with ssc, inhibiting acting mediators could be a solution for systemic sclerosis. this could be achieved by using intravenous immunoglobulin (ivig) to neutralize autoantibodies. we hypothesized that removing these culprit soluble mediators besides autoantibodies could be more efficacious than common strategies in immunomodulations. so here we report for the first time in our center and country a woman successfully treated with plasma exchange. case study we report a 39-year-old woman with skin stiffness, raynaud’s phenomenon, nail fold capillary abnormalities for about 6 months before newly diagnosed with ssc referred to us due to worsening her clinical symptoms even after initiation of routine remedies such as low dose oral ___________________________________________________________ *corresponding author: mohammad bagher owlia, md (bagherowlia@gmail.com). http://www.ajecr.org/ mailto:bagherowlia@gmail.com 134 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org fig. 1 plasma exchange in the present patient. prednisolone, calcium-channel blockers, azathioprine and pentoxyfylline. she had not significant positive medical history neither she nor her immediate family. physical examination revealed blood pressure of 145/85 mmhg and otherwise normal vitals. she had puffy and shiny face with some erythema, tethered and reduced oral aperture, pale and cold hand and foot digits with marked nail fold capillary abnormalities on both fingers and toes. tendon rub was audible in her right ankle movement. her echocardiographic study showed normal ef with normal pulmonary artery pressure. her chest x-ray was normal. hrct revealed dilated esophagus with prominent interalveolar septa. laboratory investigations showed negative rf, crp= 26.2 mg/dl (reference range (rr) <6), ana (elisa) = 5.8 (rr <1.2), negative anti-ds dna, negative anti-ccp, normal hemoglobin, esr and urine analysis. after full explanation of our plan to initiate plasma exchange, possible unwanted events for getting vascular access, and pex per se, she was hospitalized on 25 may 2015. baseline modified rodnan skin score (mrss) was 36. daily pex with 2 liters fresh frozen plasma replacement was instituted after getting a jugular venous access (fig. 1). she tolerated pex well and unexpectedly her mrss dropped to 28 in day 4 and to 18 in day 20 after 15 sessions of pex. overall clinical problems improved after first sessions of pex of them raynaud’s phenomenon and digital ischemia was more rapid and prominent. we are planning to repeat pex every six months according to the ongoing nature of such rheumatic diseases. discussion ssc is one of the most complex autoimmune disorders of unknown etiology. so, all therapeutic studies tried to find a way to combat the disease based on disease features and complications. hitherto, 77 accomplished clinical trials are registered on clinicaltrials.gov. most of the studies are focused on specific organ involvement in ssc. this shows that ssc is a multi-facet and subtle disease that is shown to be not amenable to a single holistic treatment. before new biologic agents, the only two most poorly controlled rheumatic conditions were “ankylosing spondylitis” and “systemic sclerosis”. however after history-making achievements in biologicals, meaning tnf blockers, actually the last condition that seems not to be amenable to classic treatments is systemic sclerosis. however, these new agents namely tnf blockers and b-cell depletion therapy have minimal evidence to support their clinical use in ssc. thus any novel ideas other than conventional or new drugs may be a solution. it looks that in some instances, old weapon, meaning pex, could fight old and complex diseases such as ssc more powerfully that the new ones. the superiority of pex to other therapies in immunologic and rheumatic diseases is to “eliminate” rather than “neutralize” wandering obnoxious agents or particles. because these neutralized pathogenic factors or immune complexes could still remain pathogen and make a potentially “new pathogen” itself. in most instances, ivig and pex are “liquid friends” and can be used interchangeably or even in combination in several clinical settings [8]. both pex and ivig are costly and carry potential disadvantages. several studies worked on ivig on ssc with different results [9, 19]. however future results are needed to compare short term along with long term results. mccune was one of the first clinicians who used pex in ssc as an off-label treatment and published his results in 1983 [11]. he believed that subjective improvement ensued after pex in respect to raynaud’s phenomenon and digital ulcer. there are less than 20 published articles dealing with pex in classic features other than standard indications of pex in ssc since then. the most number of patients studied in a single report belongs to guillevin in 1990 by 40 cases [12]. he concluded that pex has limited and short term results. however, we emphasize, this is fully expected that a chronic, complex and progressive disease like ssc needs a sustainable or sequential suppressive therapy and not a single course of treatment like what happens in thrombotic thrombocytopenic purpura as a standard indication for pex. as our study showed, some other reports also showed early response of raynaud’s phenomenon to pex within 3-4 days [13]. guillevin et al. [12] state poor wound healing from vanous access is a problem in ssc patients, nevertheless, we propose that the best candidates of pex are new cases of ssc who yet are in initial (edematous stage) phase of disease and not in late stages when irreversible skin stiffness has emerged. however, dramatic response of raynaud’s phenomenon and ischemic digits to pex makes it a potentially powerful tool against refractory vasospastic, digital ischemia and ulcers. in conclusion, pex could significantly modify the course of ssc as observed in our case. elimination of culprit immune mediators/cytokines/autoantibodies could be the possible mechanism of action of pex. acknowledgements the author appreciates sina owlia for his extensive review of the literature and data extraction who is working on a review in this field and also professor oliver distler for his support. conflict of interest the author declares no conflicts of interest. http://www.ajecr.org/ 135 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org references 1. vacca a, meune c, gordon j, et al. cardiac arrhythmias and conduction defects in systemic sclerosis. rheumatology 53:1172-1177, 2014. 2. pattanaik d, brown m, postlethwaite bc, postlethwaite ae. pathogenesis of systemic sclerosis. front immunol 6:272, 2015. 3. korman bd, criswell la. recent advances in the genetics of systemic sclerosis: toward biological and clinical significance. curr rheumatol rep 17:21, 2015. 4. distler o, distler j, kowal-bielecka o, gay re, muller-ladner u, gay s. chemokines and chemokine receptors in the pathogenesis of systemic sclerosis. modern rheumatol 12:107-112, 2002. 5. kill a, riemekasten g. functional autoantibodies in systemic sclerosis pathogenesis. curr rheumatol rep 17:34, 2015. 6. ribi c. novel autoantibodies in inflammatory myopathies and systemic sclerosis. revue med suisse 11:25-29, 2015. 7. saigusa r, asano y, nakamura k, et al. association of anti-rna polymerase iii antibody and malignancy in japanese patients with systemic sclerosis. j dermatol 42:524-527, 2015. 8. lefaucheur c, nochy d, andrade j, et al. comparison of combination plasmapheresis/ivig/anticd20 versus high-dose ivig in the treatment of antibodymediated rejection. am j transplant 9:1099-1107, 2009. 9. poelman cl, hummers lk, wigley fm, anderson c, boin f, shah aa. intravenous immunoglobulin may be an effective therapy for refractory, active diffuse cutaneous systemic sclerosis. j rheumatol 42:236-242, 2015. 10. cantarini l, rigante d, vitale a, et al. intravenous immunoglobulins (ivig) in systemic sclerosis: a challenging yet promising future. immunol res 61:326-337, 2015. 11. mccune ma, winkelmann rk, osmundson pj, pineda aa. plasma exchange: a controlled study of the effect in patients with raynaud's phenomenon and scleroderma. j clin apheresis 1:206-214, 1983. 12. guillevin l, amoura z, merviel p, et al. treatment of progressive systemic sclerosis by plasma exchange: long-term results in 40 patients. int j artif org 13:125-129, 1990. 13. jacobs mj, jorning pj, van rhede van der kloot ej, et al. plasmapheresis in raynaud's phenomenon in systemic sclerosis: a microcirculatory study. int j microcircul clin exp 10:1-11, 1991. http://www.ajecr.org/ 281 am j exp clin res, vol. 5, no. 2, 2018 http ://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(2):281-286 original article estimation of lippia alba antioxidants' activities in ochratoxin a intoxicated albino rats olukayode olugbenga orole1*, timothy olubisi adejumo2, adedotun adefolalu3, victor fadayomi4 1department of microbiology, federal university lafia, nasarawa state, nigeria 2department of microbiology, adek unle ajasin university, ak ungba-ak oko, ondo state, nigeria 3department of biochemistry, federal university lafia, nasarawa state, nigeria 4department of zoology, federal university lafia, nasarawa state, nigeria abstract. the p articip ation of antioxidants in body cellular p rocesses cannot be over-emp hasized as they p rotect and reduce oxidative damage within the cell and its surrounding membranes. these chemical substances act on free radicals by donating free electro ns to unstable radicals thereby haltering their cap acity to steal electrons. the cap acity of lippia alba extract to ameliorate antioxidant levels in ochratoxin a intoxicated albino rats were investigated. sixty albino rats divided into two sets were used in the study . animals were administered a single dosage of 2mg ochratoxin a p er 250 mg bw of animals, followed by the administration of the extract at 300 mg p er kg bo dy weight by gavage. results showed that intoxication with 2 mg ota/250 g bw reduced the glutathione levels in the two sets by 55.3 %, 47.1 %, 59.3 %, and 65.9 % for p re-treated (kidney ), p ost-treated (kidney ), p re-treated (liver), and p ost-treated (liver) resp ectively . sup eroxide dismutase values obtained highest recovery in group 5 p re-treated animals with increased values in the kidney and liver (51.37 ± 2.13 u/mg p rotein and 23.52 ± 1.78 u/mg p rotein resp ectively ). m alondialdehy de values reduced to 42.78 ± 4.13 mol/g kidney tissue (p re-treated set) and 53.60 ± 2.96 mol/g kidney tissues (p ost-treated set) which was a 25.1 % difference between the p re-treated and p ost-treated sets. conclusively , p re-treatment with l. alba increased antioxidant levels and it also reduced unstable radicals formed due to oxidative damage caused by xenobiotic ochratoxin a as seen in the results obtained. the extract thus imp roves the activities of antioxidant malondialdehy de, glutathione, sod, and glutathione s-transferase. keywords: antioxidants, radicals, lippia, intoxication introduction antioxidants are substances capable of neutralizing free radicals and reducing oxidative damage within the cell and surrounding membrane. these chemical substances, enzymes, elements, vitamins have the capacity to mop up free radicals and prevent them from causing cell damage [1]. they neutralize toxic radicals by bonding them through the donation of their own electrons and in the process altering the carbon-stealing reaction [2]. sen et al. [1] and jacob [2] classified antioxidant into endogen ous enzymatic antioxidants, non-enzymatic antioxidants, metabolic and nutrient oxidants, and metal binding proteins. antioxidant production by the human body decreases with age [3]; though the body produces only endogenous antioxidants which are classified into enzymatic and non-enzymatic groups in response to the destructive consequence of ros. the enzymatic antioxidant consist of superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione reductase (gr), while the non-enzymatic system are the vitamin e, vitamin c and reduced glutathione (gsh) [1, 2, 4]. free radicals are chemical substances produced in the body by the oxidative metabolism of food substances to obtain energy with capacity to induce disease conditions [1]. these are molecules or its fragment with one or more unpaired electrons formed through series of reduction oxidation reactions with capacity for independent existence and not very stable [5, 6, 7]. molecules such as rna, dna and protein enzymes are all susceptible to oxidative damage caused by free radicals within the cell. glutathione gsh a non-enzymatic antioxidant produced in the liver that produces other antioxidants, while also detoxifying toxic substances; and s trengthening the immune system [8, 9]. it is a co-factor of glutathione peroxidase (gpx) that help in the elimination of peroxides forme d during lipid peroxidation and ros [1]. superoxid e dismutase (sod) protects the cell from superoxide toxicity , act against ros by scavenging superoxide radicals to hydrogen peroxide. malondialdehyde (mda) is the decomposition product of polyunsaturated fatty acids of ___________________________________________________________ * corresponding aut hor: orole olukayode olugbenga, p hd (orolekayode@yahoo.com) http://www.ajecr.org/ mailto:orolekayode@yahoo.com 282 am j exp clin res, vol. 5, no. 2, 2018 http ://www.ajecr.org biological membranes and its increase shows the extent of membrane lipid peroxidation [10, 11]. materials and methods a) preparation of albino rat model albino rats were obtained and kept in conformity with laid down policy of “guide for care and use of laboratory animals in research and teaching” by the national academy of science, published by the national institute of health (nih) publication 86-23 [1985 revised]. b) experimental design sixty (60) animals were employed in the study. the animals were divided into two (2) sets of 30 animals with each set sub-divided into six groups of 5 animals each according to the methods of orole et al. [12]. the animals were allowed to acclimatize for two weeks (14 days) before the start of the experiment. animals in set 1: lippia alba pre-treated set were administered crude lippia alba extract at 300 mg/kg body weight for a period of three weeks before intoxicatio n depending on the group to build the immunity of the animals . group 1: negative control – animals were given dmso alone, group 2: positive control – animals were treated with 300 mg l alba per kg body weight, group 3: animals were treated with 2 mg ochratoxin a per 250 mg bw of animals only, group 4: animals were treated with 1 mg ochratoxin a per 250 mg bw of animals only, group 5: animals were treated with 2 mg ota/250 g bw + 300 mg l alba per kg body weight, group 6: animals were treated with 1 mg ota/250 g bw + 300 mg l alba per kg body weight, while set 2: lippia alba post-treated animals were administered extract at 300 mg/kg body weight after intoxication depending on the group. the animals 3 h after intoxication with observation for behavioral changes were treated with lippia alba extrac t based on body weight as in set 1. c) extracts and ochratoxin a preparation and administration to albino rats ten milligra m per milliliter (10 mg/ml) extract was prepared and administered to animals at concentration of 300 mg/kg body weight. extract administration was done every three days at 0700 hours in the morning by oral administration. administration of ochratoxin a (trilog y analytical laboratory; product number tsl-504) was through intraperitoneal route after dissolving the toxin in sterile dimethyl sulphoxide (dmso) and volume and dose adjusted to animal weight. d) organ collection and preparation of liver and kidne y homogenates seven after intoxication, animals were euthanized and subsequently sacrificed by cervical dislocation after which the liver and kidney were removed, washed with water and blotted with filter paper then weighed individually . employing the modified methods o f noori et al. [13], organs were separately perfused in saline solution then homogenized in chilled potassium chloride (1.17%). the homogenates were centrifuged to remove debris after which it was further centrifuged at 10,500 g for 20 min at 4ºc to obtain post mitochondrial supernatant needed for antioxidant assay. the kidney homogenates (1:10 w/v) were prepared by using a 100 mmol kcl buffer (ph 7.0) containing edta 0.3 mm, and the homogenates were centrifuged at 600 g for 60 minutes at 4ºc and the supernatant used for assay. e) estimation of malondiahydehyde (mda)-li pi d peroxidation lipid peroxidation level was estimated using the modified methods of nayanatara et al. [14] and kartha and krishnamurthy [15]. five millilitres of freshly prepared homogenate was incubated at 39ºc for 30 min along with “the blank” in a water bath in a separate conical flask. after incubation, 1 ml was added to the tube containing 1.5 ml of 20% cold trichloroacetic acid (tca) and then centrifuged for 10 min. after centrifugation, 2 ml of the supernatant fluid was added 2 ml of 0.7% thiobarbituric acid (tba) and kept in the boiling water bath for 10 min. the development of pink color was measured at 535 nm by using spectrophotometer. f) estimation of superoxide dismutase (sod) superoxide dismutase (sod) activity was measured using assay kit (cayman, mi, usa) according to manufacturer’s instructions. this kit utilizes a tetrazoliu m salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. one unit of sod was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radical. g) estimation of glutathione-s-transferase (gst) glutathione-s-transferase (gst) activity was assayed according to the modified methods of erejuwa et al. [16, 17]. to 2 ml of 0.3 m potassium phosphate buffer (ph 6.35), 75 μl of 30 mm cdnb solution, 725 μl of distilled water was added 0.1 ml of the organ homogenate in a test tube. the test tube was vortexed and incubated at 37ºc for 10 min . after incubation, the reaction was initiated by the addition of 100 μl of 30 mm reduced glutathione solution and measured using a spectrophotometer at 340 nm and recorded every 30 seconds for 4 minutes. h) estimation of glutathione (gsh) glutathione activity was determined by the procedure of noori et al. [13]. the assay solution contained 10% bsa (bovine serum albumin), 50 mm potassium phosphate buffer (ph 7.6), 2 mm nadph, and 20 mm oxidize d glutathione. absorbance at 340 nm was recorded at a temperature of 25ºc. the activity was calculated using the molar coefficient for nadph of 6.22 μmol-1 x cm-1 and expressed in u/g of tissue. i) statistical analysis the result of antioxidants levels were expressed as means and standard error of means (sem). analysis of variance was obtained and the means were separated using http://www.ajecr.org/ 283 am j exp clin res, vol. 5, no. 2, 2018 http ://www.ajecr.org t able 1 malondialdehyde levels in t he kidneys and livers of rat int oxicat ed wit h ochrat oxin a values are mean ± sem; values wit h different superscripts wit hin t he column are significantly different at p<0.05 by t ukey hsd t est t able 2 glut at hione levels in rat organs int oxicat ed wit h ochrat oxin a ochrat oxin a values are mean ± sem; values wit h different superscripts wit hin t he column are significantly different at p<0.05 by t ukey hsd t est tukey’s kramer post hoc test at p ≤0.05. results i) malondial dehyde levels in kidney and liver of lippia alba treated rats malondialdehyde levels as shown in table 1 obtained values ranging between 9.90 ± 0.68 mol/g kidney tissue as the lowest (group 2) and 27.60 ± 4.37 mol/g kidney tissue highest in group 3 of the pre-treated set. mda values in group 1 animals fell within the normal range then increased by 138.3 % and 158.4 % in the pre-treated and post-treated sets respectively. the values then decreased by 78.3 % and 28.7 % as seen in group 5 animals, though the effects were higher in the pre-treated set (16.60 ± 1.21 mol/g kidney tissue). in kidneys the pre-treated and post-treated sets, values were only significantly different between animals in group 3 at p<0.05. malondialdehyde values in the liver of the animals recorded about 100 % increases when compared with animals in group 3 of each set. the result showed that the extract decreased malondialdehyde values in the pre treated and post-treated sets as seen in groups 5 and 6 respectively. ii) glutathione levels in kidney and liver of lippia alba treated rats glutathione levels as shown in table 2 obtained values that increased in group 2 animals administered with lippia alba only in the pre-treated and post-treated sets when compared with the negative control g roups administered dmso only. the result showed administration of 2 mg ota/250 g bw reduced the gsh values across the two sets by 55.3 %, 47.1 %, 59.3 %, and 65.9 % for pre-treated (kidney), post-treated (kidney), pre-treated (liver), and posttreated (liver) respectively. the result also showed that gsh in kidney of group 3, 4, 5 and 6 of set 1 animals were significantly different from groups 1 and 2, while the same is applicable to kidney gsh values in set 2 animals at p<0.05. gsh values in the pre-treated and post-treated sets were significantly different in groups 1 and 2 from the rest of the groups at p<0.05. iii) superoxide dismutase activities in kidneys and livers of lippia alba treated rats table 3 presented increases in superoxide dismutase (sod) values as obtained in group 2 animals administered with 300 mg lippia alba per kg body weight across the two sets, but was contrasted by lower values obtained when group 3 animals in the two sets were administered 2 mg and 1 mg ochratoxin a per 250 g body weight respectively in the kidneys and livers of the animals. superoxide dismutase (sod) values were significantly different within the groups except in (groups 4 and 5 of kidney: pre-treated set), (groups 5, 6, and 3, 4: liver pre-treated set), and (groups 3, 4, and 5, 6 in the lippia alba post-treated set). gp 1: negative control (dmso only) 12.42 ± 1.08 a 11.84 ± 0.75 ab 32.89 ± 3.16 a 37.35 ± 2.00 ab gp 2: positive control (300 mg lippia alba / kgbw) 9.90 ± 0.68 a 10.00 ± 0.71 a 29.04 ± 1.42 a 29.00 ± 2.55 a gp 3: 2 mg ota/250 g bw 27.60 ± 4.37 c 30.60 ± 2.52 d 70.73 ± 3.99 b 73.40 ± 3.17 d gp 4: 1 mg ota/250 g bw 22 .40 ± 3.04 bc 23.20 ± 2.63 cd 65.40 ± 5.21 b 70.20 ± 2.40 d gp 5: 2 mg ota/250 g bw + 300mg/kg bw l alba 16.60 ± 1.21 ab 23.60 ± 2.45 cd 42.78 ± 4.13 a 53.60 ± 2.96 c gp 6: 1 mg ota/250 g bw + 300mg/kg bw l alba 15.80 ± 0.66 a 19.00 ± 1.92 bc 37.96 ± 4.17 a 45.01 ± 2.66 bc treatment set 1: l. alba pre-treated set 2: l. alba post-treated set 1: l. alba pre-treated set 2: l. alba post-treated mda in kidney (mol/g tissue) mda in liver (mol/g tissue) gp 1: negative control (dmso only) 6.66 ± 0.34 bc 6.80 ± 0.81 bc 11.66 ± 0.76 b 12.90 ± 0.56 c gp 2: positive control (300 mg lippia alba / kgbw) 7.80 ± 0.37 c 8.40 ± 0.75 c 13.60 ± 1.29 b 14.57 ± 0.85 c gp 3: 2 mg ota/250 g bw 2.98 ± 0.51 a 3.60 ± 0.32 a 4.75 ± 0.61 a 4.40 ± 0.51 a gp 4: 1 mg ota/250 g bw 2.92 ± 0.44 a 3.70 ± 0.55 a 3.97 ± 0.55 a 5.00 ± 0.45 a gp 5: 2 mg ota/250 g bw + 300mg/kg bw l alba 5.14 ± 0.23 a 4.44 ± 1.86 a 6.20 ± 0.49 a 6.30 ± 0.54 ab gp 6: 1 mg ota/250 g bw + 300mg/kg bw l alba 5.28 ± 0.57 ab 5.02 ± 0.13 ab 7.30 ± 0.89 a 7.95 ± 0.47 b set 1: l. alba pre-treated set 2: l. alba post-treated gsh in kidney (ug/g tissue) gsh in liver (ug/g tissue) treatment set 1: l. alba pre-treated set 2: l. alba post-treated http://www.ajecr.org/ 284 am j exp clin res, vol. 5, no. 2, 2018 http ://www.ajecr.org t able 3 sup eroxide dismut ase act ivit ies in kidneys and livers of ochrat oxin a int oxicat ed rat s values are mean ± sem; values wit h different superscripts wit hin t he column are significantly different at p<0.05 by t ukey hsd t est t able 4 glut at hione-s-t ransferase levels in kidneys and livers of ochrat oxin a int oxicat ed rat s values are mean ± sem; values wit h different superscripts wit hin t he column are significantly different at p<0.05 by t ukey hsd t est iv) glutathione s-transferase levels in kidney and liver of l. alba treated rats glutathione s-transferase (gst) values in kidneys of the two albino rats sets reduced by over 42 % (l. alba pretreated) and 48% (l. alba post-treated) when group 3 animals were differently administered 2 mg ota per 250 g bw (table 4). the results was opposed by group 2 animals with 30.5 % (l. alba pre-treated) and 18.1 % (l. alba posttreated) increment over the negative control when the animals were treated with l. alba crude extract. pre-treated and post-treated animals' results showed that improvement recorded in the values (groups 5 and 6) were significantly different at p<0.05. gst values of the liver tissue obtained the same trend as seen in groups 5 and 6 of the preand posttreated sets. values obtained between the two (group 5 and group 6) were not significantly different from each other but significantly different from other groups within the set at p<0.05. discussion ochratoxin a induces ros production which promotes oxidative damage, and thus interferes with glycolytic and glycogenetic pathways, while promoting gluconeogenesis, glycogenolysis, and membrane lipid peroxidation . malondialdehyde values in the animal kidneys in grou ps intoxicated with ochratoxin a only (both sets) were high compared to values obtained for the control groups and group administered 300 mg lippia alba/kg bw. ochratoxin a being a xenobiotic compound enhances the production of free radical and in the process create oxidative stress state in animals which might eventually cause damage to macromolecules [18]. oxidative stress state results in excessive production of reactive oxygen species (ros) which leads to lipid peroxidation characterized by malondialdehyde; a decomposition product of poly unsaturated fatty acid of membranes [10, 11]. high above normal mda values obtained in albino groups intoxicated with 1 mg and 2 mg ota per 250 g bw respectively showed that the administered ochratoxin a generated reactive oxygen species (ros) which might have led to the leaching of the cellular components of the kidney and subsequent lipid peroxidation as seen in the result obtained. chakraborty and verma [19] proposed that ochratoxin a increases cell permeability to ca2+ and enhances cellular concentration of ca 2+. in addition, hoehler et al. [20] explained that ochratoxin a breaks down oxidative phosphorylation which results in electron leakage within the cell thus altered or increased mda values. the chemo-preventive activities of l. alba is seen in lower levels of mda in the liver and kidney of pre-treated groups when compared to post-treated set (group 5 and 6). lippia alba contains limonene, perillyl alcohol, carvone, and geraiol all terpenoid which confers protective properties on the plant [21]. citral present in the plant had the capacity to inhibit cyp2b60 hydroxylase activity and suppress oxidative stress through the induction of endogenous antioxidant protein as reported by seo et al. [22] and nakamura et al. [23]. gp 1: negative control (dmso only) 67.12 ± 2.64 c 69.29 ± 1.26 d 32.62 ± 0.51 c 30.56 ± 1.11 c gp 2: positive control (300 mg lippia alba / kgbw) 82.61 ± 1.02 d 81.59 ± 1.55 e 40.82 ± 1.16 d 42.36 ± 1.80 d gp 3: 2 mg ota/250 g bw 24.46 ± 2.48 a 34.47 ± 1.42 a 13.80 ± 0.91 a 13.20 ± 1.62 a gp 4: 1 mg ota/250 g bw 28.16 ± 2.73 a 38.61 ± 1.79 ab 16.20 ± 0.58 a 15.49 ± 0.71 a gp 5: 2 mg ota/250 g bw + 300mg/kg bw l alba 51.37 ± 2.13 a 44.53 ± 1.61 bc 23.52 ± 1.78 b 23.97 ± 1.04 b gp 6: 1 mg ota/250 g bw + 300mg/kg bw l alba 53.28 ± 0.66 b 46.20 ± 1.28 c 26.49 ± 1.44 b 27.36 ± 0.85 bc set 1: l. alba pre-treated set 2: l. alba post-treated sod in kidney (u/mg tissue) gsh in liver (u/mg tissue) treatment set 1: l. alba pre-treated set 2: l. alba post-treated gp 1: negative control (dmso only) 27.00 ± 1.58 c 28.80 ± 0.58 d 48.37 ± 1.07 c 46.55 ± 1.56 b gp 2: positive control (300 mg lippia alba / kgbw) 35.24 ± 1.22 d 34.00 ± 1.18 e 64.55 ± 2.33 d 65.80 ± 3.68 c gp 3: 2 mg ota/250 g bw 14.88 ± 0.88 a 16.50 ± 0.50 a 20.38 ± 3.03 a 24.07 ± 2.05 a gp 4: 1 mg ota/250 g bw 15.08 ± 1.15 a 17.04 ± 0.62 ab 23.34 ± 1.05 a 23.89 ± 2.72 a gp 5: 2 mg ota/250 g bw + 300mg/kg bw l alba 18.60 ± 0.51 ab 19.80 ± 0.58 bc 36.31 ± 2.40 b 28.37 ± 1.81 a gp 6: 1 mg ota/250 g bw + 300mg/kg bw l alba 21.00 ± 0.71 b 21.57 ± 0.50 c 40.49 ± 2.49 b 29.61 ± 0.98 a gst in kidney (mg/ml) gst in liver (mg/ml) treatment set 1: l. alba pre-treated set 2: l. alba post-treated set 1: l. alba pre-treated set 2: l. alba post-treated http://www.ajecr.org/ 285 am j exp clin res, vol. 5, no. 2, 2018 http ://www.ajecr.org glutathione level in body organ is an indicator of cell's ability to deal with toxicity challenges [24]. results presented in this study showed that significant reductions were noticed in gsh levels in ochratoxin a treated anima l models of groups 3 and 4 for the sets. findings showed that ochratoxin a brought about reduction in gsh levels in animal models which agreed with the findings of abdelaziz et al. [25], abdel-wahhab et al. [26], and chackraborty and verma [19]. abdel-wahhab et al. [26] reported that reduction in gsh activities likely resulted fro m electrophilic neutralization of ochratoxin a radicals and other reactive oxygen species formed by ochratoxin a. results of the increasing levels of gsh in l. alba treated rats (groups 5 and 6) agreed with the result presented by young et al. [2000, 27] and analikumar et al. [2001, 28] who both suggested that the presence of polyphenol confers protection against oxidative stress leading to a corresponding increase in the level of gsh (noori et al., 2009, 13). superoxide dismutase (sod) activities showed decreasing values when comparison is done with the control group that obtained higher values. these results are in agreement with abdel-aziz et al. [25] and abdelwahhab et al. [26] who proposed that the decreasing sod values obtained might have resulted from damages to the cell of the kidney and liver as suggested by doorten et al. [2004, 29]. dismutation of superoxide radicals into molecular oxygen or hydrogen peroxide by sod decreases its levels in the kidney and liver. sod activities in the l. alba preadministered animal groups showed improved results in the kidney than in the liver. lippia alba contains vitamin c which is a potent water soluble antioxidant with th e ability to scavenge free reactive oxygen species and thus protect cells against oxidative damage (pavana et al., 2007, 30). the vitamin promotes its activities by donating electron to hydroxyl (oh-) ion and superoxide radicals thereby inactivating their reactivity and in the process restoring sod levels back to within the normal range. slight differences were however noted in sod levels of livers in groups 5 and 6 animals of the preand post-treated sets showing that the extract did not advance protectio n and healing as it did in the kidney cells. manikandar and devi [31] also reported the presence fat soluble vitamin e as an antioxidant present in lippia alba with the capacity to protect membrane fatty acids from peroxidation. the vitamin hinders peroxyl mediated chain reaction and also scavenge so in the lipid membrane. results of findings presented in this study on glutathione s transferas (gst) agrees with earlier studies by chakraborty and verma [19], soyoz et al. [32], and ve rma and chakraborty [33] who obtained significantly lowe r glutathione s transferase enzyme in ochratoxin a administered animals. chakraborty and verma [19] in their presentation proposed that the reduction in the gst level simultaneously brings about increasing activities of oxyge n species. reduced gst levels might be adduced to the toxicity of ochratoxin a and its capability to cause disruption to the membrane integrity of the liver and kidney cell respectively. albino rats in set 1 administered lippia alba only (group 2) obtained elevated levels of antioxidant gst compared to the control group within the same set. this might be attributed to overproduction of gst in response to introduction of foreign substances into the cells which is subsequently brought down by the activities o f glutathione s transferase conjugates with functionalized p450 metabolites and vitamin c which decreases the activities of several cytochrome p450 isoenzyme thus increasing gst levels in lippia alba treated rats (sorrent i et al. [34]. antioxidants in the plant which aided in prevention of formation of ros and free radicals include selenium and zinc. these brought about amelioration within the cells by altering apoptosis induced by ochratoxin a in mice liver as reported by sorrenti et al. [4] and zheng et al. [35]. chies et al. [36] explained that pre-treatment with l. alba prevents formation of malondialdehyde that promotes genotoxicity from ota-dna adduct and hinders induced sod and cat phenomena that result in the production of free oxidative reactive species within the cell membrane. conclusion the study proved reasonably that lippia alba had the potential to alter ochratoxin a induced toxicities in the form of ros and free radical production which showed in the activities of antioxidants produced in th e liver and kidney. extract of the plant significantly restore or either ameliora t e the activities of the antioxidants. conflict of interest the authors declare no conflicts of interest. references 1. sen s, chakraborty r, sridhar c, reddy ysr, bipla b d. free radicals, antioxidants, diseases and phytomedicines: current status and future prospect. int j pharmaceut sci rev res , 3(1): article 021, 91-100, 2010. 2. jacob ra. the integrated antioxidant system. nutri res , 15: 755-766, 1995. 3. goldfarb ah. 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ochratoxin a. toxicol appl pharmacol, 268: 123-131, 2013. 36. chies ce, branco cs, scola g, agostini f, gowe r ae, salvador m. antioxidant effect of lippia alba (mille r) n. e. brown. antioxidants , 2: 194-205, 2013. http://www.ajecr.org/ 74 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(4):74-77 case report vitamin d deficiency and recurrent lower respiratory tract infections: a case based discussion cenk aypak 1* , özlem türedi 1 , sümeyye ayöz 2 , mehtap acar 3 1 dışkapı yıldırım beyazıt training and research hospital, department of family medicine, ankara, turkey 2 ankara numune training and research hospital, department of family medicine, ankara, turkey 3 dr sami ulus children's hospital, department of pediatrics, ankara, turkey abstract. lower respiratory tract infections (lrti) are among the most prevalent infectious diseases. vitamin d deficiency has been found to be a risk factor for lrti. we here report a case with the diagnosis of recurrent lrti treated safely by empirical antibiotherapy and vitamin d supplementation in order to underline the importance of assessment and treatment of vitamin d deficiency in pediatric patients with the diagnosis of lrti. keywords: lower respiratory tract infections, children, rickets, vitamin d introduction lower respiratory tract infections (lrti) are among the most prevalent infectious diseases which may cause considerable morbidity and mortality during childhood [1, 2]. development of lrti is closely related with different risk factors including malnutrition, low socio-economic status, passive smoking and presence of systemic diseases (e.g immune deficiency, congenital heart diseases and prematurity) [3-8]. previous studies have shown that low serum hydroxyvitamin d3 (25(ohd) status is associated with increased risk of lrti in children [9, 10]. this issue is a particular concern because the prevalence of vitamin d deficiency is notably high in all over the world including tropical countries [9]. we here report a pediatric case with the diagnosis of recurrent lrti treated safely by empirical antibiotherapy and vitamin d supplementation. case presentation a 6-month-old boy was admitted to pediatric outpatient clinic with complaints of cough and wheezing. medical history taken from mother revealed that he was born at 39 weeks of gestation. pregnancy, labor, and delivery were uneventful. she reported that the boy was also hospitalized with the diagnosis of lrti one month ago. the infant was exclusively breastfed without vitamin d supplementation until the age of 6 months. his mother did not receive any vitamin d supplementation either. upon arrival to the hospital, the patient’s vital signs were stable, with a body temperature of 37.1°c, pulse rate of 100/min, blood pressure of 90/50 mmhg, respiratory rate of 35/min, and oxygen saturation of 96% on room air. height was 65 cm (25 percentile), weight was 7500 gr (50th percentile). at anterior fontanel was 2x2 cm and posterior fontanel was closed. the rest of physical evaluation was normal except bilateral diffuse rhonchus and rales on chest and bilateral wrist enlargement. initial laboratory data revealed a white blood cell count: 13.500/mm3, calcium: 7.5 mg/dl, phosphorus: 2.5 mg/dl, alkaline phosphatase: 421 iu/l, albumin: 4.5 g/dl, 25(oh)d: 11.7 ng/ml, parathyroid hormone: 70.5 pg/dl. urine calcium excretion was normal. anteroposterior chest radiograph showed infiltration of the right hemi-thorax (fig. 1) and the plain radiography of the wrist showed evidence of cupping, fraying, metaphyseal widening, and demineralization of the distal radial and ulnar metaphyses (fig. 2). according to the clinical, radiological and laboratory findings, the patient was hospitalized with the diagnosis of recurrent lrti and rickets. the treatment was initiated with 200 mg/kg/day ampicilin-sulbactam and 50 mg/kg/day of elemental calcium therapy. one week after this therapy he was clinically recovered and the patient was discharged along with 5000 iu/day of cholecalciferol for two weeks. calcium, phosphorus and alkaline phosphatase concentrations were normalized four weeks after initiation of vitamin d supplementation. follow up radiographs obtained at a clinic visit 3 months after the completion of treatment were found to be normal. the patient did not have lrti attack in during a follow up period of six month. ___________________________________________________________ * corresponding author: cenk aypak, md (cenkaypak@yahoo.com). this case report was presented as a poster communication at the 49 th turkish pediatric congress, july 10-13, 2014. http://www.ajecr.org/ mailto:cenkaypak@yahoo.com 75 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org figure 1 chest radiography revealed infiltration of the right hemi-thorax. figure 2 plain radiography of the wrist showed evidence of cupping, fraying, metaphyseal widening, and demineralization of the distal radial and ulnar metaphyses. discussion vitamin d deficiency is a major health problem especially in pregnant women, infants and adolescents. it has been estimated that one billion people worldwide have vitamin d deficiency or insufficiency [11, 12]. the causes of vitamin d deficiency are; increased skin pigmentation, living at higher latitudes, aging, winter time, poor vitamin d nutrition, low maternal vitamin d (in only breast-fed infants), obesity (increased sequestration of 25(oh)d in fat tissue), malabsorption (including chronic liver disease and cystic fibrosis), drugs (phenobarbital, carbamazepine, phenytoin, valproic acid, and rifampicin) [13-21]. symptomatic vitamin d deficiency (i.e. rickets) is more frequently reported in infancy and adolescence than in childhood. this is due to the increased calcium demand secondary to the rapid growth velocity during these two periods. it is well known that severe vitamin d deficiency impairs bone mineralization, leading to clinical manifestations such as rickets in children and osteomalacia in adults [22]. patients with rickets like our patient have skeletal alterations such as, cupping, fraying and irregularity of the metaphyseal regions, loss of definition of the epiphyses, widening of the epiphyseal-metaphyseal junctions, and in severe cases, pathologic fractures [23]. although 25(oh)d is primarily responsible for the regulation of calcium and bone metabolism, vitamin d receptor (vdr) and the vitamin d activating enzyme 1-αhydroxylase (cyp0b1) are expressed in many cell types such as intestine, pancreas, prostate and cells of the immune system [12, 24]. previous studies have suggested that vitamin d deficiency and insufficiency not only have negative consequences on bone health but are also likely to be a risk for many acute and chronic illnesses including autoimmune diseases (type 1 diabetes, multiple sclerosis), cardiovascular diseases, type 2 diabetes mellitus, several types of cancer (non-hodgkin lymphoma, colorectal, prostate, oesophagus and breast cancer), neurocognitive dysfunction and mental illness, infertility, adverse pregnancy-birth outcomes and infectious diseases [25-39]. vitamin d stimulates the production of antibodies and has positive effect on phagocyte activity of macrophages [40, 41]. data have shown that low 25(oh)d status was related to the increased risk of developing lrti [9, 10, 26, 42)] therefore, it was assumed that the repletion/ supplementation of vitamin d along with antibiotherapy in lrti, would reduce the length of hospital stay and possibility of recurrence of lrti over the next 3-months period of discharge [43]. however, many questions remain. what is the optimal dose of vitamin d? what is the 25(oh)d threshold for an anti-infective effect? how long does it take for vitamin d to become effective? are daily doses required or will weekly or monthly doses prevent infections? in order to prevent vitamin d deficiency and related health problems, the american academy of pediatrics (aap) recommends that all infants, children and adolescent have a minimum intake of 400ui of vitamin d per day [44], but it was shown that most us infants were not consuming adequate amounts of vitamin d according to the 2008 aap recommendation [45]. unfortunately the use of vitamin d may be neglected by the families and this might be overlooked during routine follow-up if not carefully questioned and physically examined. as vitamin d deficiency is a risk factor for recurrent lrti, all children with the diagnosis of lrti should be checked for 25(oh)d status and should receive repletion therapy in addition to antibiotic treatment if they found to be vitamin d deficient. conflicts of interest the authors declare no conflicts of interest. http://www.ajecr.org/ 76 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org references 1. unicef/who. pneumonia: the forgotten killer of children. geneva: who, 2006. 2. mulholland k. 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jm. maternal vitamin d deficiency increases the risk of preeclampsia. j clin endocrinol metab 92:3517-3522, 2007. 39. chan tyk. vitamin d deficiency and susceptibility to tuberculosis. calcif tissue int 66:476-478, 2000. 40. kaludjerovic j, vieth r. relationship between vitamin d during perinatal development and health. j midwifery womens health 55:550-560, 2010. 41. garland cf, garland fc, gorham ed, et al. the role of vitamin d in cancer prevention. am j public health. 96:252-261, 2006. 42. laaksi i, ruohola jp, tuohimaa p, et al. an association of serum vitamin d concentrations < 40 nmol/l with acute respiratory tract infection in young finnish men. am j clin nutr 86: 714-717, 2007. 43. manaseki-holland s, qader g, isaq masher m, et al. effects of vitamin d supplementation to children diagnosed with pneumonia in kabul: a randomised controlled trial. trop med int health 15:1148-1155, 2010. 44. wagner cl, greer fr. american academy of pediatrics, section on breastfeeding and committee on nutrition. prevention of rickets and vitamin d deficiency in infants, children, and adolescents. pediatrics 122:11421152, 2008. 45. perrine cg, sharma aj, jefferds me, serdula mk, scanlon ks. adherence to vitamin d recommendations among us infants. pediatrics 125:627-632, 2010. http://www.ajecr.org/ 113 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(3):113-117 original article relationship between clinical findings and spirometry parameters among patients with mild asthma amin saburi, ali hajihashemi, mostafa ghanei* chemical injury research center, baqiyatallah university of medical sciences, tehran, iran abstract. few studies have been done regarding the concordance of association of clinical findings and spirometric parameters especially in patients with mild asthma. in this study we evaluated the relationship between clinical findings and spirometric parameters among patients with mild asthma. in an analytical cross-sectional study, we evaluated patients who were referred to the baqiyatallah university hospital during the year 2009 because of mild asthma symptoms. before beginning the treatment, patients were examined by a pulmonologist and all of them underwent spirometric evaluation. a total of 68 patients were enrolled in the study. the mean age was 43.78±10.74 years and 52.9% of cases were male). regarding the bivariate analysis, there was a significant correlation between clinical findings and spirometric parameters. in multivariate analysis, a significant correlation was found between fvc values and wheezing (p=0.007, β=-0.351), fvc values and coughing (p=0.028, β=+0.272), fev1 values and wheezing (p<0.001, β=-0.440) and fev1 values and dyspnea (p=0.014, β=+0.276), also fev1/fvc values and a family history of asthma (p=0.001, β=+0.370), fev1/fvc values and wheezing (p=0.001, β=-0.365), fev1/fev values and dyspnea (p=0.009, β=-0.283) and finally for the wheezing and mef values (p<0.001, β=-0.615). in conclusion, although in the previous studies the exact relationship between any of the spirometric indices and clinical symptoms has not been described, it seems that wheezing rather than the other clinical findings may be correlated with spirometric indices, however further studies with larger sample size are necessary. keywords: mild asthma, clinical findings, spirometry indices introduction asthma is a chronic inflammatory disease of the airways that is characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli. its physiological manifestation occurs as diffused narrowing of small airways, which may improve spontaneously or as a result of treatment. it is clinically characterized by dyspnea attacks, coughing and wheezing with an incidence rate of 10% in developing countries [1-4]. diagnostic symptoms of asthma include wheezing, dyspnea and coughing, which are either spontaneously or treatment-based variable. the symptoms may worsen at nights with patients usually waking up in the early hours of the morning. patients may complain difficulty in filling their lungs with air. mucus production increases in some patients, which is usually sticky and difficult to remove. ventilation and the use of accessory respiratory muscles may increase. initial symptoms may be present before the onset, including itchy chin, feeling of discomfort between two shoulders, or reasonless fear (imminent death). the physical symptoms are mainly inspiratory, and the expiratory rhonchus can be heard significantly across the chest, and the chest becomes too windy [5]. asthma is usually diagnosed by symptoms caused by variable and intermittent obstruction of the airways; however its diagnosis is confirmed by objective measurement of lung function. the pulmonary function test (pft) criteria for diagnosis and management of asthma are patient-related. spirometry is the first choice to examine the pulmonary function. simple spirometry confirms the airflow limitation by decrease in fev1, fvc, fev1/fvc ratio, and the pef. most of the parameters measured in spirometery are result-dependent and may not show any change [1]. in asthma attacks, fev1 declines that after two times and each time two inhalations, the beta-adrenergic agonist increase in attacks by 15% or more. in mild asthma, this value may have no change or may slightly change [14]. today, the severity of asthma is classified based on clinical symptoms and spirometric changes. a few previous studies have reviewed the relationships of clinical findings of patients with the spirometric findings of which some have confirmed and some have rejected such relationships. also, it was shown in previous studies that in treatment of patients with asthma, the changes in patients’ clinical symptoms might ___________________________________________________________ * corresponding author: mostafa ghanei, md (mghaneister@gmail.com). http://www.ajecr.org/ mailto:mghaneister@gmail.com 114 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org be inconsistent with changes in spirometric findings. it was shown in a study in that the asthma symptoms are poorly correlated with spirometric findings such as fev1 and pef, which indicate the airways obstruction [6]. it has been shown in another study that there is airway obstruction in a large population of asymptomatic children with asthma, and suggested that the frequent assessment of fef is needed as daily symptom control [7]. in another study, it has shown that the differences between spirometric indices and clinical symptoms can be caused by medication and suggested the use of simultaneous evaluation of clinical symptoms and spirometric indices in order to provide a more acceptable treatment [8]. it is also said that relying on assessment of lung function by spirometry and elimination of wheezing may frequently make a wrong understanding of the real pulmonary situation of children with asthma [9]. according to above studies, which mainly mentioned a mild or uncertain relationship between the severity, symptoms type and the spirometric values in patients, review of consistency of two groups of clinical findings and spirometric findings seem to be necessary for clinical judgment (before or during treatment) about the symptoms severity. here, we intend to investigate the compliance and relationship of clinical findings in patients with mild asthma with the spirometric parameters. table 1 demographic findings of the patients variable frequency (%) education high level 18 (26.5) low level 50 (73.5) job employee 19 (27.9) household/unemployed 26 (38.2) free job 22 (32.4) student 1 (1.5) family history of asthma 22 (32.4) symptoms of allergic rhinitis 42 (61.8) materials and methods this study was conducted as a cross-sectionalanalytic study on patients referred to the pulmonary clinic of baqiyatallah hospital, tehran, iran in 2011. the inclusion criteria were the presence of clinical symptoms of mild asthma, age over 18, and the absence of recent active pulmonary diseases such as pneumonia, etc. also, the criteria for mild asthma were consistent with the international classification of mild asthma definition. in mild asthma, the patient shows the symptoms more than once a week and less than once a day; the asthma attacks may influence on the patient’s activity and sleep. symptoms of attacks at night more than twice a month and fev1 or peak expiratory flow less than 80% are expected or may show changes in response to the treatment or stimulation less than 20 to 30 % [10]. the exclusion criteria included diseases in the range of differential diagnosis of asthma such as lung parenchymal diseases, copd, panic attacks and hyperventilation syndrome. the normal spirometry was considered as fev1, fvc, fev1/fvc and mef above 80%. the patients with mild asthma were included in the study at their first visit and before the initiation of treatment. the clinical examination of the patients was performed by a lung specialist and the spirometry was performed by a technician. the patients’ information was collected through a researcher-made questionnaire, including demographic data, clinical symptoms (cough, dyspnea and wheezing on pulmonary auscultation) and the spirometric measures. the outlines of performing the research were described to the patients, and after taking their written informed consents, they were enrolled in the study. no intervention was made on patients’ treatment in this study. the methodology implementation was approved in the research committee of the chemical injuries center of the research center, baqiyatallah university of medical sciences, and the approval of plan performing was adopted from the university's ethics committee. demographic, clinical and spirometric findings of patients were entered in separate questionnaires through coding. after data-entry in spss software (v. 16), data was analyzed using the frequency, k2, independent t-test and anova tests and the logistic and linear regression models. results a total of 68 patients with mild asthma and with the mean age of 43.78±1.74 years old were included in the study, of which 36 patients (52.9%) were male. the mean duration of symptoms was 32.31 months, and the family history of asthma and the symptoms of allergic rhinitis were positive, respectively, in 22 cases (32.4%) and 42 cases (61.8%). 18 subjects (26.5%) had university education, and the most populated professional group was household individuals with a frequency equal to 26 subjects (38.2%), and next the free job group with the frequency of 22 subjects (32.4%). demographic findings are given in table 1. 56 patients (82.4%) were complaining of dyspnea; 42 patients (61.8%) were complaining of chronic cough, and 39 patients (57.4%) had abnormal findings (wheezing) on physical lung auscultation examination. the patients’ spirometric findings are as follows: mean and sd values of fev1: 90.94 ± 10.66; fvc: 86.32 ± 8.93; fev1/fvc: 84.39 ± 7.98; mef: 95.20 ± 27.65. in bivariate analysis, no significant statistical difference was found between two group of patients with and without dyspnea regarding the symptoms of allergic rhinitis (p= 0.114), family history of asthma (p= 0.936) and education (p= 0.189), although there were significant statistical differences in terms of employment (p= 0.022). no significant statistical differences were seen between the two groups of with and without wheezing on auscultation of the lungs regarding the symptoms of allergic rhinitis (p= 0.964) and education (p= 0.462); however, there were significant statistical http://www.ajecr.org/ 115 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org table 2 difference in clinical findings according to the patients’ sex confidence interval wheeze in lung auscultation 23 16 0.022 3.19 1.16-8.79 cough 25 17 0.009 3.99 1.37-11.55 asthma 30 26 0.02 5.76 1.15-28.76 symptoms of allergic rhinitis 22 20 0.264 1.76 0.65-4.76 family history of asthma 8 14 0.058 2.72 0.95-7.78 female (%)variable male (%) p-value odds ratio table 3 relationship between lung function test indexes and clinical signs and symptoms variable fev1 fvc fev1/ fvc mef wheeze in lung auscultation positive 87.29±9.29 84.19±8.05 89.19±8.28 80.64±17.21 negative 95.84±10.55 89.17±9.39 87.35±6.60 114.78±27.10 (r)p-value (3.54) 0.001 (2.35) 0.022 (2.77) 0.007 (6.34) 0.001> asthma positive 91.97±8.88 86.67±8.68 83.14±7.82 93.38±28.52 negative 86.10±16.32 84.68±10.24 90.24±6.05 103.71±22.19 (r)p-value 0.083 0.488 (2.95) 0.004 0.243 cough positive 90.48±10.60 87.28±7.94 84.03±6.99 91.63±24.45 negative 91.67±10.93 84.76±10.31 84.97±9.48 100.97±31.80 (r)p-value 0.59 0.261 0.638 0.178 differences in terms of employment (p= 0.023) as well as family history of asthma (p= 0.022). in addition, there were no significant statistical differences between two group of patients with and without cough about the symptoms of allergic rhinitis (p= 0.290), education (p= 0.618), occupation (p= 0.288) and family history of asthma (p= 0.451). in both sexes, the groups with and without the allergic rhinitis and the positive family history of asthma had no significant statistical difference in values of spirometric measurements, duration of symptoms onset and age (p> 0.05); although the frequencies of all symptoms were significantly different in both sexes, as given in table 2. no significant statistical differences were found between the group with a family history of asthma or with symptoms of allergic rhinitis and the patients without wheezing in the group with a family history of asthma (p= 0.022, or= 3.70, 95%ci= 1.17-11.74). also, among the variables, significant statistical differences were seen only between age values in group with wheezing compared to the patients group without wheezing (p= 0.001, r= -3.564). a significant statistical difference was seen between all spirometric values in patients with and without wheezing (p <0.05), and only the difference between fev1/fvc values in groups with and without dyspnea was significant (p= 0.004, r= 2.955). there was no significant difference between spirometric values in patients with and without cough (p> 0.05) (table 3). in the multivariate analysis using the linear regression test and by removing confounding factors, it seemed that the fvc values were associated only with wheezing (p= 0.007, β= -0.351) and coughing (p= 0.028, β= +0.272), but the fev1 values were only affected by wheezing detected by auscultatory finding of the lung (p <0.001, β= -0.440) and complaining of dyspnea (p= 0.014, β= +0.276). the fev1/fvc values were affected only by the family history of asthma (p= 0.001, β= +0.370), wheezing detected by auscultatory finding of the lung (p= 0.001, β= -0.365) and complaint of dyspnea (p= 0.009, β= -0.283). finally, the mef values were affected only by wheezing detected by auscultatory finding of the lung (p<0.001 β= -0.615). also with fvc values (p=0.012, exp β= 0.871) in multivariate analysis using the logistic regression test with elimination of confounding factors, the prognostic factors were related only to wheezing detected by auscultation and mef values (p= 0.008, exp-β= 0.947). however, sex was the only factor associated with cough (p= 0.005, exp-β= 5.164), and sex (p= 0.021, exp-β= 10.34) and fev1/fvc values (p= 0.006, exp-β= 0.831) were associated only with dyspnea complaint. discussion although previous studies have not determined a specific and accurate relationship between any of the http://www.ajecr.org/ 116 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org spirometric indices and clinical symptoms, but it seems the presence of wheezing in the present study compared to other symptoms in patients with mild asthma is related to spirometric indices. in addition, considering the results of this study, the sex appears to be a quite effective factor on patients’ symptoms, although it has no real influence on spirometric values. in addition to clinical symptoms, spirometric pft criteria are also required for diagnosis, which appear as 12% increase or 200 cc increase in volume of fev1 after taking two inhalations of beta-2 inhalation agonist [10-13]. this value may have no change in mild asthma or may vary slightly. spirometric criteria depend on patient in diagnosis and control of asthma. spirometry is the first choice to examine the pulmonary function. in the past, the purpose of asthma treatment was to achieve normal spirometry [10], while according to recent treatment criteria the main purpose is to improve the symptoms associated with normal spirometry [11]. specific symptoms of asthma include wheezing, dyspnea and cough, which are either spontaneously or by treatment variable. some patients, especially children may refer to the hospital with predominant symptom of coughing. when asthma is under control, there may be no abnormal physical finding. patients may have complaints about difficulty to fill their lungs with air. mucus production is increased in some patients, which is usually sticky and difficult to remove. ventilation and the use of accessory respiratory muscles may increase. pft criteria as well as the symptoms are patient-dependent for diagnosis and control of asthma. most parameters measured in spirometry depend on severity of the disease and may show no change [1]. however, as the results show, wheezing is a nonsymptomatic clinical finding that is diagnosed by the physician, and will not be presumably affected by the patient's conditions, reasoning and analysis of the disease. thus, it is expected that in conditions that the patient shows the wheezing sign in physical examination, it will be associated more with spirometric findings of the patients rather than other symptoms such as coughing and dyspnea, which mostly depend on the patients’ conditions and understanding of symptoms and the disease. the fact that the wheezing is a more reliable indicator for disease diagnosis has been somewhat reviewed in previous studies. however, the review of above subjects and association of symptoms in patients have been previously mentioned briefly [6, 14, 15]. on the contrary, the diagnosis of mild asthma diagnosis is mostly confirmed with spirometry, since it is expected that mild symptoms and non-specific complaints such as cough can also be quite efficient in diagnosis of mild form of asthma. however, the restrictive pattern is purely diagnostic for asthma in these patients. in addition, it affects the asthma symptoms except for wheezing; this means that the patients with gender differences do not reflect the symptoms of cough and dyspnea equally, which can be related to different tolerance thresholds in two sexes. but, wheezing is not affected by this underlying variable and its possible effect on the symptoms, which is due to the nature of being the sign and the dependency of this clinical symptom to physical examination [16]. an interesting point in this study is the presence of different association between spirometric values and clinical symptoms. the changes in fvc and mef values have been associated with the presence or absence of wheezing in these patients. moreover, fev1/fvc has been prognostic for presence of dyspnea. considering that these patients had a mild form of asthma previously (according to severity and frequency of symptoms and spirometric values), it is expected that all the symptoms are not seen together in all patients; hence, these patients are appropriate cases for finding the association of symptoms and spirometric findings. on the contrary, these patients had nearly normal pulmonary values and volumes based on spirometric study, but clinical symptoms facilitated the disease diagnosis in them. thus, it seems that these patients should previously had pulmonary volumes more than normal that the symptoms have emerged in them after the onset of disease process, while the spirometric values have not decreased up to the expected normal spirometric values; this can be interpreted that pulmonary volumes of all individuals are not necessarily the same that a unit cutting value can be determined for all the same patients, since the disease symptoms of coughing and dyspnea in this study have not associated with fev1 values, although this spirometric index has been emphasized more than other indices in asthma. on the other hand, the clinical wheezing finding appears to be correlated with spirometric parameters related to pulmonary volume, particularly fvc and mef and when the patients involve drop-in values of fvc and mef, the wheezing findings occur. however, the clinical findings of dyspnea have been associated with fev1/fvc mostly that these contents should be examined in studies with larger sample size. acknowledgment we, hereby, appreciate all the patients participating in the project, as well as mr. farhadi who cooperated in spirometric evaluation of patients. conflict of interest the author declares no conflicts of interest. references 1. fauci, al et. asthma. harrison'sprincipels of internal medicine 17 ed: mcgraw hill 2008. p. 480-489. 2. goerge r, rw l, ma m. essentials of pulmonary and critical care medicine. chest medicine. philadelphia: lippincott williams&wilkins; 2000. p. 132-136. 3. murray j, ja n. textbook of respiratory medicine. philadelphia: wb saunders; 1994. 4. sabbioni g, sepai o, norppa h, yan h, hirvonen a, zheng y, et al. comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene. biomarkers 123:7-21, 2007. 5. krishnan ja, lemanske rf, jr., canino gj, elward ks, kattan m, matsui ec, et al. asthma outcomes: http://www.ajecr.org/ 117 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org symptoms. j allergy clin immunol. 129:s124-135, 2012. 6. teeter jg, bleecker er. relationship between airway obstruction and respiratory symptoms in adult asthmatics. chest 113:272-277, 1998. 7. ferguson ac. persisting airway obstruction in asymptomatic children with asthma with normal peak expiratory flow rates. j allergy clin immunol 82:19-22, 1988. 8. verini m, rossi n ,dalfino t, verrotti a, di gioacchino m, chiarelli f. lack of correlation between clinical patterns of asthma and airway obstruction. allergy asthma proc 22:297-302, 2001. 9. cooper dm, cutz e, levison h. occult pulmonary abnormalities in asymptomatic asthmatic children. chest 71:361-365, 1977. 10. emad a, emad y. comparison of bronchial respon siveness to ultrasonically nebulized distilled water (undw), methacholine, and ultrasonically nebulized distilled cold water (udcw) in patients with sulfur mustard gas-induced asthma. clin toxicol (phila). [comparative study randomized controlled trial] 45:565570, 2007. 11. waked m, salameh p, attoue r, khoury n, bahous j. methacholine challenge test: correlation with symptoms and atopy. j med liban. [clinical trial] 51:74-79, 2003. http://www.ajecr.org/ 292 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(3):292-298 original article the outcome of right-siting asthma patients to primary care providers tham lai mei1,*, lathy prabhakran1, albert yick hou lim 2, sun bing 1department of nursing, tan tock seng hospital, singapore 2department of respiratory and critical care medicine, tan tock seng hospital, singapore 3clinical research and innovation office, tan tock seng hospital, singapore abstract. right-siting chronic asthma care from acute care hospitals to primary care providers (pcps) was an initiative undertaken at an institution in singapore. this study aimed to evaluate the effectiveness of right-siting asthma patients from three different clinical settings. a retrospective study was conducted from january 2012 to december 2012. a total of 460 patients were right-sited to primary care providers (pcp). of these, 392 (85.2%) were decanted to polyclinics and 68 (14.8%) to general practice (gp) settings. the asthma control test scores were significantly improved for patients who were followed up at the polyclinic within 12 months after being right-sited to the pcp (p<0 .001). out-patients had lower emergency department (ed) re-attendance rates compared to other referral sources (p<0.001) and in-patients had higher re-admission rates than other referral sources (p=0.002). re-admission patients had a statistically significantly higher mean age of 55 than non-re-admission patients at 44 (p < .001). patients with prior ed attendance and hospitalization had higher rates of re-attendance and re-admission within a year (p= 0.001, p<0.001). the risk of ed patients experiencing re-attendance at the ed within 12 months was 5 times that of out-patients (p<0.001). in conclusion, the employment of a right-siting coordinator (rso) did provide better transition for patient care between acute hospital settings and pcps. although the single intervention of connecting patients to their pcps with an appointment prior to discharge did not significantly improve patient compliance to follow-up care, it did appear to improve asthma control in patients who kept to their pcp appointments suggesting that pcp follow-up is effective in the improvement of long-term preventative care. keywords: asthma, right-siting, primary care provider, health care utilization introduction the prevalence of asthma is on the rise globally. it has been reported that 300 million people are currently affected by asthma and by 2025 this number will reach 400 million [1]. despite advanced therapies in asthma management, treatment of asthma remains common in the emergency department (ed). this reflects inefficient longterm disease control, which may be attributed to the haphazard scheduling of follow-up appointments, or lack thereof, with primary care providers (pcps) after the first index ed visit. although the global initiative of asthma (gina) guidelines highlights that the goal of treatment is to achieve and maintain asthma control for a prolonged period of time [2], this goal has not been realized for the majority of patients. in fact, a recent study in an ed at a tertiary hospital in singapore reported that only 45% of patients discharged from the ed were given formal referral letters for follow-up with a pcp [3]. the lack of follow-up care from the primary care service contributed to poor asthma control, high financial burdens and poor customer service [4]. concerns about this situation stimulated concentrated effort towards a more structured transition of care for patients. among the viable recommendations was one that suggested patients be linked to a pcp, with whom at least one appointment should be made prior to the patient’s discharge from any given acute care setting [5]. this intervention was introduced in 2011, in which a dedicated right-siting coordinator (rso) would link each patient with a pcp, according to the patient’s preferences. subsequently, this study was conducted to evaluate the effectiveness of this single pilot intervention. the primary objective of this study was to measure and compare, at 6 months and 12 months, the follow-up rates of patients from three different hospital settings who were subsequently decanted to pcps. the secondary objective of this study was to measure and compare, again at 6 months and 12 months, the emergency re-attendance and hospital re-admission rates of patients from three different hospital settings who were referred to pcps. ___________________________________________________________ * corresponding author: ms. tham lai mei (lai_mei_tham@ttsh.com.sg) http://www.ajecr.org/ mailto:lai_mei_tham@ttsh.com.sg 293 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org materials and methods role of right-siting coordinator (rso) a dedicated rso was employed for the administration of right-siting initiatives of stable and chronic asthma patients from three different sites in an institution (ed, inpatient and out-patient) to the pcp in the community [general practitioner (gp) and polyclinics (ops)]. in addition, the rso oversaw and supported the planning process of identifying patients suitable for right-siting alongside clinicians. follow-up appointment protocol was established as follows, in line with the gina guidelines [2]: a) for patients discharged from the ed, a follow-up appointment would be scheduled within 1-2 weeks. b) for patients discharged as in-patients, a follow-up appointment would be scheduled within 1-2 months. c) for patients discharged as out-patients, a follow-up appointment would be scheduled within 3-4 months. the rso would prepare all relevant documents required during patient and family counseling, to facilitate the care transfer to gps or opss, and vice versa, if necessary. she also maintained a master database of the patients who had been right-sited at 6 months and 12 months for audit and registry purposes. telephonic follow-up data all patients had a 10-minute telephone interview with the rso 6 months and 12 months after the first index date of discharge from the institution. the objective of the interview was to confirm that the patient had kept to the follow-up appointment with the pcp. compliance reschedules or defaults were only self-verified by the patient. pcps were not contacted for verification. a standardized telephone interview was conducted with each patient, to enquire about the patient’s control of asthma using the act [6]. other information obtained at 6 months and 12 months included the following: patient’s follow up status, number of times the patient sought care for asthma in the ed, and number of times the patient was hospitalized for asthma. study subjects retrospective medical records of asthma patients discharged to pcp between january and december 2012 were retrieved from the database for this study. prior to conducting this study, the protocol was approved by the institutional review board. the study was to include patients discharged from the ed, in-patient and soc. attending clinicians from these settings could discharge asthma patients to a pcp in the community for further follow-up and management of asthma. the pcps could either be from a government ops or a private gp. patients discharged to an ops were scheduled with follow-up appointments (date/time). in contrast, patients discharged to a gp were not given a specific date and time. this was because the majority of gp clinics in singapore do not use an appointment system. instead, patients discharged to gp clinics were advised to attend the assigned clinics according to the follow-up appointment protocol. prior to discharge to pcps, each patient was equipped with an individualized education and counseling session, due to the diversity of sites from which the patients were discharged (ed, in-patient and out-patient). these educations and counseling sessions were tailored according to each patient’s educational needs and their inclination for counseling. data collection demographic and health care utilization data patient demographics (age, gender and ethnicity), disposition status (ops or gp), re-attendance, rehospitalization incidents and default rates at 6 months and 12 months from the first discharge date, were retrieved for each patient through the hospital’s electronic medical records system. further information on re-attendance and/or re-hospitalization to other health care institutions was also retrieved from the system. follow-up status to pcps was provided by the patient, since the necessary data was not available via the electronic medical record system. ‘re-attendance’ was defined as an ed revisit for asthma exacerbation within 1 year from the first discharge date (of a hospital visit that did not require admission). ‘readmission’ was defined as an ed revisit for asthma exacerbation within 1 year from the first discharge date (of a hospital visit that required admission). ‘default’ was defined as a follow-up appointment that the patient had failed to attend. asthma control test (act) asthma control test (act) scores were assessed prior to patient discharge, as well as at 6 months and 12 months post-discharge, via telephone interviews. the act was a five-item questionnaire with a five-point scale that assessed asthma control. the questions assessed shortness of breath, nocturnal symptoms, interference with daily activities, and use of rescue medication over the past four weeks. the total act score indicated the effectiveness of asthma control for a given patient. the scores ranged from 5 (poor control) to 25 (complete control). statistical analysis all statistical analysis was carried out using ibm spss statistics version 19.0 (armonk, ny: ibm corp). for continuous variables, mean (sd) or median (iqr) were presented depending on the normality of the variables. categorical variables were described as frequency (percentage). we compared the act score between on decant, 6 months and 12 months using friedman test. we assessed separately the relationship of ed reattendance and hospital re-admission with respect to age, ethnicity, follow-up status, source of referral, ed visit in previous years and hospital admission before discharge, using t-test for continuous variables and chi-square tests for categorical variables. multivariable logistic regression was conducted to assess which source of referral has lower ed re-attendance and hospital re-admission rate after the adjusted odds ratio. continuous data was presented as mean and statistical deviation (sd).two-sided tests were used, and the level of significance chosen was 0.05. http://www.ajecr.org/ 294 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org figure 1 recruitment of patient right-sited to primary care providers results a total of 460 patients were right-sited to pcps from january 2012 to december 2012. a retrospective data analysis was conducted using the variables collected from the database maintained by the rso. patient characteristics the disposition status of patients after being right-sited to the appropriate pcps is presented in figure 1. 392 (85%) were right-sited to ops settings, and 68 (15%) were rightsited to gp clinics. 237 (52%) that were rightsited were from the ed setting. baseline characteristics of the patient population are shown in table 1. patients right-sited from the ed were younger, with a mean age of 37 (sd 14), compared to inpatients at 61 (sd 19) and socs at 52 (sd 19). 278 (60%) of those right-sited were female. the racial distribution of patients right-sited were as follows: chinese 181 (39%), malay 165 (36%), indian 91 (20%), and others 23 (5%). within the ed group, malay patients (47%) outnumbered chinese patients (27%). measurement and comparison of follow-up rates at pcp figures 2 and 3 depict the measurement and comparison of follow-up rates at 6 months and 12 months after patients had been right-sited to ops and gp settings. the show rate in gp settings was 45.6% at 6 months. it was higher than the show rate in ops settings (39.8%). there were no statistically significant differences (p=0.3687) found in both groups. however, long-term (12 months) follow-up monitoring showed that follow-up rates had decreased; instead, the number of patients lost to contact had increased. the results at 12 months also showed that the patients right-sited to ops settings had a higher rate of being referred back to the soc (13.3%), compared to the patients rightsited to gp settings (7.3%). however, this difference was not statistically significant (p=0.1714). patients right-sited to pcps, regardless of whether it was to ops or gp settings, showed significant improvement of act scores over the 12 months of monitoring (p< 0.001) (table 2). out of the 460 patients who were right-sited to pcps, 3 died. of the deceased, two were right-sited to the ops, and the other to a gp clinic. the three patients are elderly, aged between 72 and 79. the causes of death were attributed to other illnesses. health care utilization within 12 months post-discharge to pcp apart from the three deceased patients, 102 (22.2%) had ed re-attendance and 45 (9.8%) were re-admitted within 12 months after being right-sited to their respective pcps. factors associated with re-attendance and re-admissions within 12 months post-discharge were examined in table 3. it was found that hospital re-admission rates were positively associated with older patients (55(21) vs. 44 (19) years, p<0.001). however, the age factor was not significant in patients who re-attended the ed. malay patients were associated with higher ed re-attendance rates compared to patients of other ethnicities (p < 0.009). patients right-sited from the soc had lower ed re-attendance compared to those right-sited from the ed and in-patient units (p<0.001). patients from in-patient showed higher re-admission rates that patients from soc and ed (p=0.002) (table 3). previous ed attendance and hospital admissions were associated with a higher risk of subsequent ed reattendance and hospital re-admission within 12 months. (p<0.001 and <0.001; p=0.001 and p<0.001). from the multivariable logistic regression analysis adjusted to patients discharged from the soc, it is evident that the risk of ed re-attendance within 12 months, in comparison to patients discharged from the soc was 5 times more likely for ed patients and 3 times more likely for in-patient patients (table 4). furthermore, compared to patients discharged from the soc, patients discharged from inpatient settings were 3 times more likely to be re-admitted within 12 months (table 4). discussion the follow-up rates at ops or gp the instances of disconnected follow-up care to primary http://www.ajecr.org/ 295 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org table 1 demographic of the study population figure 2 measurement and comparison of follow up rates in outpatient service setting figure 3 measurement and comparison of follow up rates in general practitioners setting table 2 asthma contro test (act) score change over 1 year care from acute care institutions to the appointed pcp. the majority of patients discharged from the acute care settings were scheduled for follow-up appointments with pcps. both retrospective and randomized controlled studies recommended this method as an effective approach, albeit a traditional and tedious one [7-10]. using this patient centered approach, patients were successfully connected to their preferred pcps (ops 85%; gp 15%). this is unlike the results in a comparable study where 55% of asthma patients discharged from the ed were lost to follow-ups [3]. based on the appointments that were successfully scheduled, it is reasonable to conclude that the rso played a critical role in scheduling follow-up appointments and facilitating the transition of care. while this focused initiative of connecting patients to their pcps with an appointment produced measurable results, it is significant that overall compliance rates to follow-up appointments remained low (ops 39.8%; gp 45.6%). similarly, many other studies have demonstrated poor patient compliance, reporting follow-up rates from 22% to 52% following an asthma ed visit [11-14]. although a number of studies have reported high rates of compliance (60-77%) [5, 9], there were other variables involved, including co-morbid medical conditions and interventions in the ed setting. comparison of follow-up compliance rates between published studies was difficult to conduct due to confounding factors such as demographics of study populations and socioeconomic status [5]. other variables include the acuity of conditions, intervention methods, and the different departments from/to which the patients were right-sited. despite having the rso secure follow-up appointments to bridge the gap for patients to seek long-term preventive care, it did not lead to increased follow-up rates with the ops and gp. one possible explanation for this undesirable result might be that there were other complex psychological, social, financial and medical issues that kept patients from keeping to their scheduled appointments [5]. while this study did not cover aforementioned issues, it is reasonable to assume that fixed, scheduled appointments would have their own flaws. for example, patients who were linked to ops settings were given fixed, scheduled appointments, whereas patients linked to gp settings were told to follow up within a stated time period (this was due to the lack of an advanced appointment system in gp settings). at 6 months, the show rate at the gp was indeed greater (45.6%) than the show rate at the ops (39.8%), but this difference was not statistically significant (p= 0.3687). nevertheless, a reasonable explanation could be that fixed, scheduled appointments were disagreeable to patients, who were not able to exert immediate control over the scheduling of dates and/or times. in contrast, patients who were instructed to schedule their own appointments within a stated period of time may have been more amenable given the flexibility of specific date and/or time of appointment. another contributing factor may be the fact that gp clinics were operational even after office hours, while ops settings were only operational during office hours. such matters of convenience and flexibility would likely have contributed to patient adherence to follow-up recommendations [5]. this observation suggests that follow-up appointments should take into consideration the patient’s perceived convenience and flexibility in scheduling said appointments. another possible barrier to patient compliance was the variables total (n=460) in-p atient (n=88) soc (n=135) ed (n=237) p -value age (year), m ean (sd) 46(19) 61 (19) 52 (19) 37 (14) < 0.001 female, n (%) 278(60) 64 (73) 82 (61) 132 (56) 0.02 ethnics <0.001 chinese, n (%) 181(39) 36 (41) 80 (60) 65 (27) m alay , n (%) 165(36) 32 (36) 22 (16) 111 (47) indian, n (%) 91(20) 18 (21) 22 (16) 51 (22) others, n (%) 23(5) 2 (2) 11 (8) 10 (4) variables on decant 6 months 12 months p -value act, m edian (iqr) 20(18-23) 24(20-25) 24(21-25) < 0.001 act, ops, m edian (iqr) 20(18-23) 24(20-25) 24(21-25) 0.001 act, gp, m edian (iqr) 20(18-23) 24(20-25) 23(20-25) 0.03 http://www.ajecr.org/ 296 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org table 3 health care utilization within 12 months post discharged to primary care providers patient’s perception of the severity of their asthma [15]. if the patient underestimated the severity of their condition, or overestimated their own resistance to further asthmatic attacks, it is reasonable to assume that they would not fully comprehend the importance of regular, long-term follow-up. to address this barrier, telephonic coaching can be used to improve patient compliance to follow-ups. in this study, even though telephone calls were indeed made by the rso at 6 months and 12 months respectively, the purpose of these calls were primarily for outcome-monitoring and auditing, rather than addressing patient concerns. ideally, the telephone calls should be executed in a timely fashion, either before or after the patient’s scheduled appointment date. these calls would directly address the patient’s barriers to keeping their appointments, and to check the status of their experience in coping with the disease at home. bidirectional interaction such as this would enforce mutual trust that could result in higher show rates with pcps and improve overall patient satisfaction [8, 16]. in this study, long-term follow-up rates to pcps decreased over time and, at 12 months, default rates were markedly increased in both groups. similar results had been reported in other studies [8, 13]. using different models of care delivery did improve follow-up rates in the first 15 days, but they did not overcome the barriers of long-term followup after 16 days to 6 months. this result suggests that the single intervention of rso connecting patients with followup appointments to pcps may be inadequate. a more effective model of care delivery should be explored to improve patient compliance to follow-up appointments. factors affecting health care utilization the reported rates of ed re-attendance (22.2%) and readmission (9.8%) within 12 months were a combined measure from the three different sites. as such, this could have distorted the overall results of the study. the acuity of condition also differed from patient to patient, which may have contributed to misrepresentation about the effectiveness of rso intervention to reduce health care utilization. several factors were found to be associated with said health care utilization. firstly, it was found that re attendance and hospital re-admission were linked to patients who had already experienced ed attendance and hospital admission within the preceding 12 months. it has been reported that the reuse of acute care settings may be associated with poorly controlled asthma, multiple comorbidities, non-compliance, socio-economic factors, and defaults on follow-ups for ongoing preventive care [17]. secondly, the findings reveal that hospital readmissions positively correlated to age (55 vs 44 years). asthma in the elderly population is complicated not only by co-morbid diseases, but also by pathophysiological mechanisms, various psychosocial effects of aging, and the high mortality rate that is natural for ageing [18]. this inference is supported by the fact that all three deceased patients in this study died of co-morbid conditions. ideally, the elderly asthmatic patient should be given facilitated referral to a specialist clinic from the ed and in-patient units to improve their long-term asthma care. this would allow the patient to experience a smoother transition of care from acute care to yes (n=102) no (n=355) p -value yes (n=45) no (n=412) p -value age (y ears), mean (sd) 45 (19) 46 (20) 0.717 55 (21) 44 (19) <0.001 ethnic 0.009 0.768 chinese, n (%) 29(28.4) 150 (42.3) 15 (33.3) 164 (39.8) m alay , n (%) 51 (50.0) 114 (20.3) 20 (44.4) 145 (35.2) india, n (%) 18 (17.6) 72 (32.1) 7 (15.6) 83 (20.1) others, n (%) 4 (3.9) 19 (5.4) 3 (6.7) 20 (4.9) source of referral <0.001 0.002 inp atient, n (%) 21 (20.6) 64 (18.0) 17 (37.8) 68 (16.5) soc, n (%) 14 (13.7) 121 (34.1) 11 (24.4) 124 (30.1) ed, n (%) 67 (65.7) 170 (47.9) 17 (37.8) 220 (53.4) follow up status p ost discharge <0.001 <0.001 ops, n (%) 22 (21.6) 107 (30.1) 9 (20.0) 120 (29.1) gp, n (%) 2 (2.0) 24 (6.8) 0 (0) 26 (6.3) default, n (%) 9 (8.8) 82 (23.1) 3 (6.7) 88 (21.4) referred back to soc, n (%) 39 (38.2) 18(5.1) 23 (51.1) 34 (8.2) unable to verify , n (%) 30 (29.4) 124 (34.9) 10 (22.2) 144 (35.0) previous 1 y ear ed visit before discharge, yes, n (%) 53 (52.0) 63 (17.7) <0.001 23 (51.1) 93 (22.6) <0.001 previous 1 y ear hosp ital admission before discharge, yes, n (%) 20 (19.6) 28 (7.9) 0.001 17 (37.8) 31 (7.5) <0.001 ed attendance hosp ital admission variables http://www.ajecr.org/ 297 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org table 4 multivariable analysis of re-attendance at ed and hospital re-admission within 12 months note: multivariable model adjusted for follow up status post discharged, previous 1 year ed visit before discharge or previous 1 year hospital admission before discharge accordingly (data not shown). long-term support from pcps. thirdly, this study found that the risk of re-attending the ed within 12 months, in comparison to patients discharged from the soc, was 5 times more likely for ed patients, and3 times more likely for in-patient patients (table 4). furthermore, compared to patients discharged from the soc, patients discharged from in-patient settings were 3 times more likely to be readmitted within 12 months (table 4). in other words, the results showed that patients rightsited from the soc were better able to maintain asthma control. we believe that follow-ups in the soc provided more time for patients and healthcare providers to monitor and optimize treatment, review inhaler techniques, and provide education and counseling for patients [8]. patients discharged from the ed and in-patient units had shorter contact time with healthcare providers compared to patients from the soc. hence, physicians would have a limited time period to adjust medications and monitor the patient’s asthma control. additionally, educating patients during exacerbation could also be challenging in ed and in-patient settings. thus, lack of interaction due to physical and mental exhaustion could have compromised the patient’s ability for effective asthma control [19]. soc follow-ups may serve as a good platform to ensure that patients are sufficiently educated and counselled on self-management, prior to being right-sited to pcps. this in turn would lead to better care transition, potentially decreasing ed re-attendance and hospital re-admissions [20, 21]. long-term asthma control telephone call follow-ups within 12 months postdischarge recorded significant improvement of act scores in patients who followed-up with their pcps. the overall median act score was 24. taking into consideration the diversity of settings from which the patients were right-sited, there was still significant improvements of patients’ act scores in 12 months. this result reinforces the importance of ongoing long-term preventive care. limitations this study was conducted in a single acute hospital. thus, these results may not be translatable to other settings. data collection in this study also excluded patients’ comorbidities, severity of asthma, psychosocial issues, treatment compliance, and reasons for defaults to follow-up care. such data may affect the results of this study and, if collected, may provide further insight to the reasons behind the recurrent issue of healthcare. conclusion this study was a pilot investigation into the effectiveness of employing an rso to link asthma patients from the ed, in-patient and out-patient units, to their respective pcps for ongoing preventative care. the results showed that the employment of an rso did provide better transition for patient care between acute hospital settings and pcps. although the single intervention of connecting patients to their pcps with an appointment prior to discharge did not significantly improve patient compliance to follow-up care, it did appear to improve asthma control in patients who kept to their pcp appointments. this suggests that pcp followup is effective in the improvement of long-term preventative care. as a result of this study, one suggestion was that the responsibilities of an rso should be extended to include timely, telephone calls to the patient, either before or after the patient’s scheduled appointment date. this measure was recommended in order to address the patient’s potential barriers in keeping to their appointments, and to check the status of their experience in coping with the disease at home. this could potentially increase patient compliance with follow-up care. it is advised that future research should focus on the implementation of this rso strategy and consider its effectiveness post-implementation at health care institutions. acknowledgement we, hereby, appreciate ms. neo lay ping, sri siti fatimah abdul rashid, nor-ribyana binte amran for the data collection and data entry. mr. xiang wen wei for help and support. conflict of interest the authors declare no conflicts of interest. references 1. simon j. attitudes of hungarian asthmatic and copd patients affecting disease control: empirical research based on health belief model. front pharmacol 4: 135, 2013. 2. global initiative for asthma: pocket guide for health professionals. [internet]. 2015 [cited 24 january 2017]. available from: http://ginasthma.org/wp-content/uploads/ 2016/01/gina_pocket_2015.pdf; 2015. 3. prabhakaran l, vasu a, yian t, abisheganaden j, meiyi w, mun w. the current care delivery practice for asthma at the emergency department in a tertiary hospital in singapore. j asthma allergy educat 4:15-21, 2013. 4. chew ft, goh dy, lee bw. the economic cost of asthma in singapore. australian and new zealand j med 29:228-233, 1999. 5. vinson dr, patel pb. facilitating follow-up after emergency care using an appointment assignment system. j healthcare qual 31:18-24, 2009. 6. schatz m, sorkness c, li j, marcus p, murray j, nathan r. asthma control test: reliability, validity, and responsiveness in patients not previously followed by or 95% ci p -value or 95% ci p -value source of referral inp atient, n (%) 2.9 1.3-6.9 0.013 2.7 1.0-6.7 0.041 soc, n (%) ref ref ed, n (%) 4.9 2.3-10.0 <0.001 1.1 0.4-2.6 0.903 variables ed attendance hosp ital admission http://www.ajecr.org/ 298 am j exp clin res, vol. 5, no. 3, 2018 http://www.ajecr.org asthma specialists. j allergy clin immunol 117:549-56, 2006. 7. straus j, orr s, charney e. referrals from an emergency room to primary care practices at an urban hospital. am j pub health 73:57-61, 1983. 8. baren j, boudreaux e, brenner b, cydulka r, rowe b, clark s, camargo c. randomized controlled trial of emergency department interventions to improve pri mary care follow-up for patients with acute asthma. chest 129: 257-265, 2006. 9. kyriacou d, handel d, stein a, nelson r. factors affecting outpatient follow-up compliance of emergency department patients. j gen int med 20:938-942, 2005. 10. richards d, meshkat n, chu j, eva k, worster a. emergency department patient compliance with follow-up for outpatient exercise stress testing: a randomized controlled trial. can j emerg med 9:435-440, 2007. 11. leickly f, wade s, crain e, kruszon-moran d, wright e, evans r. self-reported adherence, management behavior, and barriers to care after an emergency department visit by inner city children with asthma. pediatrics 101: e8, 1998. 12. smith s, jaffe d, fisher e. improving follow-up for children with asthma after an acute emergency department visit. j ped 145:772-777, 2004. 13. zorc j, scarfone r, li y. scheduled follow-up after a pediatric emergency department visit for asthma: a randomized trial. pediatrics 111:495-502, 2003. 14. smith s, jaffe d, highstein g, fisher e, trinkaus k, strunk r. asthma coaching in the emergency department. acad emerg med 13:835-839, 2006. 15. brien g, stein m, fagan m, shapiro m, nasta a. enhanced emergency department referral improves primary care access. the am j managed care 5:1265-1269, 1999. 16. baren j, shofer f, ivey b, reinhard s, degeus j, stahmer s. a randomized, controlled trial of a simple emergency department intervention to improve the rate of primary care follow-up for patients with acute asthma exacerbations. ann emerg med 38:115-122, 2001. 17. adams rj, smith bj, ruffin re. factors associated with hospital admissions and repeat emergency department visits for adults with asthma. thorax 55:566-573, 2000. 18. gillman a, douglass j. asthma in the elderly. asia pacific allergy 2:101-108, 2012. 19. distler j. access carroll: community asthma education initiative. j am acad nurse pract 23:357-360, 2011. 20. fitzgerald j, swan d, turner m, assoc j. the role of asthma educator. can med assoc j 147:855-856, 1992. 21. mayo p, richman j, harris h. results of a program to reduce admission for adult asthma. ann int med 112:864881, 1990. http://www.ajecr.org/ 447 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2022;9(1):447-450 original article investigating the factors affecting the death rate of patients in the burn ward of rasht city hospital in 2019-2020 mohammadreza mobayen1, erfan ghanbarzadeh2, saghar samimi sadeh3, shayan pourmirbabaei3, armin soleymanpour1, amir rigi4*, fatemeh taslimi5, ali shabbak1 1guilan university of medical sciences, rasht, iran. 2gilan university of medical sciences, universal scientific education and research network (usern) 3tehran university of medical sciences, tehran, iran 4bachelor student of nursing, member of young researchers and elite club, scientific association, zahedan branch, islamic azad university, zahedan, iran. 5mazandaran university of medical sciences. mazandaran, iran abstract. burn is the fourth cause of trauma worldwide (1-3) and as a major health challenges, it is one of the most common health related accidents in different societies (4). burn is one of the important causes of injury in iran and it is responsible for 6 percent of deaths in iran, among all deaths caused by accidents (5). data was obtained by reviewing the hospital documents of patients attending to velayat burn hospital of rasht from march 2019 till 2020, regarding the possible factors affecting death among burn patients. data were then entered in spss 26 and the variants were analyzed by one sample t test and chi-square (p<0.05). there were 231 females (58.5%) and 326 males (41.5%) in our patients. the most common cause of burn was flame (49.4%). the most common area of burn was the upper limp (59.5%). the average percent of burn in patients was 23.41±19.17 %. most of the patients (45.8%) were admitted between 5 to 10 days and most burns were in the winter (28.7%). the average blood glucose in patients was 113 mg/dl, average ph was 4.4, average bun1 was 19.6 mg/dl and average creatinine was 1.2±0.7 mg/dl. a significant relationship was found between self-immolation and neuropsychiatric diseases. most common cause of death in patients was respiratory failure (54.1%). inhalation burn injury was seen in 40 patients (7.2%) and selfimmolation was 11 (1.97%) of patients. in the case of place of burn 459 patients were in home, 61 in work and 37 patients in other places. in all the patients, 47 (8.43%) were expired and 510 (91.75%) were rescued. according to current results, the most common cause of burn was flame and most common area was the upper limb. most of the burns were in winter and a significant relationship exists between selfimmolation and neuropsychiatric diseases. most common cause of death in patients was respiratory failure. lower ph and higher bun had a significant relationship with death. therefore, focusing on burn by hot liquids should be a priority in any of the high risk groups and prevention of burn and education of true usage of incendiary and hot devices and safety precautions, should be noted more than ever. keywords: burn, mortality, cause of death introduction burn is the fourth cause of trauma worldwide [1-3] and as a major health challenges, it is one of the most common health related accidents in different societies [4]. burn is one of the important causes of injury in iran and it is responsible for 6 percent of deaths in iran, among all deaths caused by accidents [5]. according to statistic in united states of america, in 2011 about 450 thousand patients (70% male, 30% female) visited to medical centers for burn treatment and mortality of thermal burns are 3500 persons annually [6]. based on statistics of 2006 country report of recorded accidents by office of accident prevention of disease management of iran ministry of health, burn consists 5% of all accidents recorded in country by the number of 56364 persons and is one of the important causes of injury in iran by the incidence of 1 in 1000 people. also it is responsible for 6 percent of deaths in iran, among all deaths caused by accidents [5]. burn is one of the most costly of diseases and its financial burden on patient, his or her family and the economy of the country has always been notable [7]. other than the expenses of burn for the patients and the health system, burn is an irreparable event and has many physical mental and social complications; but in half of the cases, this event and its complications are preventable [7, 8]. more than 95% percent of deaths of burn are occurred in low to moderate income countries. in iran burn is the 13th cause of morbidity in diseases by more than 200 thousand life years annually [9], hence studying the factors effecting the death caused by burn is of great importance. therefore in this study, we intend to survey the factors, effecting the death of ___________________________________________________________ * corresponding author: dr. amir rigi (rigi77.amir@gmail.com) http://www.ajecr.org/ mailto:rigi77.amir@gmail.com 448 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org patients attending to the burn ward of velayat hospital in rasht in 2019-2020. material and methods: this study was cross sectional in velayat burn hospital of rasht from march 2019 to 2020. data was obtained by reviewing the hospital documents of patients attending with the diagnosis of burn. the documents were surveyed regarding the possible factors effecting on death. inclusion criteria were patients with burn, attendance in burn wards (men, women, pediatrics, bicu) velayat burn hospital of rasht. burn after trauma, incompleteness of documents and inaccessibility to the documents were the exclusion criteria. data gathering data gathering was done by the checklist and all the variables including age, gender, level of consciousness at the time of attendance, percentage of burn, area of burn, length of hospital stay, external factor leading to burn and also burns caused by self-immolation, underlying disease, cardiac rhythm disturbance, vital sign disturbance, cause of death, electrolytes disturbance and other blood lab variables such as cbc, level of bun, creatinine and glucose were obtained from the patients' documents and were recorded in the designed checklist. statistical analysis data were entered in spss 26. then for the description of the quantitative data, average and standard deviation and for the qualitative data, tables and charts were used. for the comparison between two groups, one sample t-test was used in quantitative variables and chi-square in qualitative ones. the p-value of less than 0.05 was considered statistically significant. results in this study, 557 documents were surveyed. demographic and clinical information of patient are included in table 1. the average age of patients was 31±4.6 years. 326 patients (58.5%) were women and 231 patients (14.5%) were men. independent t-test did not show significant age difference between men and women (p>0.05; table 1). the causes of burn are included in table 2. most of the burns were caused by flame which consisted 49.4% (n=275) of total burns, in 33.1% (n=184) of patients scald, in 7.5% (n=42) chemicals, in 5.8% (n=32) contact and in 4.2% (n=24) electrocution were the cause of the burn (table 2). of the patients, 59.5% (n=332) had the upper limb burn, 49.1% (n=274) had lower limb, 36.7% (n=205) had head and face, 50.7% (n=283) had trunk, 13.1% (n=73) had genitalia and 7.5% (n=42) had whole body burn. therefore, the most areas of burn are respectively the upper limb, trunk, lower limb, head and face, genitalia and whole body (table 2). the average percentage of burn in patients was 23.41±19.17 %. in terms of the degree of burn, the most common type was the 2nd and 3rd degree together after that, 2nd, 3rd and 4th degree respectively (table 3). the length of hospital stay in respect to the prevalence were 5 to 10 days, 1 to 5 days, 10 to 15 days and more than 15 days (table 4). most of the burns occurred in winter and after that respectively in summer, autumn and spring (table 2). the average blood glucose in patients was 113 mg/dl, average ph was 4.4, average bun was 19.6 mg/dl and average creatinine was 1.2±0.7 mg/dl (table 5). inhalation burn injury was seen in 40 patients (7.2%) and self-immolation was 11 (1.97%) of patients. in the case of place of burn 459 patients were in home, 61 in work and 37 patients in other places. in all the patients, 47 (8.43%) were expired and 510 (91.75%) were rescued. more than half of the patients had some kind of underlying disease such as neuropsychiatric disorders, cardiovascular diseases, hypertension, diabetes, pulmonary diseases and gastrointestinal disorders. also, from 16 out of 53 self-immolation patients had neuropsychiatric disorders. ki-square had shown a significant relationship between selfimmolation and underlying neuropsychiatric disorder (p=0.04; table 6). the most common cause of death was respiratory failure and after that cardiac arrest, bradycardia and gastrointestinal bleeding respectively (table 3). in comparison of patient who died and who did not, the chi-square analysis showed significant difference in cause of burn and length of hospital stay (p=0.00); in rescued patients hot liquid and in expired patients fire was the most important cause of burn. also, the length of hospital stay in rescued patients was significantly higher in comparison to expired patients. the average percentage of burn in rescued patients was 44.68% and was 66.25% in expired patients, but this difference was not statistically significant (p=0.31). data survey by independent t-test showed that in biochemical variables, lower ph (p=0.00) and higher bun (p=0.04) were related to death, but no significant difference were seen in serum creatinine of rescued patient in comparison to expired patients (p=0.41). in table 7 comparison of biochemical variables such as blood glucose, ph, creatinine, and bun, underlying disease, percentage and degree of burn, length of hospital stay, and area and cause of death, by mortality are shown (table 7). discussion burn is one the most destructive injuries and is one of the major concerns of global public health [10]. more than 300,000 people die of burn annually and millions of people suffer from handicap and social, mental and economic disabilities of burn [11, 12]. mortality rate of burn is variable in different age groups. for example, burn caused by fire is the 6th important cause of mortality in 5 to 14 year old people in low income countries [13]. even though the average age of patients in the study by abdulwahab [14] and kadri [15] were 16.5 and 52.9 ± 18.1 years respectively, our findings were more consistent with results of chorlip [16] which the average age was 35.6 ± 15.72. in our study, the average age of patients were 31 ± 4.6 years. it seems that this age group which is the most active age group and considered as the community's work force are more exposed to burn. therefore, the immunity in work place in order to lower the burn related to different occupations could be an effective approach in lowering the burn injuries in this age group and could be important from the economic point of view. in the study of sharma et al. 46% of burn patients were http://www.ajecr.org/ 449 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org male and 54% were female [17]. tabiee et al. [18] surveyed the epidemiology of burn in attended hospital in burn ward as a descriptive cross sectional study. document of 342 patients were surveyed. the results of that study suggested that of total burn injured patients attending to the emergency ward, 55.6% were men. in our study, 231 patients (58.5%) were women and 326 patients (41.5%) were men. in the study by amani et al. [19], explosion and boiling water were introduced as the most common causes of burn. statistical analysis of the study by sharma et al. [17] suggested that burn with boiling water is an important risk factor for mortality. in the study of kabirzadeh et al. [20], the most common external factor leading to burn (57.9%) were fuels such as oil, petrol and gasoline. ibrahimian [21] suggested that most patients (32%) were in the age group zero to 9 years old and among them, 20.7% were burned by hot liquids and 52.1% by flaming oil and petrol. moreover berry et al. [22] suggested in their study that burning with flammable liquid were effective in mortality. in our study, burning with flame (275 patients (49.4%)) and hot liquids (184 patients (33.1%)) consisted the most common causes of burn. hence, necessary education of parents and children should be conducted about burning with boiling water which could be due to overthrow of kettle, samovar and pot. possibly, increasing the safety standards in gas burning devices by production companies and on the other hand, increased precaution of consumers could be effective in reducing the mortality from explosion. in the study of bhansali et al. [23], it was reported that whole body burn had a significant relationship with mortality (p=0.000). also, tarim et al. [24] studied on the factors influencing the mortality in burn patients attending in intrusive care unit and suggested that whole body burn was more common in expired patients [25]. yen et al. suggested in their study that patients with whole body burn and inhalation burn injury have higher risk of mortality [26]. study of kasenda et al. at 2018 suggested that higher surface of burn and burn with boiling water are consistent with higher mortality [27]. in the current study, the most common areas of burn in patients were respectively upper limb, trunk, and lower limb. head and face, genital area and whole body; in rescued patients, hot liquids and in expired patients, fire was the most common cause of burn. study of bhansali et al. [23] suggested that the average length of hospital stay in burn patients were 5 days and there was a significant relationship between whole body burn and duration of hospital stay (p<0.001). the study of kasenda suggested that the higher duration of hospital stay was correlated with higher mortality [27]. by assessing the electronic documents of burn patients, cheung et al. suggested that duration of hospital stay was an effective factor on predicting mortality [28]. de macedo et al. [1] suggested that lower hospital stay was consistent with lower mortality. in the current study, the duration of hospital stay was 5 to 10 days, 1 to 5 days, 10 to 15 days and more than 15 days in respect to prevalence. william et al. [29] conducted a study on mortality of burn patients and reported that having diabetes, pulmonary disease and cardiovascular diseases were related to higher mortality rate. in the current study, expired patients were diagnosed with neuropsychiatric diseases, hypertension, diabetes, cardiovascular diseases, pulmonary disease, gastrointestinal disorders, stroke and cancer. also, a significant relationship was found between self-immolation and neuropsychiatric diseases. bolemsma et al. [30] reported in their study that the most common cause of death were multi organ failure (64.9%) and septic shock (45.9%). in our study, the most common cause of death was respiratory failure and after that cardiac arrest, bradycardia, gastrointestinal bleeding, and hypotension. it can be stated that with the underlying disease, the resistance of body in reduced and the mortality was increased. conclusion according to the results of current study, the most common cause of mortality was flame and most common area of burn was upper limb. most burns were occurred in winter and a significant relationship was found between self-immolation and neuropsychiatric disorders. the most common cause of mortality was respiratory arrest. lower ph and higher bun had a signification relationship with mortality. therefore, focusing on burn by hot liquids should be a priority in any of the mentioned high-risk groups and prevention of burn and education of true usage of incendiary and hot devices and safety precautions, should be noted more than ever. conflict of interest the authors declare no conflicts of interest. acknowledgement we would like to thank the staff of the medical records department of the velayat burns hospital of rasht for their sincere efforts and cooperation. references 1. macedo jlsd, santos jb. predictive factors of mortality in burn patients. revista do instituto de medicina tropical de são paulo 49:365-370, 2007. 2. peck m, pressman ma. the correlation between burn mortality rates from fire and flame and economic status of countries. burns 39:1054-1059, 2013. 3. murray cj, lopez ad, organization wh. the global burden of disease: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to 2020: summary: world health organization; 1996. 4. tabiee s, nakhaee m. epidemiology of burn patients in emam reza hospital, birjand, 1998–2002. j shahrekord uuniv med sci 2004;2006. 5. vasee n, badoohi n, molavi m, jahangiri k, babaee a. to determine la50 in shahid motahari burn hospital, tehran, iran. payesh (health monitor) 8(3):297-301, 2009. 6. association ab. burn incidence and treatment in the united states: 2011 fact sheet. chicago: american burn association. 2011. 7. rouzbahani r, zamani a, omranifard m, rouzbahani a, faragzadegan z, rezaei f. an epidemiological study on burned patients admitted to imam mousa kazem hospital, isfahan, 2003-2004. j shahrekord uuniv med sci 2005;7. http://www.ajecr.org/ 450 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org 8. rafiei m, memarzadeh m, hosseinpour m. evaluation of burn epidemiology in children hospitalized in esfahan province during the recent two years. 2007. 9. azizi aa, zarei j, nabovati e, vakili-arki h, abbasi e, razavi ar. determining of the factors affecting mortality in burn patients using a decision tree data mining algorithm. journal of management of health 16:34-45, 2018. 10. forjuoh sn. burns in low-and middle-income countries: a review of available literature on descriptive epidemiology, risk factors, treatment, and prevention. burns 32:529-37, 2006. 11. peck md. epidemiology of burns throughout the world. part i: distribution and risk factors. burns 37:10871100, 2011. 12. peck md, kruger ge, van der merwe ae, godakumbura w, ahuja rb. burns and fires from nonelectric domestic appliances in low and middle income countries: part i. the scope of the problem. burns 34:303311, 2008. 13. peck md. epidemiology of burns throughout the world. part ii: intentional burns in adults. burns 38:630-637, 2012. 14. patel pn, abdelwahab m, most sp. a review and modification of dorsal preservation rhinoplasty techniques. facial plast surg aesthet med 22:71-79, 2020. 15. kadri ss, miller ac, hohmann s, bonne s, nielsen c, wells c, et al. risk factors for in-hospital mortality in smoke inhalation-associated acute lung injury: data from 68 united states hospitals. chest 150:1260-1268, 2016. 16. tan chor lip h, tan jh, thomas m, imran f-h, azmah tuan mat tn. survival analysis and mortality predictors of hospitalized severe burn victims in a malaysian burns intensive care unit. burns & trauma 7:s41038-018-0140-1, 2019. 17. sharma pn, bang rl, ghoneim ie, bang s, sharma p, ebrahim mk. predicting factors influencing the fatal outcome of burns in kuwait. burns 31:188-192, 2005. 18. tabiee s, nakhaee m. epidemiology of burn patients in emam reza hospital, birjand, 1998–2002. j shahrekord univ med sci 6:43-51, 2004. 19. amani l, soleymanzadeh moghadam s, roudbari m, roustapoor r, armat m, rastegar lari a. epidemiology and mortality of burned patients referred to motahari hospital, tehran. rjms 21:31-38, 2015. 20. kabirzadeh az, zamani kiasari a, bagherian farahabadi a, mohseni saravi b, kabirzadeh agh, tavasoli ashrafi a. burn death rate among hospitalized patients in zare' teaching hospital of mazandaran medical university, sari, iran (2002-04). j gorgan univ med sci 9:79-82, 2007. 21. ebrahimian m. causes of burning and its mortality rate in shiraz. j inflammat dis 3:97-102, 2000. 22. berry cc, wachtel tl, frank ha. an analysis of factors which predict mortality in hospitalized burn patients. burns 9:38-45, 1982. 23. bhansali ca, gandhi g, sahastrabudhe p, panse n. epidemiological study of burn injuries and its mortality risk factors in a tertiary care hospital. indian journal of burns 25:62, 2007. 24. tarim a, nursal tz, yildirim s, noyan t, moray g, haberal m. epidemiology of pediatric burn injuries in southern turkey. j burn care & rehab 26:327-330, 2005. 25. tarim ma. factors affecting mortality in burn patients admitted to intensive care unit. eastern j med 18:72, 2013. 26. yen c-i, chiou m-j, kuo c-f, liao h-t. determination of risk factors for burn mortality based on a regional population study in taiwan. burns 44:1591-1601, 2018. 27. kasenda s, mategula d, manda ge, chokotho tk. risk factors of mortality of hospitalised adult burn patients a malawian tertiary hospital burns unit. biorxiv 2018:421982. 28. cheung m, cobb an, kuo pc. predicting burn patient mortality with electronic medical records. surgery 164:839-847, 2018. 29. williams fn, strassle pd, knowlin l, napravnik s, van duin d, charles a, et al. sex-based differences in inpatient burn mortality. world j surg 43:3035-3043, 2019. 30. bloemsma g, dokter j, boxma h, oen i. mortality and causes of death in a burn centre. burns 4:1103-1107, 2008. http://www.ajecr.org/ 273 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(2):273-280 original article neuroprotective effect of co-administered vitamin e isoforms in sciatic nerve crushed injury of diabetic rats bijo elsy 1 , aijaz ahmed khan 1, *, veena maheshwari 2 1department of anatomy, jn medical college, aligarh muslim university, aligarh, india 2department of pathology, jn medical college, aligarh muslim university, aligarh, india abstract. diabetic peripheral neuropathy is believed to be due to vascular deficit, altered antioxidant defense mechanism and glycemic status. this study is designed to explore the effect of co-administration of some natural antioxidants e.g. vitamin e isoforms, on the regeneration of crush-injured sciatic nerve in healthy and diabetic rats. diabetes was induced through single subcutaneous injection of alloxan at the dose of 100 mg/kg. twenty four albino rats were divided into four groups; healthy control, diabetic control, healthy treated and diabetic treated. treated groups received 100 mg/kg of d-α-tocopherol and d-δ-trf each, orally and daily for three weeks. a horizontal skin incision was made on shaved right mid-thigh region and sciatic nerve was approached by splitting and retraction of surrounding muscles followed by the crushing of sciatic nerve proximal to its division with kocher’s forceps. skin wound was closed with an absorbable suture. sciatic functional and static indices were used to assess the functional recovery. the histopathology, histomorphology of crushed nerve and serum biochemical study were performed at the end of third week. one-way anova followed by tukey’s test and student’s t test were used for statistical analysis of data. all results revealed that vitamin e isoforms on co-administration synergistically improve the antioxidant status, glycemic level; promote neovascularization, regeneration and remyelination of nerve fibers and matrix remodeling after crushed injuries of sciatic nerve. it is concluded that these vitamin e isoforms are potent therapeutic dietary supplements on peripheral nerve regeneration in both healthy and diabetics. keywords: crushinjury, d-α-tocopherol, d-δ-tocotrienol rich fraction, diabetes, rats, sciatic nerve introduction persistent hyperglycemia, oxidative stress and vascular impairment in diabetes are known causative factors in the development of peripheral neuropathy [1]. in diabetes reactive oxygen species (ros) have been implicated for direct damage to the peripheral neurons and altered the antioxidant defense mechanisms [2]. hence antioxidant treatments appear to be promising therapeutics that can prevent or correct the oxidative stress, motor and sensory nerve conduction velocity in diabetic rats [3-5]. the use of antioxidant vitamin c or steroids reduces the postinjury nerve dysfunction and improves nerve regeneration of crushed sciatic nerve in healthy rats [6, 7]. vitamin e has a central role in maintaining neurological structure and function [8]. free-radical scavenging effects of tocopherol and tocotrienols revealed that tocotrienols appear superior because of their better distribution in the fatty layers of the cell membrane [9]. in treating of diabetes and its complications including neuropathies, a suitable treatment must contain agents that have both antioxidant and blood glucose decreasing properties [10]. as such no individual treatment has proven to have both antioxidant and blood flow enhancing effect in diabetic neuropathy [1]. our previous studies [11, 12] also revealed that the individual supplementation of d-α-tocopherol and d-δ-trf helped to accelerate the peripheral nerve regeneration in both healthy and diabetic rats. hence the present study is to analyze the effect of co-administration of these isoforms in healthy and diabetic rats on peripheral nerve repair and regeneration by using functional, histopathological, histomorphological and biochemical methods. materials and methods twenty four albino rats of either sex each weighing 230-320g was obtained from central animal house of jn medical college, amu, aligarh. the study was approved by institutional animal ethical committee (no. 8937/2014). diabetes was induced to the diabetic groups after deprivation of food for 4 hours, followed by single subcutaneous injection (hip region) of alloxan (100 mg/kg; alloxan monohydrate from sigma-aldrich). food and water were provided after one hour of injection. blood was ___________________________________________________________ * corresponding author: prof aijaz ahmed khan (aijazahmedkhan7@live.com) http://www.ajecr.org/ mailto:aijazahmedkhan7@live.com 274 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org figure 1 a. arrow () pointing to sciatic nerve crushed parts. b. shows complete paralysis of right foot after sciatic nerve crushed injury。 figure 2 photographs of the hindlimb foot prints of all groups at the end of 3rd week. note: in diabetic control (dc) prints are not measurable. better foot prints in co-administered (hxt and dxt) groups as compared to healthy control (hc) group. figure 3 showing values (mean ± sd) of sciatic functional index (sfi) and sciatic static index (ssi) in all groups at the end of 3rd week. note that in diabetic control (dc) group both sfi and ssi values indicate total impairment, in healthy control (hc) these values were significantly (p<0.01) negative compared to treated groups. obtained via tail vein for monitoring glucose level by using glucometer (dr. morepen gluco one) on the 4th day of alloxan injection. animals with blood glucose level at 250 mg/dl and above were selected as diabetic for this study. weight and blood glucose levels of all animals in each group were monitored at weekly intervals [13, 14]. statistical analysis all the data were statistically evaluated and the significance calculated using one-way ‘anova’ followed by tukey’s test. student’s t-test was used for comparing the initial and final mean body weight of diabetic control (dc) and blood glucose level in diabetic administered (dxt) group before and after treatment. all results were expressed as mean ± standard deviation (sd) and p<0.05, in case of anova and p<0.0001, in case of student’s t-test was considered as statistically significant. results body weight and blood glucose level during the experimental period, typical clinic manifestations of the diabetes such as polyphagia, polydipsia and polyuria were observed in diabetic control rats while these clinical signs were reduced in diabetic treated groups after three weeks co-administration of d-αtocopherol and d-δ-trf. weight and blood glucose levels of all animals in each group were monitored at weekly intervals. mean body weight in diabetic control (dc) group showed significant (p < 0.0001) reduction whereas in all other groups it remained stable at the end of study period (table 1). mean blood glucose levels of healthy groups (hc and hxt) remained within normal limits. in diabetic coadministered (dxt) group the mean blood glucose level was significantly (p<0.0001) reduced after three weeks treatment while in dc it remained  500 mg/dl throughout the experimental period (table 2). gross observations after sciatic nerve crushed injury, complete paralysis of the right side foot was observed in all rats (figure 1). since autotomy is commonly seen to begin with the nibbling of toenails, this was prevented by application of anti-nail-bite substance on the experiment side in those who showed tendency to bite. thus none of the rats had frank autotomy or nibbling of toenails, edema, infection or ulceration on the foot [11, 12]. functional analysis on completion of 3rd week the better footprints were observed in treated groups compared to control groups (figure 2). both sfi and ssi mean values in diabetic control (dc) showed total impairment while in healthy control (hc) values were significantly (p<0.01) negative compared to treated groups (figure 3). microscopic observations at the end of 3rd week in longitudinal sections 1. degenerating changes and fibrosis the control groups showed increased vacuolization of nerve sheath and numerous atrophic fibers with macrophages and degenerative debris whereas the treated groups had decreased vacuolization of nerve sheath and few atrophic fibers with macrophages and little degenera http://www.ajecr.org/ 275 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org figure 4 representative images from all groups on completion of 3rd week showing vacuolization (), lipid droplets (), debris (), macrophages () and regenerated nerve fibres (, red colour). stain: masson’s trichrome, initial magnification x1000. figure 5 representative images from all groups at the end of 3rd week showing nerve fibres () and arrangements of collagen fibres (red colour). stain: luxol fast blue with picrosirus red, initial magnification x1000. figure 6 representative images from all groups on completion of 3rd week showing elastin fibres (, violet colour). stain: aldehyde fuchsin with fast green, initial magnification x1000. tive debris (figure 4). more collagen fibers were observed in healthy control (hc) and these fibers were disorganized in diabetic control (dc) whereas in treated groups these fibers were few but figure 7 representative images of all groups at the end of 3rd week showing bands of bungner (), proliferated fibroblasts () and in diabetic control only proliferated schwann cells (). stain: haematoxylin and eosin, initial magnification x 1000. figure 8 representative images of diabetic control group on completion of 3rd week showing a: multinucleated giant cell (), g: granuloma, b: inflammatory cells (). haematoxylin and eosin stained longitudinal sections at initial magnification x400, inset image of a and verhoeff van gieson stained transverse sections at x1000 of initial magnifications. more organized (figure 5). 2. regenerating changes a. reappearance of elastin fibers the control groups revealed only few elastin fibers in the epineurium and absence of these fibers in the other connective tissue coverings whereas in treated groups these fibers were obvious in all three connective tissue coverings (figure 6). http://www.ajecr.org/ 276 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org figure 9 number (mean ± sd) of blood capillaries in transverse sections of all groups the end of 3rd week. note that in control groups number of blood capillaries were significantly (p<0.01) less as compared to all treated groups. figure 10 representative images of all groups on completion of 3rd week showing blood capillaries (). stain: masson’s trichrome, initial magnification of transverse sections at x200 and longitudinal section at x400 (inset) in diabetic control group (dc). b. cellularity the healthy control group showed moderate degree of infiltration of inflammatory cells and had only few bands of bungner whereas the treated groups had only mild degree of inflammatory cells but more bands of bunger (figure 7). in diabetic control group bands of bungner was deficient but they had few schwann cells’ proliferations (figure 7). in addition to above features diabetic control group had also more inflammatory cells especially around capillaries and presence of multinucleated giant cells with granuloma formation (figure 8). in general the proliferated fibroblasts were more in treated groups than control groups (figure 7). c. regenerated nerve fibers the healthy control had less number of thin non myelinated nerves whereas the diabetic control had few short distance running neurofibrils. presence of long distance running both myelinated and nonmyelinated nerve fibres were noticed only in treated groups (figures 4 and 5). histomorphology neovascularization on completion of 3rd week in control groups transverse sections the number of capillaries were significantly (p<0.01) less compared to treated groups (figure 9). endoneurial arteriolar walls were thicker in diabetic control group compared to all other groups (figure 10). biochemical analyses at end of 3rd week a. enzymatic antioxidant and oxidative stress parameter serum catalase activity and total antioxidant capacity in treated groups were significantly higher (p < 0.01, p < 0.05) compared to control groups. these analyses values in dc were significantly lower (p < 0.05) compared to healthy control group (table 3). discussion in hyperglycemia enhanced generation of reactive oxygen species (ros) is one of the known reasons of neuronal damage which leads to the development of diabetic neuropathy [1, 19 and 20]. therefore, antioxidants administration may be potentially attractive as clinically applicable neuroprotective agents against such oxidative stress [3]. beneficial effect of vitamin e supplementation has also been shown in diabetic neuropathy [21]. in the present study, in diabetic control group the mean body weight was significantly reduced at the end of experimental period. reduction of body weight in diabetes is considered mainly due to the progressive muscle wasting and breakdown of tissue proteins [22]. after three weeks supplementation of vitamin e isoforms the diabetic co-administered group showed stable body weight with respect to their initial body weight. vitamin e has also anti-hyperglycemic effect thereby it maintains the body weight in diabetic treated animals [23]. another related four week study [24] reported that, diabetic group without tocotrienol rich fraction (trf) supplementation showed significantly lower body weight than that of diabetic rat treated with trf. mean blood glucose level was reduced in diabetic treated group after three week oral co-administration of dα-tocopherol and d-δ-trf but in diabetic control group showed hyperglycemic state throughout the study period. it has earlier been shown [25] that d-δ-trf has potency to maintain the glycemic level in diabetes. this result is in agreement with other study [26] reporting that tocotrienol supplementation significantly increases the insulin levels and reduces the blood glucose in diabetic induced rats in a dose dependent manner. crush injury induces axonotmesis which causes severe http://www.ajecr.org/ 277 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org sensorimotor impairments and functional disabilities [27]. nerve regeneration and functional recovery after peripheral nerve injury even today remains a clinical challenge [28]. evaluation of functional recovery after injury is essential in assessing the nerve regeneration [29, 30], axonal reinnervation and restitution of the nerve muscle interaction [31]. methods used in this present study to evaluate the functional recovery of sciatic nerve after crushed injury are sciatic functional index (sfi) and sciatic static index (ssi). the sfi is a non-invasive method to assess the overall functional recovery of the sciatic nerve during the regeneration process because proper walking requires coordinated function involving sensory input, motor response and cortical integration [32, 33]. at the end of 3rd week better and assessable hind limb foot prints were recorded in treated groups than healthy control group. these prints were not measurable in diabetic control group. the ssi is an effective and accurate method for the assessment of the functional recovery after sciatic nerve injury in rats [16]. in the current study better indices values in treated groups support the faster functional recovery as compared to healthy control. increased axonal repair at injured area is more likely to enhance the successful functional recovery [34]. but in diabetic control group these values indicate total impairment and the functional motor recovery is slower in the presence of persistent hyperglycemia [35]. commonly the histological parameters are the predictors of peripheral nerve damage and regeneration [36, 37]. on 3rd week healthy control showed moderate degenerating changes whereas in diabetic control presence of numerous atrophic fibers with histiocytes and increased vacuolization of nerve sheath indicates that the wallerian degeneration which is prerequisite for nerve regeneration is delayed in experimental diabetic rats [38-40]. another study [41] revealed that in wallerian degeneration of optic nerve of rabbit the degenerative debris was only partly removed even on 3rd month. the treated showed faster removal of debris thus providing appropriate environment for regeneration thereby also reducing the diabetic neuropathic complications. the control groups showed more deposition of collagen fibers which is an indicator of more fibrosis [42] and these fibers were arranged in a disorganized manner in diabetic control. over all reduced fibrosis and organized collagen fibers in treated groups was apparent as a result of combined effect of d-α-tocopherol and d-δ-trf for three weeks. after three weeks supplementation of these isoforms the treated groups showed only minimal and organized collagen fibers, which maintains the structural and functional integrity of the nerves [43]. the viscoelastic properties of the peripheral nerve are due to its connective tissue supporting elements like elastin and collagen [44]. elastin fibers are present in epineurium consisting of thick and thin fibers, perineurium with thicker band of fibers and endoneurium with thinner fibers [45]. the control groups revealed few elastin fibers only in the epineurium but in treated groups these fibers were obviously seen in all three connective tissue coverings. presence of endoneurial elastin fibers in treated groups may provide sufficient force to impart the wavy appearance of the individual axons within the fascicle [45]. during axonal degeneration schwann cells proliferate and dedifferentiate. proliferations were induced by the loss of axonal contact and also stimulation by macrophages releasing growth factors [46, 47]. this dedifferentiated schwann cells align as longitudinal columns inside the basal lamina forming bands of bunger [48, 49]. in this present study bands of bungner were deficient in the diabetic control and they contained only few proliferated schwann cells and fibroblasts. but treated groups showed presence of numerous bands of bungner and more proliferated fibroblasts than healthy control group. these bands of bungner provide supportive environment and guide for successful axonal regeneration [47]. more infiltration of inflammatory cells was seen in control groups. in addition to above, presence of multinucleated giant cells with granuloma formation were noticed in diabetic control group. therefore, in control groups severe immune response appears to worsen the wallerian degeneration and consequently impair the repair and regeneration [50, 51]. reduced inflammatory responses noticed in treated groups were possibly due to the anti-inflammatory effect of vitamin e [52]. during axonal regeneration sprouts (neurofibrils) arise from remaining part of axon that moves distally along the endoneurial tube within the basal lamina [48, 53]. in healthy control group less number of thin newly regenerated nerve fibers was noticed whereas the diabetic control showed few thin short distance running neurofibrils, indicating a partial regeneration of the nerve fibers [42]. the treated groups showed presence of thin nonmyelinated and myelinated nerve fibers. this finding is correlates with many other previous studies [21, 54 and 55] which reported the beneficial effects of vitamin e supplementation in diabetic neuropathy, sensory neuronal loss and sciatic nerve regeneration after nerve crush. the newly regenerated fibers appear to be thinner due to remodeling [56] and these fibers initially lack myelin even when the parent axon is a myelinated fiber. with time, these unmyelinated fibers will become thick and myelinated [57]. most of the regeneration and re-establishment of normal tissue architecture during healing occurs by vessel pruning [58]. in diabetic neuropathy the thickening of arteriolar endoneurium is due to increased deposition of basement membrane material [59], these features were observed in diabetic control. treated groups had more numbers of capillaries than control groups. changes in capillary number and permeability and over all increased vascularization enhance successful axonal regeneration [60]. tocotrienols are promising anticancer agent for minimizing tumor angiogenesis, tocopherol did not inhibit angiogenesis [61]. another study [62] stated that treatment of tocomin 50 (tocotrienol-rich oil) did not show any negative effects in preexisting vessels. in our previous studies [11, 12] number of capillaries was significantly more in d-δ-trf administered groups than d-α-tocopherol treated groups. some other studies [63, 64] reported that http://www.ajecr.org/ 278 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org the anticancer agents like vasostatin and endostatin may not have any inhibitory effect on new vessel formation but possibly induce vessel maturation. catalase is a preventive antioxidant which inhibits the initial production of free radicals and removes the excess h2o2 [65]. the present study showed that serum catalase activity value was lower in diabetic control group which is in agreement with other studies [66, 67]. this activity was normalizing in control group after vitamin e treatment [67].the three weeks co-administration of d-α-tocopherol and d-δ-trf helped to increase the serum catalase activity in treated groups [25]. the antioxidant capacity of plasma is the primary measure and marker to evaluate the status and potential of oxidative stress in the body [68].the present work observed that serum total antioxidant level in diabetic control was significantly lower (p<0.05) compared to healthy control which is in agreement with the findings of other study [69]. improved serum antioxidant capacity was observed in treated groups by coadministration of d-α-tocopherol and d-δ-trf for three weeks as reported earlier [25]. conclusion based on the findings of the present study it is concluded that co-administration of d-α-tocopherol and dδ-trf synergistically enhances the antioxidant level, maintains the glycemic status, accelerate neovascularization, regeneration, remyelination and matrix remodeling in crush-injured sciatic nerve. hence these vitamin e isoforms appear to 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and clinical research am j exp clin res 2019;6(3):355-358 original article urethral stricture; investigation of demographic characteristics in two tertiary hospitals in isfahan/iran hamid mazdak, abdoul karim khorrami, zahra tolou-ghamari* isfahan kidney transplantation research center, department of urology, isfahan university of medical sciences, isfahan, iran abstract. according to previous publication, urethral stricture is a common pathology with dissimilar etiologic features in diverse age groups and societies. the aim of this study was to investigate demographic characteristics in two tertiary hospitals in isfahan/iran. all patients with urethral strictures were obtained from 2008 to 2018 from urology wards located at the two tertiary hospitals in isfahan/iran. according to icd-10, demographic and clinical information were recorded in excel and analyzed by spss. age, as a continuous variable, was expressed as mean ± standard deviation (sd). a p value of <0.05 was considered as significant. for a period of 10 years there were a total of1792 patients comprised of 433 females and 1359 males with urethral strictures. the mean ± sd of age was 41.9 ±24.5 years old. with a between age of 30 to 70 in 71% females showed disease at younger duration of life (p<0.001). urethral strictures was occurred in 30% under the age of 10 years old. the pattern of attendance for most patients was once and in ranged from 2 to 5. intermediate time for those with the pattern of recurrence ranged from 8 to 3074 days after surgery. urethral strictures categorized as an undertreated disease both nationally and internationally. in this study for over a period of ten years, around 1792 patients’ visited two tertiary hospitals in isfahan/iran due to urethral strictures disease. therefore in order to reduce the severity of disease and associated costs, identifying risk factors for occurrence or progression in addition to pharmacotherapy approach recommended to be advantageous. keywords: urethral, strictures, urethrotomy, demographic, isfahan introduction urethral strictures frequently occur, and their risk increases with age. they are also reflected to be the most stimulating and demanding problem that urologists are required to manage [1]. patients with urethral strictures experience progressive narrowing of the urethral lumen such as weak steam, imperfect emptying and many other related symptoms that may look like those of other causes of bladder outlet obstruction such as benign prostatic hyperplasia. the disease needs to be ruled out in patients presenting with fournier’s gangrene, especially when there is urinary extravasation, and in young patients with recurrent epididymitis or prostatitis [2]. an associated prevalence of 229-627 per 100,000 males was reported for urethral strictures [3]. these values ranged from 10 to 100 in the united kingdom [2]. study of urethral strictures characteristics obtained from 1439 male patients in italy showed a mean age of disease presentation around 45.1 years (range 2-84 years) with a mean length of 4.2 cm comprise 92.2% occurring in the anterior urethra [4]. the leading cause of urethral stricture was mentioned as infection urethritis, trauma and iatrogenic or idiopathic. however as an easy procedure, internal urethrotomy might offered as an optimal strategy for treatment associated to urethral strictures but it could not be considered as the firstline of treatment due to its poor success rate of around 20% [5-8]. the aim of this study was to investigate demographic and clinical characteristics associated with urethral strictures in two tertiary hospitals in isfahan/iran. materials and methods the study was conducted at the isfahan kidney transplantation research centre (iktrc) and approved by the institutional review board (no. 396454). data were obtained from 30 november 2008 to 4 aug 2018associated to patients with urethral strictures those attended urology wards located at the two tertiary hospitals; khorshid and alzahra. the urethral strictures were defined according to the international classification of diseases (icd-10). in the first step all patients with urethral strictures were defined from (icd-10) linked to code n35.9 and in the next step patients with internal urethrotomy selected for further analysis according to (icd-10) by the code n58.6. ___________________________________________________________ * corresponding author: prof. zahra tolou-ghamari (toloeghamari@pharm.mui.ac.ir). http://www.ajecr.org/ 356 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org figure 1 distribution of age in patients with urethral strictures in two tertiary hospitals. figure 2 distribution of age in population studied at khorshid hospital. figure 3 distribution of age in population studied in al-zahra hospital. statistical analysis microsoft excel was used to arrange raw data before being inputted into the statistical package for social science (spss® version 20; ibm corp., armonk ny, usa) for analysis. age, as a continuous variable, was expressed as mean ± standard deviation (sd). the normality distribution of age was tested using the kolmogorov–smirnov test. variables such as gender, urethral stricture recurrence, year of report and hospital stay were expressed by frequency and percentage. figure 4 distribution of age in patients with internal urethrotomy. (n=349). results table 1 shows demographic characteristic in patients with urethral strictures at the two tertiary hospitals (alzahra&khorshid) from 2008 to 2018 in isfahan/iran. in all, 1792 patients with urethral strictures comprised of 875 patients in alzahra and 917 patients in khorshid hospital were identified. of the total population studied 24% werfemales with urethral strictures. figure 1 shows the distribution of age in total population studied. with a mean±sd age of 41.9 ±24.5 years old, age related to urethral stricture disease was ranged from 30 to 70 years in 71% of females and 42% of males (42%) that was significantly different (p<0.001). approximately 30% of patients comprised of 7% females and 23% males were diagnosed at the age under 10 years old.figure 2 shows the distribution of age according to gender at khorshid hospital. with a minimum of 1 and a maximum of 96, the mean age of population was 49 .1 ± 22.9 years old. figure 3 shows the distribution of age according to gender. with a minimum of 1 and a maximum of 93, the mean age of population with urethral strictures was 34 .7 ± 26.1 years old. as could be seen 31% of males aged less than 10 years old. regarding to hospital attendance 800 patients attended hospital for once, 57 attended for two, 11 for three, 4 for four and 2 for 5 times. duration of attendance for 126 patients was 1 day after surgery and in 11 patients ranged from 8 to 3074 days after surgery. among this population 1 female attended hospital after 60 days of surgery. in a further attempt, 349 patients with internal urethrotomy comprised of 324 males and 25 female were studied (table 2). figure 2 shows the distribution of age in patients with internal urethrotomy. with a minimum of 0 and maximum of 13 days, the mean duration of hospital stay was 1.4 ± 0.07 days. discussion the natural history of urethral stricture has been well clarified. urethral stricture could lead to major morbidity comprising urinary retention, urethral abscess, trabeculated bladder, hydronephrosis and urinary tract calculi [8, 9]. it signifies a distinctive opportunity for specialized surgical management that enormously improves long term morbidity [10], but there is limited data regarding to prevalence of urologic disease globally in general and in isfahan/ iranin particular [11]. according to previous http://www.ajecr.org/ 357 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org table 1 demographic characteristics in patients with urethral stricture table 2 demographic characteristics in patients with internal urethrotomy (n= 349) publication, a significant portion of undertreated global disease is urologic—urinary retention, urethral stricture, malignancy, and urolithiasis [12, 13].extensive anterior urethral stricture is common in patients as study of 42 patients those surgically managed for adult acquired buried penis, 13 had urethral stricture disease (31.0%). stricture location was universal in the anterior urethra [14]. in this study from 2008 to 2018, there were 1792 patients those attended two tertiary hospitals. in addition to lower frequency of females with urethal stricture (24%), the higher proportion of females (71%) age ranged from 30 to 70 years old. previous publications confirmed that the true incidence of female urethral stricture is not known. bladder outlet obstruction in women is rare, and it was reported to be in less than 8%. the main pathogenic causes for developing female urethral stricture disease mentioned as; blunt trauma, infection, chronic irritation, prior dilatation, difficult catheterization, urethral surgery, urethral diverticulae, and iatrogenic injury [15-18]. in this study, the mean ± sd age of patients was 41.9 ± 24.5 years old that is significantly younger when compared to previous reported data a mean age of 53.43±16.5 years [20] and 69.5 years [21]. in agreement with previous published article, urethral strictures affected 30% of population comprised of 7% females and 23% males under the age of 10 years old. urological disease disturbs the young and old, male and female with a wide‐ranging variety of pathology such as; non-invasive, invasive, severe, long-lasting, reconstructive, pharmacotherapy and surgical strategies [13]. conclusion in isfahan/iran there were 1792 patients with urethral stricture over a period of ten years that attended two tertiary hospitals. these number of cases suggested that it could be relatively a common urologic disorder, and in many cases seem to be devastating. as study of demographic characteristics of patients confirmed a mean age of 41.9 years that was younger than previously reported age from other countries. further research is needed to better define the cause of disease. recognizing origin in these cases may support to classify pharmacotherapy and surgery management that could prevent disease development. conflict of interest the authors declare no conflict of interest. acknowledgement thanks to isfahan university of medical sciences for the ethical code no. of 396454. references 1. almannie rm, alkhamis wh, alshabibi ai. management of urethral strictures: a nationwide survey of urologists in the kingdom of saudi arabia. urol ann 10: 363-368, 2018. 2. alwaal a, blaschko sd, mcaninch jw, breyer bn. epidemiology of urethral strictures. translandrol urol 3:209-213, 2014. 3. santucci ra, joyce gf, wise m. male urethral stricture disease.j urol 177:1667-1674, 2007. 4. palminteri e, berdondini e, verze p, et al. contemporary urethral stricture characteristics in the developed world.urology 81:191-196, 2013. 5. stein dm, thum dj, barbagli g, et al. a geographic analysis of male urethral stricture aetiology and location. bju int 112:830-834, 2013. 6. mazdak h, izadpanahi mh, ghalamkari a, kabiri m, khorrami mh, nouri-mahdavi k, alizadeh f, zargham m, tadayyon f, mohammadi a, yazdani m. internal urethrotomy and intraurethral submucosal injection of triamcinolone in short bulbar 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radiographic knee osteoarthritis mohammad bagher owlia 1 , golnaz malekzadeh 2 * 1 department of medicine, shahid sadoughi university of medical sciences, yazd, iran 2 school of medicine, shahid sadoughi university of medical sciences, yazd, iran abstract. primary osteoarthritis (oa) is known historically to be a non inflammatory condition but recent observations indicate that a low grade inflammation is encountered in pathophysiology of oa's symptoms and progression. we enrolled 150 female patients aged between 50-70 years old diagnosed as oa. exclusion criteria were any recent infection, trauma, and proved rheumatologic disease. bilateral standing knee joint radiography was taken and all were categorized according to kellgren and lawrence scale. markers of inflammation consisting of esr (1st hour), crp, anti-ccp and igm rf were measured in harvested blood samples. laboratory results from the patients with low radiographic knee oa (grade i and ii) were compared to those in high grade patients (grade iii and iv). the mean serum level of esr in low and high grade groups was 12.85±18.65 and 13.65±15.25 respectively (p = 0.77(. as for the anti-ccp, the values of 13.87±43.75 and 23.42±58.87 were obtained for low and high grades respectively (p = 0.26). the differences between the positivity of rf (p = 0.51) and crp (p = 0.56) in both groups was also not statistically significant. but inflammation score was significantly higher in high grade group than low grade group (p = 0.03). we conclude that although the differences weren't remarkable but severity of inflammation in higher radiographic grades should be taken into consideration of oa's progression. keywords: radiographic knee osteoarthritis, kellegren and lawrence scale, inflammatory markers, esr, crp, rf, anti-ccp introduction primary knee osteoarthritis (oa) is a common chronic degenerative disease characterized by the loss of articular cartilage components due to an imbalance between extracellular matrix destruction and repair [1]. oa is the main cause of arthralgia and also the most important rheumatologic underlying cause of disability [2] which lead to premature retirement [3]. pathological mechanism in oa consists of destruction of articular cartilage, increased activity of sub-chondral bone and creating osteophytes. in progressive oa, synovitis and thickening of articular capsule also exist. by the progression of pathology, the radiologic signs appear [4]. the principal risk factors for oa are age, gender and weight [5]. age has the strongest relation with oa among others. about 80% of patients over the age of 70 years old have radiologic changes compatible with oa [6]. in some countries because of some specific activities in their life style, their knees are under pressure to a greater extent and oa represent in very early ages [7]. other less important risk factors for oa are biomechanical stress on articular cartilage, genetic factors, alignment disorders, repeated trauma to the ligaments and meniscus [8]. another risk factor which can be added to this list is systemic inflammation. most known rheumatologic conditions can cause secondary osteoarthritis but hidden spectrum of systemic inflammation is the case. inflammation influences the synnovium. it then synthesizes biological stimulators such as cytokines and growth factors that lead to destruction of articular cartilage. inflammation not only destructs the matrix of cartilage but also prevent any reconstruction [9]. one of the mechanisms that can lead to oa is systemic low grade inflammation. oa in non-weight bearing joints of obese patients is a good example to explain the role of inflammation. adipose tissue secretes leptin and growth factors. leptin proliferates t-cells, monocytes and neutrophils which secret interleukin 6 (il6), tumor necrosing factor (tnf), c1 inhibitor, c3 and etc and finally destruct the joint [10-14]. recent studies on molecular pathogenesis of oa revealed new pathways that contribute to inflammation. by activating these pathways, phenotypic shift occurs in chondrocytes and normal homeostasis deregulates which finally all these pathways lead to upregulation of metalloproteinase 13 that causes the damage in joint. along with this the antagonist of il-1 cannot be produced due to increase in nitric oxide concentration which exacerbates the destruction process of joint [15, 16]. destruction process in oa is usually known to be a non-inflammatory event to be discriminated from rheumatoid arthritis. however, the results of recent studies and the considerable amount of patients showing clinical ___________________________________________________________ * corresponding author: golnaz malekzadeh (golnaz_malek_zade@yahoo.com). http://www.ajecr.org/ mailto:golnaz_malek_zade@yahoo.com 94 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org and laboratory signs and symptoms related to inflammation such as morning stiffness and therapeutic effect of nsaids, bring this idea to the forefront to search for any possible trace of inflammation in oa. in this study we evaluated the markers of inflammation including esr (1st hour), crp, anti-ccp and igm rf in different grades of radiographic knee oa and compared the high grade and low grade groups to each other. materials and methods in this comparative diagnostic study, 150 female patients between the ages of 50 to 70 years old, diagnosed as oa and were under treatment at least for 6 months who admitted to shahid sadoughi rheumatology clinic were selected. exclusion criteria were the history of any recent infection, anemia, any trauma to knees, any kind of proved rheumatologic disease such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, cppd. after getting the inform consents, bilateral standing knee joint radiography was taken in an antero-posterior view in patients. all radiographs were categorized according to kellgren and lawrence scale [17] into 4 grades by an expert radiologist as follows: grade 0: normal, grade 1: doubtful narrowing of joint space and possible osteophytic lipping, grade 2: definite osteophytes, possible narrowing of joint, grade 3: moderate multiple osteophytes, definite narrowing of joints space, some sclerosis and possible deformity of bone contour, and grade 4: large osteophytes, marked narrowing of joint space, severe sclerosis and definite deformity of bone contour. in the sequence order, markers of inflammation consisting of esr (1st hour), crp, anti-ccp and igm rf were measured in harvested blood samples. at least one month before doing the lab tests patients should not use any non-steroidal inflammatory drugs (nsaids). westergren tube was used to measure esr (1st hour) and less than 30mm/h was assumed to be normal. augglutination method was used to measure both crp and igm rf. the results were reported as positive or negative. anti-ccp was measured by elisa and less than 15mg/dl was assumed to be normal. laboratory results from the patients with low radiographic knee oa (grade i and ii) were compared to those with high radiographic knee oa (grade iii and iv). in addition patients of both groups were matched according to their age and also presence of diabetes mellitus to reduce any possible bias in the results of measuring inflammatory markers. for each patient in both groups inflammation score was also calculated in such a way that normal serum level of anti-ccp and esr (1st hour) and negative result of rf and crp receives the score of zero for each of the lab tests, so patients with all normal lab tests receive a 0 scores for inflammation. having higher serum level of anti-ccp, esr and positive rf and crp, receives 1 score for each abnormal laboratory test. inflammation scores of patients ranged from 0 to 4. all patients’ data were analyzed using spss software version 18 (spss, chicago, il) and t-test and chi-square test were used. a p value <0.05 was considered significant. results this study was carried out on 150 female patients who were diagnosed as oa at least for 6 months and were under treatment. of these 75 patients who had knee radiography compatible with grade i and ii by kellgren and lawrence scale were allocated to low radiographic knee osteoarthritis grade group and 75 patients with grade iii and iv were allocated to high grade group. figure 1 comparison of anti-ccp in low and high grade radiologic knee osteoarthritis (p=0.26). figure 2 comparison of esr (1st hour) between low and high grade radiologic knee osteoarthritis (p= 0.77) figure 3 comparison of positivity rate of igm rf (p=0.51) and crp (p=0.56) in low and high grade knee osteoarthritis. the mean age of patients in low grade group was 54.45±9.05 years old and this was 56.24±10.45 years old in high grade group. the difference between the ages of both groups was not statistically significant (p= 0.73). the mean serum level of anti-ccp in low and high http://www.ajecr.org/ 95 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org grade radiographic knee oa was 13.87±43.75 and 23.42±58.87 mg/dl respectively. although a difference of 10 mg/dl was seen between the groups but this difference was not statistically significant (p=0.26) (fig. 1). 12 patients (16%) in low grade radiographic knee oa and 25 patients (33.3%) in high grade radiographic knee oa had higher serum level of anti-ccp than normal range. this difference was significant statistically (p=0.01). the mean serum level of esr (1st hour) was 12.85±18.65 in low grade radiographic knee oa group and 13.65±15.25 in high grade group. the value of esr (1st hour) was also higher in high radiographic knee oa group but was not statistically significant (p=0.77) (fig. 2). the number of patients showing higher serum level of esr (1st hour) than normal range in low and high radiographic knee oa was 11 (14.7%) and 14 (18.7%) respectively which was not statistically significant (p=0.33) the frequency of igm rf positivity in low grade radiographic knee oa group was 14.7% and in high grade group was 18.7%. the difference between both groups was not statistically significant (p= 0.51) (fig. 3) the frequency of crp positivity in low and high grade radiographic knee oa group was 21.3% and 25.3% respectively, showing a 4% increase in high grade group but this difference was not statistically significant (p=0.56) (fig. 3). the mean inflammation score in low and high grade radiographic knee oa was 0.67±0.87 and 0.96±0.79 respectively. statistical analysis showed that the difference between two groups was significant (p=0.03) (see table 1). therefore, considering four markers of inflammation for each patient showed a higher trace of inflammation in higher radiologic oa grades. discussion in recent studies systemic inflammation has been introduced to have a role in pathogenesis and progression of oa, thus in this study we evaluated the inflammatory markers consisting of anti-ccp, esr (1st hour), crp and igm rf among female patients between the ages of 50 to 70 years old who were diagnosed as oa and were under treatment. the results of this study showed higher serum level of inflammatory markers in high grade radiologic knee oa (grade iii and iv) than low grade group (grade i and ii) but this difference was not statistically significant. but the number of patients showing abnormal lab test for anti-ccp and also the inflammation score which was based on the frequency of abnormality of four lab tests together showed significant difference. the result of early studies showed elevated inflammatory markers in oa compared to normal serum and synovial samples but not exclusive to rheumatoid arthritis [18]. in recent studies at least in some cases the trace of inflammation in oa has been proved. the theory of "wear and tear" was questioned and this disease is no longer assumed to be completely non-inflammatory [19]. in a review by berenbaum, oa is introduced not to be osteoarthrosis and low grade systemic inflammation caused by metabolic syndrome, innate immunity and inflammaging are not in favor of assuming oa as a noninflammatory disease anymore [20]. in another review by liu-bryan, inflammatory complement of innate immune system has a key role in progression of oa [21]. the results of a study done by pearl et al. showed that patients with oa who had cartilage inflammatory infiltration in pathology had a higher crp that can be representative for synovial inflammation. our results also indicated a higher rate of crp positivity in higher grade radiographic knee oa group. of all patients, 23.3% had positive crp. if crp in our study would be quantitatively measured, a better comparison could be defined. in addition in our study high sensitivity crp was measured [13]. stannus et al. showed that crp is positively associated with total knee pain adjusted for radiographic oa or mri defected structural abnormalities [22]. they concluded that systemic inflammation can predict worsening of knee pain independently. it should be mentioned that crp measured in this study also was high sensitivity crp. the study by smith et al. showed that patients with higher crp had more destruction in their knees and this can be a good marker to predict the severity of oa [23]. the results of a study by dolzani et al. underlines the lack of inflammatory markers consisting of anti-ccp and highly sensitive crp in different radiologic grades of hand oa that corresponds to our results. in this study only one patient in erosive hand oa had positive anti-ccp but in our study a total of 37 patients had positive anti-ccp [24]. although reaching the same results but they just investigated the patients who had only hand oa without hip or knee involvement. selecting patients just to have hand oa is somehow against systemic inflammation. because systemic inflammation cannot have local effects only restricted to hand, other factors might be involved in the pathogenesis and progression of hand oa alone. in another study done by sumihisa orita, the relation of inflammatory markers consisting of tnfα, il-6, ngf (nerve growth factor) were evaluated in different radiologic grades of oa and the results showed higher inflammatory markers in lower radiologic grades. it has been vindicated that in higher radiologic grades of oa, due to destruction of cartilage there is no more active cartilage to secrete any inflammatory agents. the results of this study are somehow against our results but considering the differences it can be explained. first, they have measured different inflammatory markers than ours and second these inflammatory markers have been measured in synovial table 1 frequency of inflammation score in low and high grade radiographic knee osteoarthritis* radiologic grade inflammation score 0 1 2 3 high (%) 53.3 33.3 6.7 6.7 low (%) 29.3 49.3 17.3 4.0 * mean inflammation score: high grade vs. low grade, p = 0.03. http://www.ajecr.org/ 96 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org fluid that is local but our inflammatory markers represented systemic inflammation [25]. in the study of dan caspi et al., inflammatory markers in oa, rheumatoid arthritis and psoriatic arthritis were compared. although inflammatory markers were significantly higher in rheumatoid arthritis than oa but the mean serum level of iga rf in patients with oa has been reported more than the normal upper limit which corresponds to our results. of our patients, 16.6% showed higher level of igm rf [26]. vangsness’s study investigated 21 inflammatory markers in patients who underwent knee arthroscopy and their results indicated no significant difference in synovial fluid of kellgren and lawrence grades of knee oa but it was significant by the international cartilage repair society (icrs) classification [27]. in vlad’s study, 17 different markers of inflammation has been measured but none of them were significant in various grades of oa. their results were consistent with our findings but the difference was that the blood samples were harvested 5 years before taking the radiographs, so almost all of the patients may not have oa or were developing it at the time of measuring markers of inflammation [28]. in haywood’s study, 31% of synovial tissue samples of patients with oa showed severe inflammation and it was not confined to end stage patients [29]. glucosamine is a drug introduced as a remedy for oa in different studies. this drug accelerates the reconstruction of synovial fluid and may increase the synthesis of articular cartilage. another mechanism is the inhibitory effect on neutrophils which in result prohibits the inflammation. the effectiveness of this drug on oa can explain the inflammatory basis of oa [30-36]. the markers of esr, anti-ccp, crp and rf are the inflammation markers which can be used in clinical practice readily and according to the result of this study can represent the systemic inflammation in oa for evaluating the progression of this disease. conclusion although inflammatory markers were higher in high grade radiologic oa than the low grade ones but it was not statistically significant. on the other hand, the number of patients with abnormal lab tests which is representative for inflammation is remarkable in apparently primary oas and should be taken into consideration in future discussion and studies. acknowledgments the authors want to thank dr soleimani, dr dehghan and dr farrahifard for their useful discussion. this article is the result of thesis of ms. golnaz malekzadeh of doctorate degree in medicine. conflict of interest the authors declare no conflicts of interest. references 1. garnero p, piperno m, gineyts e, christgan s, delmas pd, vignon e. cross sectional evaluation of biochemical markers of bone, cartilage and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage, ann rheum dis 60:619-626, 2001. 2. urwin m, symmons d, allison t, brammah t, estimating the burden of musculoskeletal disorders in the community. ann rheum dis 57:649-655, 1998. 3. felson dt, naimark a, anderson j, kazis l, castelli w, meenan rf, the prevalence of knee osteoarthritis in the eldery, the framingham osteoarthritis study. artheritis rheum 30:914-918, 1987. 4. jan dequeker, paul dieppe. disorders of bone, cartilage and connective tissue. john h. klippeland paul dieppe rheumatology, 2th edition. london mosby 1998, vol 2, chap 8. 5. bjordal jm, johnson mi, lopos martin rab, bogen b, chow b, ljunggren ae. short term efficacy of physical interventions in osteoarthric knee pain, asymmetric review and meta analysis of randomized placebo controlled trials. bmc muscluskelet dis ord 8:51, 2007. 6. felson dt, zhang y. an update on the epidemiology of knee and hip osteoarthritis with a view to prevention. arthritis rheum 41:1343-1355, 1998. 7. shane anderson, richard f. leoser. why is osteoarthritis an age related disease? best pract res clin rheumatol 24:15-26, 2010. 8. ray a,ray bk. an inflammation responsive transcription factor in pathophysiology of osteoarthritis biorheology 45:399-409, 2008. 9. goldring mb, otero m, tsuchimochi k, ijiri k, li y. defining the roles of inflammatory and anabolic cytokines in cartilage metabolism. ann rheum dis 67:75-82, 2008. 10. staab ca, maser e. 11beta hydroxyl steroid dehydrogenase type 1 is an important regulator at the interface of obesity and inflammation. j steroid biochem molbiol 119:56-72, 2010. 11. strohacker k, mcfarlin bk. influence of obesity, physical inactivity and weight cycling on chronic inflammation. frontiers in bioscience 2:98-104, 2010. 12. hotamisligil gs., inflammation and metabolic disorders. nature 444:860-867, 2006. 13. pearle ad, scanzello cr, s. george ba, mandl la, dicarlo ef, peterson m, sculco tp, crow mk. elevated high-sensitivity c-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis. osteoarthritis cartilage 15:516-523, 2007. 14. ulf muller-ladner, klaus w. formmer, elena neumann, what fat does to arthritis. the rheumatologist 5:36-39, 2011. 15. pelletier jp, martel pelletier j. role of synovial inflammation, cytokines and igf1 in the physiopathology of osteoarthritis. rev rheum ed fr 15; 61:103s-108s, 1994. 16. goldring sr, goldring mb., the role of cytokines in cartilage matrix degradation in osteoarthritis. clin orthop relat res (suppl) 427:27-36, 2004. 17. kellgren jh, lawrence js. radiological assessment of osteoarthrosis. ann rheum dis 16:494-502, 1957. http://www.ajecr.org/ 97 am j exp clin res, vol. 2, no. 2, 2015 http://www.ajecr.org 18. nettelbladt e, sundbl l, protein patterns in synovial fluid and serum in rheumatoid arthritis and osteoarthritis. arthritis rheum 2:144-151, 1959. 19. jeremy sokolove, christin m. lepus, ba. role of inflammation in the pathogenesis of osteoarthritis. ther adv musculoskel dis 5:77-94, 2013. 20. berenbaum f. osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). osteoarthritis cartilage 21:16-21, 2013. 21. liu-bryan r. synovium and the innate inflammatory network in osteoarthritis progression. curr rheumatol rep 15:323, 2013. 22. stannus op, jones g, blizzard l, cicuttini fm, ding c. associations between serum levels of inflammatory markers and change in knee pain over 5 years in older adults: a prospective cohort study. ann rheum dis 72:535-540, 2013. 23. jessica w. smith, meng, thomas b. martins, ms, evelyn gopez, troy johnson, harry r. hill, thomas d. rosenberg. significance of c-reactive protein in osteoarthritis and total knee arthroplasty outcomes. ther adv musculoskel dis. 4:315-325, 2012. 24. dolzani p, assirelli e, pulsatelli l, addimanda o, mancarella l, peri g, mantovani a, facchini a, meliconi r. systemic inflammation and antibodies to citrullinated peptides in hand osteoarthritis. clin exp rheumatol 29:1006-1009, 2011. 25. sumihisa orita, takana koshi, takeshi mitsuka,masayuki miyagi, gen inoue, gen arai,tetsuhiro ishikawa, eiji hanaoka, keishi yamashita, masaomi yamashita, yawara eguchi, tomoaki toyone, kazuhisa takahashi, and seiji ohtori. associations between proinflammatory cytokines in the synovial fluid and radiographic grading and pain-related scores in 47 consecutive patients with osteoarthritis of the knee. bmc musculoskelet disord 12: 144, 2011. 26.dan caspi, marina anouk, itzhak golan, daphna paran, ilana, kaufman, irena wigler, david leartovsky, ori elkayam. synovial fluid levels of anti-cyclic citrullinated peptide antibodies and iga rheumatoid factor in rheumatoid arthritis. psoriatic arthritis and osteoarthritis. arthritis rheum 55: 53-56, 2006. 27. c. thomas vangsness, wendy s. burke, steven j. narvy, robert d. macphee, and alexander n. fedenko. human knee synovial fluid cytokines correlated with grade of knee osteoarthritis. bulletin nyu hosp joint dis 69:122-127, 2011. 28. vlad sc, neogi t, aliabadi p, fontes jd, felsom dt. no association between markers of inflammation and osteoarthritis of hands and knees. rheumatol 38:16651670, 2011. 29. haywood l, mcwilliams df, pearson ci, gill se, ganesan, wilson d, walsh da. inflammation and angiogenesis in osteoarthritis. arthritis rheum 48:21732177, 2003. 30. setnikar i, giacchetti c, zanolo g. pharmacokinetics of glucosamine in the dog and in man. arzneimittelforschung 36:72935, 1986. 31. mccarty mf. enhanced synovial production of hyaluronic acid may explain rapid clinical response to highdose glucosamine in osteoarthritis. med hypotheses 50:50710, 1998. 32. chan ps, caron jp, rosa gj, orth mw. glucosamine and chondroitin sulfate regulate gene expression and synthesis of nitric oxide and prostaglandin e2 in articular cartilage explants. osteoarthritis cartilage 13:387-394, 2005. 33. bruyere o, honore a, rovati lc, giacovelli g, henrotin ye, seidel l, et al. radiologic features poorly predict clinical outcomes in knee osteoarthritis. scand j rheumatol 31:13-16, 2002. 34. das ak, hammad ta. efficacy of a combination of fchg49tm glucosamine hydrochloride trh 122tm low molecular weight sodium chondroitin sulfate and manganese ascorbate. cartilage 8: 343, 2000. 35. noack w, fischer m, forster kk. glucosamine sulfate in osteoarthritis. cartilage 2: 51, 1994. 36. setnikar i, cereda r, pacini ma, revel l. antireactive properties of glueosamine sulfate. arzneimittelforschung 41:157-161, 1991. http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/pubmed?term=ding%20c%5bauthor%5d&cauthor=true&cauthor_uid=22580582 http://www.ncbi.nlm.nih.gov/pubmed?term=dolzani%20p%5bauthor%5d&cauthor=true&cauthor_uid=22032420 http://onlinelibrary.wiley.com/advanced/search/results?searchrowcriteria%5b0%5d.querystring=%22d.%20a.%20walsh%22&searchrowcriteria%5b0%5d.fieldname=author&start=1&resultsperpage=20 267 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2018;5(2):267-272 original article domestic violence among infertile women: a study in north of iran fatemeh alijani 1 , afsaneh keramat 2 , zeinab hamzeh gardeshi 3 , ahmad khosravi 4 *, mansoureh afzali 5 , fatemeh habibi 6 1midwifery counseling, student research committee, shahroud university of medical sciences, shahroud, iran 2department of reproduction health, shahroud university of medical sciences, shahroud, iran 3sexual and reproductive research center, mazandaran university of medical science, sari, iran 4center for health related social & behavioral sciences research, shahroud university of medical sciences, shahroud, iran 5midwifery counseling, student research committee, nassibeh faculty of nursing and midwifery, sari university of medical sciences, sari, iran 6midwifery, infertility center, imam khomeini hospital, sari, iran abstract. infertility is associated with emotional problems, marital distress and domestic violence (dv). it might have a substantial consequence on women, families, and society as a whole. this study was conducted to determine the prevalence of domestic violence and its related risk factors among infertile women in the north of iran. a total of 379 infertile women between october 2015 and march 2016 were included in the current study, using consecutive sampling. socio-demographic characteristics were assessed via a structured questionnaire. the revised conflict tactics scale (cts2) was used to evaluate domestic violence. the data were statistically analyzed and a p <0.05 was considered as the significance level for all tests. of 379 infertile women, 88.9% reported domestic violence. psychological violence was the most common type of violence. there were no significant relationships found between violence and women’s educational status, men’s jobs, place of residence, alcohol consumption, and drug addiction. men smoking and women with younger age were risk factors of violence against women (p <0.05). the prevalence of domestic violence is considerably high in sari, iran. being a smoker and having a younger age may increase violence against infertile women. health staff should identify at-risk women and support them and their husbands via educational programs and counseling. keywords: domestic violence, infertility, women, prevalence, risk factors introduction epilepsy infertility is defined as the failure of clinical pregnancy after one year or more of regular unprotected sexual intercourse [1]. it may be primary or secondary, the former refers to the inability to bear any children, whereas in secondary infertility couples are unable to have an additional live birth [2]. the rate of infertility in one year is approximately 3.5% to 16.7% for couple worldwide3 however, infertility rate varies among different countries. in iran, the overall prevalence of lifetime primary infertility was reported from 17.3% to 20.2% [4, 5]. although the who has recognized that infertility equally affects men and women [6], it is commonly considered a female disorder and women generally bear the burden of couple's infertility [7]. the disorder is associated with emotional and financial problems, economic deprivation, social stigma, marital distress, divorce and domestic violence [8, 9]. violence is a public health issue, which affects the lives of millions of women all over the world regardless of ethnicity, culture, religion, socioeconomic status and educational levels [9]. the united nation adopted the declaration on the elimination of violence against women, which defines violence against women as “any act of gender-based violence that results or likely to result in physical, sexual or psychological harm or suffering to women including threats of such harm, coercion or arbitrary deprivation of liberty whether occurring in public or private life” [10]. domestic violence (dv) might have substantial consequences and threatens not only the physical and emotional health of women, but also affects families and society as a whole. the who estimates that almost onethird of women who have intimate partners have experienced physical and/or sexual violence [10]. furthermore, it has been found that infertility increases the risk of dv compared to women who have children [11]. ___________________________________________________________ * corresponding author: ahmad khosravi, msc. (mahdieh.shojaa_mw@yahoo.com) http://www.ajecr.org/ mailto:mahdieh.shojaa_mw@yahoo.com 268 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org prevalence of dv among infertile women varies widely based on socioeconomic status, geographical location, and cultural and religious differences. for example, the prevalence of dv among some countries are as follows: 31.6% in turkey [9], 64 % in pakistan [12] and 77.8% in india [13]. in iran, researchers cite various percentages varying from 14% to 61.8 % [8, 15, 16]. results of some research studies indicate that women who are victims of violence are more likely to have depression, anxiety, reduced self-esteem, stress disorder, attempted suicide, injuries, sexual and physical health problems [9, 10]. although there is a wide range of research about domestic violence, there was a lack of evidence regarding the violence against infertile women. therefore, considering the importance of the issue and its adverse effects among this vulnerable group, the current study was performed to determine the prevalence of dv and its related risk factors among infertile women seeking fertility treatment at the only infertility clinic of the teaching hospital affiliated with mazandaran university of medical sciences in sari, north iran. materials and methods the current cross-sectional study was carried out among infertile women who were referred to the only infertility clinic of the teaching hospital affiliated with mazandaran university of medical sciences, located in the north of iran between october 2015 and march 2016. the formula for calculating the required sample size by considering type 1 error 0.05 and prevalence of domestic violence to be 60 %8 revealed that 375 subjects were needed: n= 𝑧2(1−∝/2)𝑝𝑞 𝑑2 = 4∗𝑜.4∗0.6 0.052 = 375 women who consecutively reported to the infertility clinic and who consented to be a part of the study were included. the eligibility criteria included: recognition of infertility by obstetricians, minimum one year of recognition, married for more than one year, and a lack of physical and mental diseases. participants were excluded in the event of pregnancy or unwillingness to continue participating in the study. sociodemographic characteristics including age, duration of infertility and marriage, residential place, the level of education, employment status, smoking, drug addiction, and alcohol consumption were recorded via a structured questionnaire. domestic violence was also measured during the last twelve months using the revised conflict tactics scale (cts2) [17], which consists of 39 questions. the cts2 covers five aspects of spousal conflict including physical violence with 12 items (e.g. my partner grabbed me); sexual violence with 4 items (e.g. my partner made me have sex without a condom); negotiation with 6 items (e.g. my partner explained his side of a disagreement to me); psychological violence with 8 items (e.g. my partner shouted or yelled at me); and physical injury with 6 items (e.g. had a sprain, bruise, or small cut because of a fight with my partner). the questionnaire has been revised and modified by behboodi moghadam et al. [14]. due to social figure 1 prevalence of domestic violence in infertile women (n=379). and cultural differences, researchers disregarded the following three questions concerning sexual acts: he insisted that i engage in oral or anal sex (but did not use physical force); he used force (such as hitting me, holding me down, or using a weapon) to make me engage in oral or anal sex; he used threats to make me engage in oral or anal sex [8]. references confirmed the reliability and validity of the scale for the iranian population. the affirmative answer to any of these questions was deemed as violence. a trained midwifery master’s student completed the questionnaires utilizing private, face-to-face interviews in the absence of their husbands or family members. the ethics committee of the mazandaran university of medical sciences approved the study. written informed consent was obtained from all subjects prior to their participation in the study. in the event that the participants were illiterate, the interviewer read the informed consent aloud and they used their fingerprint instead of their signature. for confidentiality reasons, the respondents’ names were not recorded on the questionnaire and they could withdraw from the study without any consequence at any time. statistical analysis the data were analyzed using the spss-16 software. a t-test and chi-square were used to evaluate the association between dv and other variables for continuous and categorical variables respectively. multivariate logistic regression was also utilized to determine odds ratios (±95%ci) for independent risk factors of dv. p <0.05 was considered as the significance level for all tests. results a total of 379 infertile women with a mean (sd) age of 31.66 ± 6.46 years participated in this study. the mean age (sd) of their spouse was 35.99 ± 8.40) years. a 88.9 % of the participants reported having experienced domestic violence. at 85.8%, psychological violence was the most frequently reported form of violence against women. the frequency of various forms of violence is depicted in figure 1. table 1 demonstrates the prevalence of domestic violence based on the characteristics of infertile women and their husbands. there was no significant relationship 85.8 48.3 28.2 25.9 0 10 20 30 40 50 60 70 80 90 100 psychological verbal sexual physical % type of violence http://www.ajecr.org/ 269 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org table 1 sociodemographic variables for women with and without domestic violence and their partners (n = 379) *using t-test; **using the χ2 test. between violence and the women’s educational statuses, the men’s occupations, place of residence, the men’s alcohol consumption and drug addictions. however, other variables revealed a significant relationship with violence (p < 0.05). the results of the multivariate logistic regression revealed that as the age of the women increased, the odds of dv decreased. each year increment in age decreased the odds of dv by 9% (or: 0.91, ci: 0.85-0.99, p = 0.03). additionally, smoking cigarettes by men was a significant risk factor of domestic violence against women (or: 8.12, ci: 1.87-35.21, p = 0.005). men’s age, length of marriage, infertility duration and women’s occupation were not risk factors for domestic violence, as shown in the univariate analysis in table 2. table 2 association between domestic violence against women and other variables using logistic regression analysis (n = 379) or: odds ratio; ci: confidence interval. *for all demographic characteristics, **adjusted for women age, men age, marriage and infertility duration, women job and men smoking. discussion our study found a high prevalence of dv among infertile women at a rate of 88.9%. an explanation for the high rate of violence in our study may be due to the inclusion of emotional violence and it was also conducted in the public fertility clinic, which provides services to people mainly coming from lower educational and socioeconomic status, while other studies in iran, turkey, pakistan, india and nigeria found it to be between 31-76% among infertile women [8, 9, 12, 15, 16, 18, 19]. the result of a study conducted in india reported that 76.3% of the infertile women interviewed had experienced violence [13]. however, none of the studies reported rates of dv as high as our study. this difference may stem from methodological complexities, using different questionnaires, and sociocultural variations among women in these countries. in our study, psychological violence, at 85.8%, was the most common type of violence followed by verbal violence that was seen in almost half of the women. sexual violence occurred less frequently and physical violence was reported in one-quarter of women who experienced violence. although the prevalence of overall violence is considerably high, no one reported experiencing physical injury. it is worth noting that different types of violence are connected to each other. for instance, sexual violence may lead to physical and verbal violence simultaneously and these can cause psychological violence. similar to our findings, numerous studies reported psychological violence as the most common form of violence [8, 15, 16, 18, 20, 21]. comparable to our result, psychological violence rates against women were found in previous studies to be 87.3%, 82% and 74.3% in iran [16, 20, 22]. a higher prevalence was reported in nigeria at 94% among infertile women, which is slightly greater than the findings of this study [18]. in contrast, yildizhan et al. found verbal abuse to be the most common type of violence with rates of 63.4 %; however, they did not include psychological violence in their study [9]. verbal violence was found to be the second most common type of violence in our study. a similar result was obtained in nigeria, where the prevalence of verbal abuse was women age (y), mean (sd) 31.66 ± 6.46 31.08 ± 6.22 36.29 ± 6.54 < 0.001* men age (y), mean (sd) 35.99 ± 8.40 35.22 ± 7.70 42.12 ± 11.02 < 0.001* marriage duration (y), mean (sd) 7.08 ± 4.82 6.91 ± 4.64 8.43 ± 6.1) 0.05* infertility duration (y), mean (sd) 5.56 ± 4.40 5.38 ± 4.13 7.05 ± 6.0 0.02* women educational status illiterate 21 (5.5) 20 (6) 1 (2.4) < diploma 100 (26.4) 85 (25.2) 15 (35.7) diploma 153 (40.4) 134 (39.8) 19 (45.2) academic degree 105 (27.7) 98 (29) 7 (16.7) men educational status illiterate 26 (6.9) 26 (7.7) 0 < diploma 114 (30.1) 94 (27.9) 20 (47.6) diploma 146 (38.5) 135 (40) 11 (26.2) academic degree 93 (24.5) 82 (24.3) 11 (26.2) women occupation housewife 317 (83) 277(82.2) 40(95.2) employed 62 (16.4) 60(17.8) 2 (4.8) men occupation eemployed 372(98.2) 331(98.2) 41(97.6) unemployed 7(1.8) 6(1.8) 1(2.4) residential place urban 254 (67) 225(66.8) 29(69) rural 125 (33) 112(33.2) 13(31) men smoking no 281 (74.1) 241(71.5) 40 (95.2) yes 98 (25.9) 96 (28.5) 2 (4.8) men alcohol use no 375 (98.9) 333 (98.9) 42 (100) yes 4 (1.1%) 4 (1.1) 0 (0) men addiction to drug no 332 (87.6) 292 (86.7) 40 (95.2) yes 47 (12.4) 45 (13.3) 2 (4.8) 0.47** 0.11** 0.005** 0.02** 0.03** 0.19** 0.78** 0.76** variables total (%) domestic violence yes no p-value variables crude odds ratio (95% ci)* p-value adjusted odds ratio (95% ci) ** p-value women age (y) 0.88 (0.84-0.93) 0.001 0.91 (0.85-0.99) 0.03 men age (y) 0.92 (0.890.95) 0.001 0.95 (0.91-1.01) 0.16 marriage duration (y) 0.94 (0.89-1) 0.05 1.09 (0.96-1.23) 0.16 infertility duration (y) 0.93 (0.87-0.99) 0.02 0.90 (0.79-1.03) 0.15 women occupation housewife 0.23 (0.05-0.98) 0.04 0.26 (0.06-1.15) employed reference men smoking no reference yes 7.96 (1.88-33.61) 0.005 8.12 (1.87-35.21) 0.07 0.005 http://www.ajecr.org/ 270 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org recorded at 39.2% [21]. in the current study, sexual violence was behind verbal abuse in terms of prevalence violence was behind verbal abuse in terms of prevalence followed by physical violence, which is in line with the finding of abadi et al. showing a 51.4% prevalence of sexual violence [20]. a higher prevalence of sexual violence at 82.9% was found in nigeria [18]. this study had no reports of physical injury, which is in contrast to ardebily et al. where 6 % of the respondents reported injury [8]. according to our knowledge, the number of studies that report physical injury among infertile women was quite low. this discrepancy might be due to different understandings of violence, reporting issues, and variations in research design in the different studies. the results of this study found that no significant relationship exists between dv and women’s educational status, men’s employment status, place of residence, drinking alcohol and drug addiction by men. although education might be a protective factor, the lack of a significant relationship between age, women’s education, men’s occupation, place of residence and violence against infertile women indicates that dv is a global problem and widespread regardless of economic development and educational level. moreover, associations between violence and sociodemographic characteristics are controversial due to differences among various studies or even same country. for instance, in some studies, there was no association between violence and women's educational and employment status [8, 12, 19] while, the result of a study conducted in nigeria found a significant relation between violence and education and occupation [18]. in a similar study, ardabily and colleagues reported a significant relationship between violence and an unemployed spouse.8 in the present study, domestic violence was associated with women’s employment status, infertility duration, length of marriage, women and men age, men smoking, and men’s educational status. in contrast, akyüz et al. found no correlation between violence with age and occupational status of the women [19]. similarly, some studies reported no significant association between age and violence among infertile women.9 regarding the place of residence, some studies reported being a resident in rural areas increases the risk of dv [20, 23], whereas, in line with our finding, few studies reported no association between violence and residential place [24, 25]. however, a study reported living in urban areas was related to higher rate of violence [26]. this discrepancy might be due to the number of differences between rural and urban areas in various societies. supporting our finding, akyuz et al. demonstrated significant relationships between domestic violence and infertility duration [27], which contradicts other studies conducted in iran and nigeria [8, 21]. despite the lack of association between violence and addiction in our study, in a similar study addiction problems of a spouse were seen in more than half of the women who experienced violence [20]. of all included variables, only smoking by men and women with younger ages were risk factors of violence. the result of our study showed that smoking increases the chance of violence eightfold. similar to findings of the present study, some studies reported smoking as a risk factor of dv [28, 29]. in contrast to this study, some studies found higher rates of violence among women with the lower level of education and housewives [18, 20]. the difference in the current study might be due to the cultural and economic factors that may neutralize the effect of other possible factors. unlike the result of our study, some studies found alcohol consumption as a factor increasing the likelihood of violence [26, 30]. a plausible explanation is the low levels of alcoholism in two groups of the current study. limitation there are some limitations to our study. due to cultural reasons, we removed three questions of the cts2 questionnaire. the participants were recruited only at the infertility clinic of the teaching hospital at mazandaran university of medical sciences, therefore, the result of our study is not generalizable to the general population of infertile women. furthermore, the study was based on selfreported data of the female participants. due to the sensitivity of the topic, it should be interpreted with caution as an underestimation or overestimation of the true rate of the problem might exist. despite these limitations, it is important to mention that the quite large sample size has provided valuable data as risk factors of domestic violence. although several studies have investigated dv against women as a general population, the number of studies that focus on infertile women is not adequate and shows the necessity of conducting more research studies among this vulnerable group of women. furthermore, according to our knowledge, most of the similar studies in iran focused on emotional, physical, and sexual violence, while the current study also includes verbal and physical injury of dv. conclusion the result of our study demonstrated a high prevalence of dv among infertile women. psychological violence was found to be the most common type of violence, revealing that women mainly suffer from emotional problems. male smoking habits and younger females were risk factors of domestic violence. it is necessary that health staff identify at-risk women and provide special attention to them and their husbands via educational programs such as communication skill, problem-solving, and coping strategies. due to the use of different instruments and methodological approaches in previous studies, there are inconsistencies in the reported violence rate. therefore, further studies will be performed among infertile and fertile women with the same instrument to compare the rate of violence among them. it should also include the private fertility clinic as well. violence against women may lead to depression, stress, anxiety disorders, injury or even suicide [10]. due to the importance of women’s role in society, domestic violence against infertile women should not be neglected. unfortunately, cultural barriers in developing countries make interventions very difficult, hence raising awareness http://www.ajecr.org/ 271 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org and training of spouses regarding mutual rights and anger management via counseling centers and social media is highly suggested. acknowledgement this research is based on a research project with the code of ethics 9303, which was approved on april 23, 2014 by the financial support of shahroud university of medical sciences. conflict of interest the authors declare no conflicts of interest. references 1. zegers-hochschild f, adamson gd, mouzon j, ishihara o, mansour r, nygren k, et al. international committee for monitoring assisted reproductive technology (icmart) and the world health organization (who) revised glossary of art terminology. fertil steril 92:1520-1524, 2009. 2. mascarenhas mn, flaxman sr, boerma t, vanderpoel s, stevens ga. national, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. plos med 2012; 9:e1001356. doi: 10.1371/journal.pmed.1001356. 3. boivin j, bunting l, collins j a, nygren k g. international estimates of infertility prevalence and treatment seeking: potential need and demand for infertility medical care. hum reprod 22:1506-1512, 2007. 4. kazemijaliseh h, ramezani tehrani f, behboudigandevani s, hosseinpanah f, khalili d, azizi f. the prevalence and causes of primary infertility in iran: a population-based study. glob j health sci 7:226-232, 2015. 5. akhondi m m, kamali k, ranjbar f, shirzad m, shafeghati s, behjati ardakani z, et al. prevalence of primary infertility in iran in 2010. iran j public health 42:1398-1404, 2013. 6. world health organization. gender and genetics: assisted reproductive technologies (arts) (2015). http://www.who.int/genomics/gender/en/index6.html. accessed november 25. 7. dyer s j, abrahams n, mokoena n e, lombard c j, van der spuy z m. psychological distress among women suffering from couple infertility in south africa: a quantitative assessment. hum reprod 20:1938-1943, 2005. 8. ardabily h e, moghadam z b, salsali m, ramezanzadeh f, nedjat s. prevalence and risk factors for domestic violence against infertile women in an iranian setting. int j gynaecol obstet 112:15-17, 2011. 9. yildizhan r, adali e, kolusari a, kurdoglu m, yildizhan b, sahin g. domestic violence against infertile women in a turkish setting. int j gynecol obstet 104: 110112, 2009. 10. world health organization. violence against women: fact sheet n°239. geneva: (2016). [updated nov 2014;cited 2015 apr] available from: http://www.who.int/ mediacentre/factsheets/fs239/en/ 11. stellar c, garcia-moreno c, temmerman m, van der poe s. a systematic review and narrative report of the relationship between infertility, subfertility, and intimate partner violence. int j gynaecol obstet 133:3-8, 2016. 12. sami n, ali ts. domestic violence against infertile women in karachi, pakistan. asian rev soc sci 1:15-20, 2012. 13. pasi a, hanchate m, pasha m. infertility and domestic violence: cause, consequence and management in indian scenario. biomed res 22:255-258, 2011. 14. behboodi moghadam, z, eftekhar ardabily h, salsali m, ramezanzadeh f, nedjat s. physical and psychological violence against infertile women. j family reprod health 4:65-67, 2010. 15. farzadi l, ghasemzadeh a, bahrami asl z, mahinib m, shirdel h. intimate partner violence against infertile women, j clin res gov 4:147-151, 2014. 16. sheikhan z, ozgoli g, azar m, alavimajd h. domestic violence in iranian infertile women. med j islam repub iran 28:152, 2014. 17. straus ma, hamby sl, boney-mccoy s, sugarman db. the revised conflict tactics scales (cts2) development and preliminary psychometric data. j fam issues 17:283-316, 1996. 18. iliyasu z, galadanci h s, abubakar s, auwal m s, odoh c, salihu h m, et al. phenotypes of intimate partner violence among women experiencing infertility in kano, northwest nigeria. int j gynaecol obstet 133:32-36, 2016. 19. akyüz a, şahiner g, seven m, bakır b. the effect of marital violence on infertility distress among a sample of turkish women. int j fertil steril 8: 67-76, 2014. 20. abadi m p, mahdavi s, esmaeili k, amighi m, hashemian ah. the amount of domestic violence in kermanshahi (a provincial center in west iran) women given birth in 2011-2012. wjmmsr 11:202-206, 2014. 21. ameh n, kene ts, onuh so, okohue je, umeora ou, anozie ob. burden of domestic violence amongst infertile women attending infertility clinics in nigeria. niger j med 16:375-377, 2007. 22. guruge s, roche b, catallo c. violence against women: an exploration of the physical and mental health trends among immigrant and refugee women in canada. nurs res pract volume 2012: article id 434592, 2012. 23. faramarzi m, esmailzadeh s, & mosavi s. prevalence and determinants of intimate partner violence in babol city, islamic republic of iran. east mediterr health j 11:870-879, 2005. 24. parish wl, wang t, laumann e o, pan s, & luo y. intimate partner violence in china: national prevalence, risk factors and associated health problems. int fam plan perspect 30: 174–181, 2004. 25. dosary ah. health impact of domestic violence against saudi women: cross sectional study, int j health sci (qassim) 10:166-173, 2016. 26. sinha a, mallik s, sanyal d, dasgupta s, pal d, mukherjee a. domestic violence among evermarried women of reproductive age group in a slum area of kolkata, indian j public health 56:31-36, 2012. 27. akyuz a, seven m, şahiner g, bakır b. studying the effect of infertility on marital violence in turkish women. int j fertil steril 6: 286-293, 2013. http://www.ajecr.org/ 272 am j exp clin res, vol. 5, no. 2, 2018 http://www.ajecr.org 28. abbaspoor z & momtazpour m. domestic violence and its related factors based a prevalence study in iran, glob j health sci 8:1-7, 2016. 29. aklimunnessa k, khan m, kabir m, mori m. prevalence and correlates of domestic violence by husbands against wives in bangladesh: evidence from a national survey, j men's health & gen 4:5263, 2007. 30. dalal k, rahman f, jansson b. wife abuse in rural bangladesh. j biosoc sci 41:561-4573, 2009. http://www.ajecr.org/ 68 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(4):68-71 original article ctla-4 polymorphism in iranian patients with systemic lupus erythematosus mahdieh shojaa 1 , mahsa amoli 2 , mehrdad aghaie 3 *, patricia khashayar 4 . naemeh javid 1 , fatemeh shakeri 1 , abbasali keshtkar 4 , mostafa qorbani 5 , ramin mohebbi 6 , zahra mohammadi 7 1 golestan university of medical sciences, tehran, iran 2 endocrinology and metabolism clinical sciences institute, tehran university of medical sciences, tehran, iran 3 bone joint and connective tissue disease research center (bjcrc), golestan university of medical sciences, gorgan, iran 4 osteoporosis research center, tehran university of medical sciences, tehran, iran 5 department of public health, alborz university of medical sciences, karaj, iran 6 shahed university of meical sciences, tehran, iran 7 azad university of medical sciences, tehran, iran abstract. cytotoxic t lymphocyte-associated antigen-4 (ctla-4) is an important negative regulator of t-cell responses. ctla-4 polymorphisms have been confirmed to be associated with several autoimmune diseases such as systemic lupus erythematosus (sle). we analyzed the role of +49ag polymorphism in exon1 of the ctla-4 gene in iranian patients suffering from sle. a cohort of 180 sle patients and 304 ethnically and age matched healthy controls were studied. polymerase chain reaction restriction fragments length polymorphism (pcr-rflp) was used to analyze the genotype and allele frequencies of these polymorphisms. we found that the aa genotype was significantly higher in sle patients (67.2% vs. 41.1%, p=0.0001). the ag genotype frequency, on the other hand, was more frequently reported in the controls (49.7% vs. 27.8%, p=0.0001). the gg genotype was also more common in the control group than sle patients but the difference was not significant (p=0.06). the frequency of g allele was significantly higher in sle patients: 34% versus 18.9% than in control (p=0.0001). there was no significant correlation between the risk of developing sle and the individual’s age, parental consanguinity, and family history of sle. we didn’t observe any significant association between genotype and the clinical features of sle. we conclude that the +49ag polymorphism of ctla-4 gene appear to play a significant role in the development sle in the iranian patients, but not to be associated with clinical features of sle. keywords: systemic lupus erythematosus, ctla-4, exon1, 49ag polymorphism introduction systemic lupus erythematosus (sle) is a complex inflammatory disease characterized by autoantibody production [1]. the disease is more common in women but found in different racial and ethnic groups. it is more frequently reported in individuals in the second, third or fourth decades of life [2]. sle affects more than 1 million individuals in the us and 3.2 to 14.1 cases per100000 in women of european descent [3, 4]. the disease is reported in is 40 per 100000 of the iranian people [5]. the etiology of the disease is unknown but is thought to be caused by both genetic and environmental factors [6]. the expression of ctla-4 is increased in patients with active sle [7]. cytotoxic t lymphocyte-associated antigen-4 (ctla-4) is an important negative regulator of t-cell responses, and its dysregulation has the potential to affect the pathogenesis of sle by altered activation of t cells to self-antigens [1]. inappropriate t-cell dependent expansion of autoreactive b cells is considered to play a role in the production of pathogenic autoantibodies [8] in multiple organs, including kidneys, heart, lung, joints and immune system [9]. the ctla-4 gene is located within the risk region on chromosome 2q33 and several polymorphisms have been reported in this gene [10]. ctla-4 polymorphisms have been confirmed to be associated with several autoimmune disorders such as, graves’ disease, type i diabetes, celiac disease, autoimmune thyroid disease, rheumatoid arthritis and multiple sclerosis and sle [11]. one of these is located in exon 1 at position +49(a/g). using a case-control study design, we have determined the role of +49ag polymorphism in sle pathogenesis. ___________________________________________________________ * corresponding author: mehrdad aghaie, md (shojaamahdieh@yahoo.com). http://www.ajecr.org/ mailto:shojaamahdieh@yahoo.com 69 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org materials and methods patients in this study, 180 sle patients (15 males and 165 females) with a mean age of 32.99 ±10.45 years (ranging from 13 to 70 years) were enrolled. in addition, 304 ethnically and age matched healthy controls (23 males and 281 women) with no history of any autoimmune diseases were recruited from the azar 5th teaching hospital affiliated to gorgan university of medical sciences, gorgan, iran. all the sle patients fulfilled the american college of rheumatology 1997 revised criteria for sle [12]. the study was approved by the local ethics committee and a written informed consent was obtained from each patient. dna extraction & genotyping the dna of from the patients and the controls was extracted from their peripheral blood with a dna extraction kit (roche applied science) according to the standard protocol from the manufacturer. polymerase chain reaction-restriction fragment length polymorphism (pcrrflp) was used to analyze the +49ag polymorphism in exon1. +49ag polymorphism was genotyped as following using a single primer set: forward 5ʹ gctctacttcctgaagacct-3ʹand reverse 5ʹ agtctcactcacctttgcag-3ʹ. amplification was carried out after initial denaturation at 94°c (2 min), followed by thirty cycles at 94°c (30 s), 60°c (30 s), 72°c (1 min), and a final extension at 72°c (2 min). the pcr products were digested using restriction enzyme bbvi (new england biolabs) at 65◦c for 3 h and then were analyzed on 3% agarose gel using ethidium bromide staining. the amplified dna for was 162 bp fragment (a allele) or two fragments of 91 and 71 bp (g allele). statistical analysis the frequency of alleles and genotypes were assessed using direct counting. chi-square and fisher’s exact test were used to compare the distributions and association of alleles and genotypes in the patients and the controls. a pvalue <0.05 was considered as statistically significant. the strength of the association between different groups and alleles or genotypes of polymorphism was estimated using odds ratios (or) and 95% confidence intervals (ci). statistical analysis was conducted with stata (v8) software. linkage disequilibrium and haplotype analysis estimated haplotype frequencies and testing for linkage disequilibrium between pairs of polymorphisms in the cases and controls were calculated using the ehplus program, which provides log likelihood, chi-square and the number of degrees of freedom. to test for heterogeneity in haplotype frequencies between the cases and controls, the likelihood ratio test was used. results blood samples from 180 sle patients and 304 controls were genotyped for the +49 ag in exon 1 region of the ctla-4 gene. 37.2 percent of patients had consanguineous parents. positive family history of sle was reported in 15 percent of the patients. there was no significant correlation between the risk of developing sle and the individual’s age, parental consanguinity, and family history of sle (table1). the genotype and allele frequencies of the +49ag polymorphism are seen in table 2. the frequency of aa genotype was significantly higher in sle patients (67.2% vs. 41.1%, p= 0.0001; or=2.93). the ag genotype frequency, on the other hand, was more frequently reported in the controls (49.7% vs. 27.8%, p=0.0001; or=0.39). gg genotype was also more common in the control group than sle patients but the difference was not statistically significant (p= 0.06; or=0.51). the frequency of the g allele significantly increased in sle patients: 34% versus 18.9% in control (p=0.0001; or=0.34). however, the a allele frequency increased in patients but we observed no significant difference in the frequency of the a allele between patients and control (p=0.06). table 3 shows the relationship between 49ag genotypes and clinical features. although, all manifestations were more frequently reported in individuals with aa genotype, however, there was no association between ctla-4 genotypes and clinical features of sle (table 3). table 1 association between ctls-4 +49 genotype and sle risk factors risk factor aa (%) ag (%) gg (%) total (%) p value age <15 15-45 >145 3 (1.6) 101 (56.1) 17 (9.5) 1 (0.5) 47 (26.1) 2 (1.1) 0 7 (3.9) 2 (1.1) 4 (2.2) 155 (86.2) 21 (11.6) 0.31 consanguineous parents yes no 50 (27.8) 71 (39.5) 15 (8.5) 35 (19.4) 2 (1.1) 71 (3.9) 67 (37.2) 113 (62.8) 0.24  family history yes no 21 (11.7) 100 (55.5) 6 (3.3) 44 (24.5) 0 9 (5) 27 (15) 153 (85) 0.29 table 2 genotypic distribution and allelic frequencies of ctla-4 +49ag polymorphysms in the iranian sle patients and healthy controls exon 1 sle n=180 (%) control n=304 (%) p value or (95% ci) genotype aa ag gg allele a g 1 21 (67.2) 50 (27.8) 9 (5) 292 (81.1) 68 (18.9) 125 (41.1) 151 (49.7) 28 (9.2) 401 (66) 207 (34) 0.0001 0.0001 0.06 0.06 0.0001 2.93 (1.99-4.32) 0.39 (0.26-0.57) 0.51 (0.23-1.12 1.92 (0.88-4.18) 0.34 (0.23-0.5) http://www.ajecr.org/ 70 am j exp clin res, vol. 1, no. 4, 2014 http://www.ajecr.org discussion although the definite etiopathogenesis of sle remains unclear, evidence indicates that ctla-4 polymorphisms play an important role in susceptibility to sle. some studies have reported a significant correlation between ctla-4 polymorphisms and sle (13-16). on the other hand, several studies have failed to show any association between sle and ctla-4 polymorphisms (16-20). in this study, a strong association was observed between ctla-4 polymorphism and sle in the iranian population. in line with our results, a significant correlation has been reported between 49ag polymorphism and sle among asian and caucasian population (14-15, 21-22). on the other hand, several studies reported no association between this polymorphism and sle in the different racial groups (1721, 24-25). possible reason behind these controversial results may arise from many aspects such as different ethnic populations, gender, sample size and age at the onset of disease. our results indicated an association between the aa genotype and susceptibility to sle. corroborating with our results, ulker et al reported that aa genotype is found more frequently among sle patients (15). this comes while the contrary was reported for the korean patients (14). moreover, the ag genotype and g allele were more common in control group. this comes while previous studies had reported controversial results (14, 20-22, 26). we also investigated some of sle risk factors such as age, having consanguineous parents and a positive family history of sle. having consanguineous parents was more common in patients with aa genotype. the majority of the patients with the vary genotypes (56.1%) were aged between 15 and 45 years old; the correlation however was not significant. our study also failed to show any correlation between ctla-4 exon 1 position 49 polymorphism and clinical features. the clinical features though were more likely to be reported in patients with aa genotype. corroborating with our results, ulker et al reported that patients with aa genotype were more likely to manifest sle symptoms (15). in line with our result, the correlation was not statistically significant. several studies have reported ctla-4 exon 1 position + 49 gene polymorphisms in sle patients but we could not find any study to report association between 49ag polymorphism, risk factors and clinical features of sle patients. we found age, having consanguineous parents and a positive family history of sle are not associated with none of the genotypes. our study is the first study to report association between +49ag and sle among the iranians. hence, further studies on other populations especially in the middle eastern nation are needed in this regard. acknowledgment this study was funded by the department of research affairs of golestan university of medical sciences. the 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screening method. tissue antigens 54:578-584, 1999. http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22parks%20cg%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22hudson%20ll%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22cooper%20gs%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22dooley%20ma%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22treadwell%20el%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22st%20clair%20ew%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22gilkeson%20gs%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22pandey%20jp%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'lupus.'); 349 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2019;6(3):349-354 original article comparison of the esr and crp levels and their relationship with psa in benign prostate hyperplasia and prostate adenocarcinoma patients: a retrospective analytical study shokouh taghipour zahir1, mohammad-sadegh raeeszadeh2, farzan safi dahaj3*, koorosh rahmani4, mehrdad roozbeh5* 1department of pathology, shahid sadoughi university of medical sciences, yazd, iran. 2department of medicine, shahid sadoughi university of medical sciences, yazd, iran 3student research committee, shahid sadoughi university of medical sciences, yazd, iran 4department of neurology, shahid beheshti university of medical sciences, tehran, iran abstract. prostate cancer is the most common malignancy (excluding skin malignancies) in men. chronic inflammation has been shown to be associated with cancer. although this association has not been proven for prostate cancer, evidence shows that inflammation has a possible role in prostate cancer. erythrocyte sedimentation rate (esr) and c-reactive protein (crp) are the two key indices in assessing inflammation. it was therefore decided to investigate esr and crp in patients with prostate cancer and assess their relationship with psa level at initial diagnosis. in this retrospective-analytical study, hospital records of all patients referred to mortaz and shahid rahnemoon general hospitals during 2013-2018 and undergoing prostatectomy with pathology reports of benign prostatic hyperplasia or prostate adenocarcinoma were extracted by census method. the required variables including patient’s age, psa, esr and crp levels were extracted from hospital records. extracted data were analyzed by anova and chi-square tests in spss software version 18. p-value <0.05 was considered statistically significant. according to the results, patients' mean age was 70.71±10.19 years, mean esr 38.86±31.28, and mean crp 1.28±1.01. there was a significant difference between the two groups in mean values of esr and crp (p<0.05). the results also showed meaningful correlations between age and psa, esr and psa, crp and psa, and crp and esr in patients with prostate cancer (p<0.05). as inflammatory factors, esr and crp increase in patients with prostate cancer, they can be used in the initial diagnosis of prostate cancer as an adjunct diagnostic assay and prognostic factor (only esr) in conjunction with psa. keywords: neoplasm, prostate, blood sedimentation, inflammation introduction prostate cancer is the most common malignancy (other than skin) in men in the united states of america (usa), and the second leading cause of cancer-related death in the western world [1]. there are no accurate statistics on the prevalence of prostate cancer in iran, but investigations suggest that its prevalence is lower in iran compared to western countries and usa [1, 2]. although available information for treatment of prostate cancer has increased [3-5], prognosis for advanced stage patients is unfortunately associated with mean survival of 2.5 years [6]. long-term chronic inflammation is associated with infectious cancers such as stomach, liver, and colon cancers, which are commonly seen in patients with intestinal inflammation [7]. it is thought that chronic inflammation stimulates cancer through a variety of mechanisms, including irreversible cell and dna damage by producing free radicals, and accelerated cell development through dna and cell transcription [8]. although the role of chronic inflammation or return of inflammation in the progress of prostate cancer has not been proven, a number of reports suggest the possible role of inflammation in prostate cancer through various interrelated mechanisms [9]. inflammation may be associated with prostate carcinogenesis and is often seen in prostate biopsy, radical prostatectomy samples and tissues taken to treat bph. in histological terms, inflammatory cells are commonly found in and around the center of atrophy and identified by increased proliferation index. this center that is also known as proliferative inflammatory atrophy, maybe the initial indicator of prostate cancer or an indicator of an appropriate environment for development of prostate cancer [7]. serum inflammatory markers had significantly elevated ___________________________________________________________ * corresponding author: dr. farzan safi dahaj (safi.farzan@gmail.com) http://www.ajecr.org/ mailto:safi.farzan@gmail.com 350 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org table 1 mean variables by groups (prostate adenocarcinoma (pad) & benign prostatic hyperplasia (bph) table 2 mean psa by age group in patients with prostate cancer table 3 mean psa in different age groups of patients with prostate cancer *lsd test in patients with prostate cancer, especially in those with higher gleason and psa scores [10]. measurement of erythrocyte sedimentation rate (esr) is a simple and inexpensive laboratory test, and is used in assessing the acute phase [11]. esr has prognostic value in several cancers, including prostate cancer [12-18]. esr is an indicator of increased risk and death in prostate cancer, and is likely to reflect the relationship between tumor and the host [19]. esr has been shown to predict survival rate in early stages of localized prostate cancer [20]. c-reactive protein (crp) is a general inflammation marker, and is associated with prostate cancer. increased crp indicates poor prognosis [21, 22], and is high in men with bone metastasis [23]. there is also a strong relationship between crp and psa, irrespective of the stage of the tumor, which suggests that inflammation may be the basis of prostate cancer [24, 25]. measurement of plasma crp level does not support diagnosis of benign conditions in patients with increased psa levels, even though plasma crp levels have a good correlation with plasma psa levels in patients with prostate cancer, and suggest a strong relationship between inflammation and prostate cancer [26]. the present study investigates esr and crp levels in patients with prostate cancer and their relationship with psa in initial diagnosis, and the results will be used for better treatment and management of prostate cancer. materials and methods study design in this retrospectiveanalytical study, hospital records of all patients referred to mortaz and shahid rahnemoon general hospitals during 2013-2018 and undergoing prostatectomy with pathology reports of benign prostatic hyperplasia or prostate adenocarcinoma were extracted by census method. the required variables including patient’s age, psa, esr and crp levels were extracted from hospital records. extracted data were analyzed by anova and chi -square tests in spss software version 18. p-value <0.05 was considered statistically significant. exclusion criteria patients with inflammatory diseases (arthritis, vasculitis…), rheumatoid arthritis, gout, asthma, chronic lung diseases, heart attacks or apoplexies, and those using nsaid were excluded. data sources/measurement patients' demographic details and psa, esr, and crp levels were extracted from their hospital records and inserted in a checklist. pathology reports of bph and prostate adenocarcinoma patients were included in the checklist. all glass slides were reassessed by two pathologists for the coordination of grading based on the new gleason grading of the world health organization (2016). statistical methods data collected were analyzed in spss-18 using ttest, lsd, anova, and correlation tests. logistic regression was used to eliminate the effect of confounding variables and determine the relationship between independent variables and cancer. p value less than 0.05 was considered statistically significant. ethical considerations the study was given ethical approval with ethical committee of the medical school of shahid sadoughi university of medical sciences and shahid rahnemoon educational hospital and mortaz general hospital and all the patients had given written informed consent as a role in first admission and hospitalization. results a total of 290 patients were enrolled in the study by census method. one hundred fortyfive benign prostatic hyperplasia patients and also 145 with prostate adenocarcinoma. according to the results, patients' mean age was 70.71±10.19 years ranging from 34 to 102 year. mean values of esr and crp were significantly higher in the group with benign prostatic hyperplasia compared to the patients with prostate adenocarcinoma the results relating to different variables (age, esr, and crp) by groups showed mean age of 69.97±11.18 years in benign prostatic hyperplasia (bph) patients and 71.44±9.07 years in patients with prostate adenocarcinoma (pad). other data are presented in table 1. anova test results showed significant differences between two groups in mean values of esr (p=0.001) and crp (p=0.001). in other words, mean values of esr and crp were pad (n=145) bph (n=145) total (n=290) age 70.71±10.19 69.97±11.18 71.44±9.07 0.22 esr 38.86±31.26 19.39±7.51 58.32±33.84 0.001 crp 1.28±1.01 0.57±0.49 1.99±0.9 0.001 variable mean ± sd p-value* 34-64 (n=44) 65-74 (n=40) 75-102 (n=61) total (n=145) psa 43±18.94 40.98±23.21 53.31±25.14 46.78±23.41 0.014 p-value* age range (yrs) variable 34-64 65-74 75-102 34-64 0.686 0.024 65-74 0.686 0.009 75-102 0.024 0.009 age group p-value* http://www.ajecr.org/ 351 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org table 4 mean value of variables by gleason score in patients with prostate cancer *anova test table 5 correlation between study variables in patients with prostate cancer *logistic regression significantly higher in the pad patients compared to the bph. no significant difference was observed between two groups in terms of mean age. there were significant differences in mean psa by age group in patients with prostate cancer the results relating to mean psa by three age groups of 34-64, 65-74, and 75-102 years in patients with prostate cancer showed mean psa of 43±18.94 in 34-64 age group, 40.98±23.21 in 65-74 age group, and 53.31±25.14 in 75102 age group. other data are shown in table 2. anova test results showed significant differences in mean psa by age group in patients with prostate cancer (p=0.014). thus, lsd test was used to assess two-by-two relationships between groups. lsd test results showed no significant difference between first and second age groups (p=0.686), but the difference between first and third groups and also between second and third age groups was significant (p=0.024, p=0.009 respectively), as shown in table 3. no significant differences in mean values of esr and crp by age group in patients with prostate cancer were seen the results relating to mean values of esr and crp by three age groups of 34-64, 65-74, and 75-102 years in patients with prostate cancer showed mean esr of 61±29.93 in 34-64 age group, 56.68±27.91 in 65-74 age group, and 57.46±39.9 in 75-102 age group. anova test results showed no significant differences in mean values of esr and crp by age group in patients with prostate cancer (p=0.82, p=0.168 respectively). in other words, the difference between mean values of esr and crp in different age groups was not statistically significant. significant differences between the esr, crp, psa in terms gleason score in patients with prostate cancer were seen the results relating to the frequency of gleason grading score in patients with prostate cancer showed that of the 145 patients, 32 (22%) were in grade 8. the results relating to mean values of esr, crp, and psa in terms of well (16), moderate (7-8), and poorly differentiated (9-10), gleason scores in patients with prostate cancer showed mean esr of 42.74±17.81 in well differentiated group, 55.08±18.16 in moderately differentiation group, and 62.77±38.34 in poorly differentiation group. anova test results showed significant differences between these three variables in terms of gleason score in patients with prostate cancer (p=0.032, p=0.001, p=0.001 respectively for esr, crp, psa in terms of gleason score). thus, lsd test was used to assess two-by-two relationships between groups. lsd test results showed significant differences between well and poorly differentiated groups in mean esr, between well and poorly differentiated and well and moderately differentiated in mean crp, and between well and poorly and well and moderately differentiated in mean psa (p<0.05), as shown in table 4. positive correlations were found between psa and age, esr and psa, crp and psa, and crp and esr in patients with prostate cancer pearson correlation coefficient test showed a positive correlation between psa and age with r=0.254 and p=0.002. also, positive correlations were also found between esr and psa, crp and psa, and crp and esr in patients with prostate cancer (p<0.05). other data are shown in table 5. esr relationship with prostate cancer became significant binary logistic regression was used to determine the effect or relationship of each independent variables of age, esr and crp with prostate cancer after eliminating the effect of other variables, which confirmed significance of the model (p<0.05). in other words, cancer prediction power of the model significantly increased with inclusion of these three variables in regression model. correlation coefficient square was found r2=0.877, which is high and shows that 87.7% of variations in the dependent variable are covered by independent variables included in the model. cancer diagnosis sensitivity and specificity of the above three variables were 91.7% and 97.9% respectively, with accuracy of 94.8%, which are high. hence, after elimination of the effects of age and crp on cancer (not significant), the relationship of esr with prostate cancer became significant (p=0.001), which means that for every unit increase in esr, risk of cancer increases by 1.38 times in relation to bph. the above model showed that risk of cancer was not related to age or crp (p>0.05). the above results will be exactly confirmed with elimination of age and crp, and the only difference will be in the increase in specificity of cancer diagnosis to 100% and in accuracy to 95.9% probability of developing cancer was assessed by log adds using logistic regression data, and then esr with significant relationship was plotted (figure 1). according to the above figure, probability of developing prostate cancer with esr between 0-3 is close to zero. this probability exponentially increases with increasing esr, welldifferentiated (n=23) moderately differentiated (n=24) poorly differentiate d (n=98) total (n=145) esr 42.74±17.81 55.08±18.16 62.77±38.34 58.32±33.84 0.032 crp 1.3±1.02 1.92±0.77 2.17±0.82 1.99±0.9 0.001 psa 29.87±15.7 47.46±20.85 50.58±23.92 46.78±23.41 0.001 variable gleason score p-value variable statistics age psa esr pearson correlation 0.254 0.191 p-value 0.002 0.021 pearson correlation -0.019 0.191 p-value 0.82 0.021 pearson correlation -0.115 0.214 0.662 p-value 0.168 0.01 0 esr crp psa http://www.ajecr.org/ 352 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org especially when esr is 30-50. this probability approaches 100% when esr=50. the graph becomes plateau-like at esr>50, and further increase in esr results in no change in probability of cancer. figure 1 esr relationship with probability of developing prostate cancer in study subjects discussion the present study aimed to assess esr and crp levels in patients with prostate cancer and their relationship with psa level at initial diagnosis. significant differences were found between: 1mean serum psa in different age groups of patients with prostate cancer; 2mean serum esr and crp levels in pad and bph patients; and 3 mean serum esr, crp, and psa and gleason score (found at well, moderate, and poorly differentiated degrees). no significant difference was found between different age groups of patients with cancer in terms of esr and crp levels. the increasing risk of developing prostate cancer was found to have no correlation with age or crp level. while increasing esr level is associated with increasing risk of cancer. according to evidence, chronic inflammation is the underlying factor for 20% of all cancers in adults. this inflammation can be induced by infectious or environmental factors. according to previous studies, inflammation has a major role in pathogenesis of prostate cancer, and crp and esr are regarded as prognostic inflammatory factors in this cancer [20, 27]. studies have shown that many cancers (including stomach, liver, and colon cancers) are associated with chronic inflammation [7]. esr and crp are two diagnostic tests for inflammation, and are used in diagnosis of acute and chronic inflammation. the role of inflammation in prostate cancer is not fully understood. although a number of studies have been conducted in this area, and most have used esr and crp as diagnostic inflammation tests [9]. the majority of these studies have investigated the prognostic value of esr and crp, and some have used esr and others crp as prognostic tests in patients with prostate cancer, with high prognostic value. bing et al. investigated the role of esr and psa in prognosis of patients with advanced prostate cancer and reported positive results [28]. in another study, graff et al. found that increased crp implies poor prognosis in patients with prostate cancer [29, 30]. the results obtained by borre et al. showed that esr is a predictive factor for survival of patients with primary localized prostate cancer (organ limited) [20]. the study by johansson et al. also referred to the prognostic value of esr in patients with prostate cancer [19]. the present study results showed that esr has a high prognostic value, which agrees with some of the above studies [19, 31]. but, no significant relationship showing prognostic value of crp was found, which disagrees with some of the above studies [32]. in the present study, mean serum psa was significantly different in different age groups. in a study conducted by putra et al. on patients with bph, psa level was 4.29 in patients younger than 60 years, 4.61 in patients aged 61-69 years, and 4.8 in patients over 70 years of age, which shows that psa increases with aging. these results can be matched with increasing levels of this marker with aging in the present study, except that putra et al. study was conducted on patients with bph [33]. comparing the results of the present and the above studies suggests that the possible tenfold increase compared to the normal range in psa levels can help determination of type of neoplasm, but this has to be confirmed in future studies. the present study results showed positive correlations between age and psa, esr and psa, crp and psa, and crp and esr. another study conducted by yun et al. on healthy people attending for check-ups revealed a significant relationship between systemic inflammatory markers such as: esr and psa [34]. according to the present study results, esr and crp levels are significantly higher in prostate cancer groups compared to bph group, which is similar to the results obtained in previous studies. in a study conducted by kim et al. serum crp level was higher in prostate cancer group compared to bph group [32]. in agreement with the present study results, aldemir et al. found that serum inflammatory markers had significantly increased in patients with prostate cancer (especially in those with higher gleason score and psa level) [10]. the present study results also showed positive correlations between age and psa, esr and crp, psa and crp, and crp and esr in patients with prostate cancer. in their study, chang et al. found a relationship between plasma crp and serum psa levels in patients with prostate cancer [26]. given higher mean values of esr and crp in patients with prostate cancer compared to bph group, and a significant difference between them, it can be concluded that as inflammatory factors, esr and crp increase in patients with prostate cancer, and thus they can be used as diagnostic test in diagnosing prostate cancer. moreover, the positive and significant correlations found between esr and psa, crp and psa, and crp and esr in patients with prostate cancer show that esr and crp can have the same diagnostic value as psa in diagnosing prostate cancer. considering the significant relationship found between psa and age groups, psa can be said to http://www.ajecr.org/ 353 am j exp clin res, vol. 6, no. 3, 2019 http://www.ajecr.org increase with aging in patients with prostate cancer, and this increase in the over 75-year age group is statistically significant compared to other groups. acknowledgments authors would like to thanks dr. mahmood akhavan tafti for his special guidelines. conflict of interest the authors declare that there is no conflict of interest or financial support regarding the publication of this paper. references 1. siegel r, naishadham 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hajime shimizu1, ramin ghazizadeh2, mohammad ghazizadeh3* 1department of molecular pathology, institute of development and aging sciences, graduate school of medicine, nippon medical school, kawasaki, japan 2podiatric medicine, weiss memorial hospital, chicago, illinois, usa 3world academy of anti-aging and regenerative medicine, imperial tower, tokyo, japan abstract. keloid is a fibro-proliferative lesion that develops as a result of abnormal wound healing in susceptible individuals. many factors such as skin tension, wound infection, racial difference and genetic predisposition have been implicated in the etiology of keloid. micrornas are highly conserved noncoding small rna sequences and are key posttranscriptional gene regulators that contribute to the maintenance of differentiated cell phenotype. in this review, we outline the results of the studies on the expression of various mirornas in keloid as well as their role in pathogenesis of this lesion. keywords: microrna, keloid fibroblast, normal dermal fibroblast, mechanism, pathogenesis, type ⅰ procollagen keloid is a fibro-proliferative lesion characterized by excessive collagen deposition. it develops as a result of abnormal wound healing in susceptible individuals. keloid is defined as a scar within the skin that grows beyond the confines of original wound. many factors such as skin tension, wound infection, racial difference and genetic predisposition have been implicated in the etiology of keloid lesions [1]. they are characterized by overproduction of extracellular matrix (ecm) and invasiveness beyond the confines of original wound [2]. excess deposition of ecm as collagen by fibroblasts is responsible for keloid but the etiology and mechanism are unknown still now. a number of treatment modalities have been employed to overcome keloid. in general, surgical excision alone has proved unsatisfactory because of a recurrence rate of 45100% [3]. this has led to the development of various adjuvant therapies including radiotherapy [4]. recently, early postoperative electron beam (eb) irradiation has proved to be a well-tolerated and effective method in reducing the recurrence rate of keloid [5]. furthermore, the molecular mechanisms behind the effect of eb irradiation in reducing keloid recurrences revealed that the involvement of the interleukin 6 (il-6) synthesis and ecm gene expression in keloid was inhibited in response to eb irradiation [5]. however, continuous efforts are needed to explore more effective and less hazardous modes of therapy and prophylaxis for this lesions. understanding the mechanisms behind the development of the keloid scar is highly desirable in order to establish such approaches. we determined a functional role of il-6 signaling in keloid scars. keloid fibroblasts (kf) and counterpart normal fibroblasts (nf) were subjected to induction or inhibition of il-6 or its specific receptor il-6 receptor alpha and detection of their effects on ecm gene expression [6]. it was concluded that il-6 plays a pivotal role in the developmental mechanism(s) of keloid. mirnas, highly conserved noncoding small rnas of 19 to 26 nucleotides, are key posttranscriptional gene regulator that contribute to the maintenance of differentiated cell phenotype [7]. mammalian mirnas usually bind to the 3’ utr of target mrnas, promoting mrna degradation and/or inhibiting translation of the protein-code genes [8]. in mammals, mirnas are predicted to control the activity of approximately 30% of all protein-coding genes and have been shown to participate in the regulation of almost every cellular process. it is also highly likely that mechanisms that control translation initiation will have a significant impact on mirnas-regulated gene expression [9]. recently, some mirnas have been reported to participate in fibrosis and ecm metabolism. a number of mirnas, such as the mir21 and mir-29 families are emerging as common regulators of fibrosis and that directly target the translation of ecm components [10-14]. mir-21 ratios are selectively increased in fibroblasts of the failing heart by the extent of interstitial fibrosis and cardiac hypertrophy. these findings ___________________________________________________________ * corresponding author: m. ghazizadeh, md, phd (mghazizadeh1@gmail.com). http://www.ajecr.org/ 338 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org reveal that mir-21 as a disease target and antagomir-21 as a therapeutic target in heart failure in mice model [14]. administration of mir-21 antisense probes diminished the severity of experimental lung fibrosis in mice with bleomycin-induced fibrosis [15]. in addition, comparison of different mir-21 inhibitor as 8-mer or 22-mer oligonucleotide, treatment with 22-mer anti-mir-21 is ineffective in preventing cardiac disease in a mouse model [16]. thus, mirnas have important roles which suggest a new approach using mirna therapeutics in fibrotic diseases. the mechanisms behind keloid pathogenesis remains unclear. in a study, we attempted to clarify the differential analysis of mirna expression in primary cell culture from kfs and nfs using mirna microarray and then explored the function of mir-10a which showed the lowest expression level in kfs compared with nfs (unpublished data). we confirmed that mir-10a was statistically significantly under-expressed in kfs compared with nfs. suppression and activation study of mir-10a by microrna inhibitor and retinoic acid in kfs and nfs from the culture supernatant revealed that il-6 and procollagen type ⅰ (picp) secretions were significantly inhibited by mir-10a inhibitor and activated by retinoic acid (ra) treatment. kf and nf derived cells were transfected with microrna oligonucleotide mimic and inhibitor for mir10a. the secretion of picp was reduced in mir-10a transfected kfs and nfs. hoxa1 was expressed at significantly higher levels in kfs compared to nfs. in ra treatment experiments, hoxa1 was increasing in kf at lower levels, but in mir-10a inhibitor treatment, hoxa1 expression was significantly inhibited. downregulation of mir-10a was consistent with direct hoxa1 dependent regulation. mir-10a expression was inversed to that of hoxa1 expression. we found that hoxa1 is a direct target of mir-10a. on the ra treatment, mir-10a was significant overexpressed in kfs and nfs. treated fibroblasts in each well were harvested for mir-10a qpcr assay and the culture supernatant in each well was analyzed for il-6 and picp secretion by elisa method. these elisa assays indicated that il-6 concentrations were markedly increased in kfs compared with nfs in ra treatment. the molecular mechanisms of ra-mediated transcription regulation is not well-defined and little is known about the influence of ra signaling on mirna regulation. our own data demonstrate mir-10a regulates the secretion of collagen synthesis in kfs and nfs. the secretion of type ⅰ collagen was reduced in mir-10a transfected kfs and nfs. especially, remarkably reduction was shown in mir-10a transfected kfs. downregulation of mir-10a may be one of the mechanisms by which collagens are highly deposited in keloid tissues. these results suggested that mir-10a regulates the secretion of collagen synthesis in kfs and nfs. we concluded that mir-10a may be a promising new effective strategy for targeting keloid lesions. keloid scaring develops as a result of abnormal wound healing that grows beyond the wound margins and exhibits inflammation, fibroblastic cell proliferation, and accumulation of excessive collagen deposits. our previous study have shown that il-6 secretion is significantly increased in kf compared to nf. il-6 induces collagen synthesis in fibroblasts. several downstream targets including jak1, atat3, raf1, and elk1 are upregulated at mrna and protein levels in kfs compared to nfs. in the il-6 signaling pathway are upregulated at transcriptional and translational levels [6]. it is possible that deregulation of the il-6 signaling pathway might represent a common molecular mechanism that contribute to the pathogenesis in keloid. from a therapeutic point of view, the appreciation of dysfunction of il-6 signaling as the underlying molecular mechanism of keloid immediately suggests that stabilization of il-6 activity would have a potential therapeutic impact on keloid [17]. we have also shown postoperative adjuvant electron beam (eb) irradiation as successful tool to reduce keloid recurrences. the molecular mechanism behind the effect of eb irradiation might hinder keloid formation by regularizing disturbances in the homeostatic equilibrium between inducer and inhibitor activities in the matrix system most likely through the il-6 pathway [5]. recent evidence has shown that mirna-21 had the highest fold change (6.87-fold) and mirna-203 had the lowest expression level in keloid samples [18]. another report demonstrated that downregulation of mirna-196a may be one of the mechanisms by which collagens are highly deposited in keloid tissues [19]. mirna-19a/b played important role in the regulation of il-6 and mmp3 release in rheumatoid fibroblast-like synoviocytes [20]. similar to mrna expression, mirna expression has been found to be dysregulated in disease tissues in comparison with normal tissues. these dysregulated mirna represent a novel pool of therapeutic targets and biomarkers, including those in tissue fibrosis. for example, the mir-29 family of mirna is down-regulated in a mouse model of cardiac fibrosis following myocardial infarction [21]. in comparison with the normal skin tissues, mirnas were aberrantly expressed in limited cutaneous scleroderma and diffuse cutaneous scleroderma skin tissues. mirnas whose expressions were correlated with systemic sclerosis fibrosis; mir-21, mir-31, mir-146, mir-503, mir-145, and mir-29b were predicted to be involved [22]. the mirnas, mir-10a and mir-10b, are close homologs, differing by a single central nucleotide only. the mir-10a interacts with the 5, untranslated region of mrnas encoding ribosomal proteins to enhance their translation [23]. down regulation of mir-10a may increase upstream stimulatory factor 2 and contribute to the increase in cell proliferation of cml. implicating a mirna in the abnormal behavior of cml [24]. mir-10a is sharply down-regulated during megakaryocytic differentiation [25]. significant low endothelial expression of mir-10a detected in regions of athero-susceptibility was investigated by a combination of genomic profiling, mirna manipulations and molecular analyses in freshly isolated arterial endothelium and in cultured cells [26]. on the contrary, mir-10b is highly expressed in metastatic breast tumors initiate robust invasion and metastasis. the mir-10b induced by the transcription factor twist http://www.ajecr.org/ 339 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org proceeds to inhibit translation of the messenger rna encoding homeobox d10, resulting in increased expression of a well-characterized pro-metastatic gene, rhoc. significantly, the level of mir-10b expression in primary breast carcinomas correlates with clinical progression [27]. a close relative of mir-10b, mir-10a has been recently reported to target hox1, a gene that plays an oncogenec role in human mammary carcinoma cells [28], indicating that mir-10a might have an opposite rather than similar function in breast cancer. homeobox a1 (hoxa1) is an experimentally validated downstream target of mir-10a, as shown by direct hoxa1 3, utr-dependent suppression during megakaryocytopoiesis. during human megakaryotic differentiation, the main finding was down-regulation of mir-10a. hypothetically, the down-regulation of micrornas unblocks target genes involved in differentiation. mir-10a expression is inverse to that of hoxa1, and we showed that hoxa1 is a direct target of mir-10a [25]. hoxa1 was expressed at significantly higher levels in kfs compared to nfs. in ra treatment, hoxa1 was increasing in kf at lower levels, but mir-10a inhibitor treatment, hoxa1 expression was significantly inhibited. down-regulation of mir-10a consistent with direct hoxa1 dependent regulation. on the ra treatment, little is known about the influence of ra signaling on microrna regulation. upregulation of mir-10b was previously reported in rainduced neuronal differentiation of human embryonal carcinoma nt2/d1 cells [29], and an increase in mir-10a could be detected during ra-induced differentiation of mouse embryonic stem cell into smooth muscle cells [30]. mir-10a is a key mediator of metastatic behavior in pancreatic cancer. inhibition of mir-10a expression (with retinoic acid receptor antagonists) or function (with specific inhibitor) is a promising attracting point for antimetastatic therapies [31]. new technologies are emerging that utilize artificial mirna target sites to exploit or inhibit endogenous mirna regulation. this approach has been used to improve cell-specific targeting for gene and stem cell therapy studies and for animal transgenics and also to reduce the toxicity of oncolytic viruses and to attenuate viral vaccines [32]. whereas microrna-21 regulates the erk-map kinase signaling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function, mir-21 levels are increased selectively in fibroblasts of the failing heart, augmenting erk-map kinase activity through inhibition of sprout homologue 1. this mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. silencing of mir-21 by specific antagomir reduces cardiac erk-map kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction [14]. if the future proves that mirnas are major players in inflammatory disorders, a new generation of rna drugs could include small antisense rnas that antagonize mirna function [33]. such antagomirs can be synthetically made [34] or encoded by dna [35] and may specifically target an overexpressed mirna. as an example of such strategy, stabilized rna antagomirs has been recently followed up by the use of mir-10b-directed antagomirs in cancer treatment including anti-metastatic therapy [36]. downregulation of mir-10a may be one of the mechanisms by which collagens are highly deposited in keloid tissues. in this regard, both mir-10a inhibitors and ra receptor antagonists are promising effective strategy for targeting keloid therapies. conclusion in conclusion, micrornas regulate gene expression and modulate critical processes involved in keloid development. in keloid, a number of micrornas act as pro-fibrotic molecules and their upregulation induce fibroblast proliferation and collagen synthesis. others have anti-fibrotic function and their downregulation promote fibroblast proliferation and collagen synthesis. thus a balance between pro-fibrotic and anti-fibrotic micrornas has to be maintained in order to prevent keloid formation. various inflammatory processes involving il-6 signaling, tgf-beta signaling, ecm deposition, fibroblast proliferation and differentiation, and emt play important role in keloid pathogenesis. therapeutic strategies should consider methods to upregulate anti-fibrotic mirnas or downregulate pro-fibrotic mirnas or both in the management of keloid. conflict of interest the authors declare no conflict of interest. references 1. ma s, chang wp, fang rh. measurement of radiation-induced dna double-strand breaks in human diploid fibroblasts from keloid and normal skin by singlecell gel electrophoresis. plast reconstr surg 5:821-826, 1996. 2. syed f, ahmadi e, iqbal sa, singh s, mcgrouther da, bayat a. fibroblasts from the growing margin of keloid scars produce higher levels of collagen i and iii compared with intralesional and extralesional sites: clinical implications for lesional site-directed therapy. br j dermatol 164:83-96, 2011. 3. berman 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scherr m, venturini l, battmer k, schaller schoenitz m, schaefer d, dallmann i, ganser a, eder m. lentivirus-mediated antagomir expression for specific inhibition of mirnafunction. nucleic acids res 35:e149, 2007. 36. ma l, reinhardt f, pan e, soutschek j, bhat b, marcusson e, teruya-feldstein j, bell gw, weinberg ra. therapeutic silencing of mir-10b inhibits metastasis in a mouse mammary tumor model. nat biotechnol 28:341-347, 2010. http://www.ajecr.org/ 152 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(2):152-156 original article clinical assessment and echocardiography follow-up results of the children with acute rheumatic fever ahmet basturk1*, kazim oztarhan 2, sultan kavuncuoglu 2, cemal polat 3 1department of pediatric gastroenterology, faculty of medicine, akdeniz university, dumlupinar bulvari-campus, antalya, turkey 2department of pediatrics, istanbul kanuni sultan süleyman research and teaching hospital, i stanbul, turkey 3department of biochemistry, public health laboratuary, kütahya, turkey abstract. acute rheumatic fever (arf) is an inflammatory collagenous tissue disease which shows its cardinal signs in joints, heart, skin and nervous system while affecting whole connective tissue system more or less. this study was conducted in order to investigate the clinical pattern and severity of arf, echocardiographic findings and the course of the patients with heart valve involvement by studying the clinical and laboratory aspects of the patients diagnosed with arf according to updated jones criteria. the study included 214 patients diagnosed with arf for the first time between january 2005 and may 2008. all patients were scanned with doppler echocardiography (echo) between certain intervals. severity of carditis was grouped into 3 groups of mild, moderate and severe. the frequency of carditis was 57.9%, arthritis was 73.4%, chorea was 11.7% and erythema marginatum was 0.9% but no subcutaneous nodules. recovery was observed in 22% of the cases of isolated aortic insufficiency (ai), 50% of the cases with isolated mitral insufficiency (mi) and 80% of the cases with mitral and aortic insufficiencies together (mi+ai). recovery in isolated mi was significantly much more than recovery in isolated ai. however, recovery in ai was significantly much more than in mi in cases of mitral and aortic insufficiencies together. in conclusion, arf is a cause of acquired and preventable heart disease and it can be reversed through right diagnosis and appropriate treatment. isolated mitral insufficiency, isolated aortic insufficiency and both mitral and aortic insufficiency are observed during a valvular disease. remission among valvular diseases are most commonly in those with mitral insufficiency and remissions in both mitral and aortic insufficiency occur most commonly in aortic ones. regular prophylaxis is the key element for long term prevention of patients with arf. keywords: acute rheumatic fever, echocardiography, children, carditis, mitral insufficiency, aortic insufficiency introduction acute rheumatic fever (arf) is a systemic inflammatory disease that can involve heart, joints, skin, subepidermal tissues and brain. it is a result of a delayed reaction to acute pharyngeal infections caused by group a ß-hemolytic streptococci. arf can manifest with arthritis, carditis, subcutaneous nodules, and erythema marginatum and sydenham chorea in a certain amount of time (approx. 3 weeks) after the infection. arf doesn’t necessarily develop in every individual who had streptococcal pharyngitis. probability of arf developing is 0.5-3%. as the most important cause of mortality and morbidity of the disease is carditis, it can cause heart failure and death due to pancarditis during acute phase. however, most common result is development of permanent dysfunction of the affected heart valves like stenosis or insufficiency which happens years after the episode [1, 2]. although incidence is getting low in developed countries during the century, it is still a significant public health problem in developing countries [3]. t. duckett jones established criteria concerning arf and recurrences in 1944. these criteria, which are used today, were reviewed preserving the core features in 1965 and 1984 and in 1992 they took their latest form [4]. this study was conducted in order to investigate the clinical pattern and severity of the disease, echocardiographic findings and the course of the patients with heart valve involvement by studying the clinical and laboratory aspects of the patients diagnosed with arf according to updated jones criteria. materials and methods this study included 214 patients diagnosed with acute arf for the first time between january 2005 and may 2008 at the pediatric clinic of istanbul bakırköy obstetrics and pediatrics research and training hospital. approval by the ethics committee of the istanbul bakırköy obstetrics and pediatrics research and training hospital ___________________________________________________________ * corresponding author: ahmet basturk (drahmetbasturk@hotmail.com). http://www.ajecr.org/ mailto:drahmetbasturk@hotmail.com 153 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org figure 1 echocardiography follow-up findings in patients with carditis. (ref. no. 131). patients were diagnosed as arf according to the jones criteria updated in 1992. findings were recorded from the patient follow-up file. in all patients, complete blood count (cbc), erythrocyte sedimentation rate (esr), anti-streptolysin o (aso), c-reactive protein (crp) and throat swab cultures were studied. a 12-lead electrocardiogram (ecg) and teleradiography (tele) were done. cardiomegaly was graded according to the cardiothoracic ratio in teleradiographic study. cardiothoracic ratio over 0.5 was defined as cardiomegaly in older children. all patients were scanned with doppler echocardiography between certain intervals. echo at the time of the diagnosis was recorded as echo1, one after acute inflammation was recorded as echo2 and the last one done during follow-up was recorded as echo3. all echocardiographies were done by the same pediatric cardiology specialist. carditis was diagnosed according to physical examination and auscultatory findings. any valve insufficiency detected by doppler echo but normal according to physical examination or auscultation was not used as a diagnostic criteria alone. severity of carditis was grouped into 3 groups as mild, moderate and severe. carditis without cardiomegaly was considered as mild carditis, carditis with cardiomegaly without heart failure was assigned as moderate carditis and carditis with advanced cardiomegaly and congestive heart failure and/or pericardial involvement was set as severe carditis [5, 6]. grading of the valve insufficiency was done by measurement of the aortic and mitral jet length detected by doppler echo. grade 1 was ≤ 1.5 cm, grade 2 was 1.52.9 cm, grade 3 was 3-4,4 cm and grade 4 was ≥4,5cm [7]. grade 1 was grouped as mild, grade 2 as moderate and grade 3 and 4 as severe. other factors were fever (xillary temperature of 38oc or above), crp (≥5 g/l positive and < 5 g/l negative), prolonged pr interval (pr interval in ecg being longer than upper limit according to age and heart rate), prolonged qtc interval (qtc interval in ecg being longer than upper limit according to age and heart rate), elevation of aso (above 250 todd units), elevation of esr (20 mm/h or above) and leukocytosis (white blood cell count over 10000/mm3). statistical analysis the spss 15.0 statistical software (spss inc., table 1 distribution of major criteria table 2 distribution of minor criteria and supporting signs chicago, il) was used to analyze the data. during the analysis of the data, chi-square and analysis of variance was used in comparison along with definitive statistical methods (average, standard deviation, frequency). results were in 95% confidence interval and statistical significance was p <0.05. results age at presentation of patients with arf was between 5 and 15, average 11.09±2.61 and male to female ratio was 1/1. season of the presentations was most often in spring (36.9%) and least often summer (10.7%). family history of the disease was seen in 5.1% of cases and 94.9% of the cases had no family history. as reviewing distribution of the cases according to the major jones criteria, we’ve seen carditis in 57.9%, arthritis in 73.4%, chorea in 11.7%, erythema marginatum in 0.9% but no subcutaneous nodules (table 1). as reviewing distribution of the cases according to the minor jones criteria, we’ve seen fever in 65.4%, arthralgia in 50.5%, prolonged pr interval in 23.4%, elevation of one and/or two acute phase reactants in 69.6. aso elevation was seen in 93% of the patients, and throat cultures were isolated ai isolated mi mi+ai n o . o f p a ti e n ts major crit eria no. (%) isolat ed cardit is 31 (25.0) art hrit is 75 (35.0) chorea 15 (7.0) eryt hema marginat um 1 (0.5) subcut aneous nodules 0 (0) art hrit is-cardit is 82 (38.3) cardit is-chorea 10 (4.7) cardit is-eryt hema marginat um 1 (0.5) combined minor crit eria no. (%) fever 140 (65.4) art hralgia 108 (50.5) p rolonged p r int erval 50 (23.4) crp posit ivit y 63 (29.4) elevat ion of esr 149 (69.6) leukocyt osis 80 (37.4) elevat ion of aso 199 (93.0) p osit ive t hroat cult ure 23 (10.7) acut e phase react ant s support ing signs http://www.ajecr.org/ 154 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org positive in 10.7% of the patients. reviewing the distribution of the presenting symptoms, 86.9% of them had joint problems, 11.7% had involuntary movement, 9.3% had chest pain, 7.5% had trouble in breathing and 1.4% had racing heartbeat (table 2). we detected that carditis was accompanied by cardiomegaly in 30.6% of the patients, heart failure in 16.9% of them and pericardial effusion in 4% of the patients. of the total of 124 cases who had carditis, 69.3% had mild, 12.1% had moderate and 18.5% had severe carditis. as reviewing distribution according to echo1 findings in patients with carditis, 62.2% of them had isolated mitral insufficiency (mi), 32% had mitral and aortic insufficiencies together (mi+ai) and 5.7% had isolated aortic insufficiency (ai). while of the patients with aortic insufficiency, 90% had mild and 10% had moderate carditis, and of the patients with mitral insufficiency, 56.7% had mild, 24.7% had moderate and 18.6% had severe carditis. as reviewing distribution according to echo2 findings after acute inflammation in patients with carditis, 4.4% of the patients had isolated aortic insufficiency, 62.3% had isolated mitral insufficiency, 24.5% had mitral and aortic insufficiencies together and 8.8% had recovered. as reviewing distribution according to echo3 findings in patients with carditis, 4.4% of the patients had isolated aortic insufficiency, 62.9% had isolated mitral insufficiency, 19.5% had mitral and aortic insufficiencies together and 19.5% had recovered (fig. 1). we detected recurrences in 2 patients who had regular prophylaxis and 25 patients who had irregular prophylaxis. rate of recurrence was above 90% in patients who had irregular prophylaxis (p<0.05). one patient who had regular prophylaxis and 4 patients who had irregular prophylaxis were operated on. incidence of recurrence and surgery was higher in patients who had irregular prophylaxis (p<0.05). discussion acute rheumatic fever is responsible for many of the acquired heart diseases. sequela seen in adults holds its significance among valvular heart diseases [3]. the disease is commonly seen in ages between 5 to 15 years old. however, attacks have been reported in ages between 2 and 65 years old [2, 8]. different studies conducted in different times reported an incidence higher in males as though some studies reported an incidence higher in females [9]. in our study (108 females and 106 males) no significant difference between sexes were seen. arf is seen more commonly in winter and spring seasons when streptococcal infections are more common [10]. in our study, we had 79 patients seen in spring and 78 patients seen in winter, comprising 73.3% of all patients. arthritis is the most common sign of arf. typically, it affects bigger joints like knee, elbow, wrist and ankle. more than one joint is affected and arthritis is migratory [11]. in our study, arthritis was the most common major sign. arthritis was seen in 57.9% of the patients and affected different joints such as ankle in 80.3% of the patients, knee in 55.4%, elbow in 10.2% and others (hip, shoulder, and wrist) in 26% of them. carditis is the major sign, which is seen in 45%-50% of the patients and defines the progress of the disease, it can affect all layers of the heart. involvement is from endocardium to pericardium; without endocardial involvement, there cannot be pericardial or myocardial involvement [12]. endocardial involvement is seen as valvular insufficiency. while insufficiency is seen during acute illness, stenosis occurs due to fibrosis after years. most commonly affected valve is mitral valve. mitral insufficiency murmur is heard due to mitral involvement. second most commonly affected valve is aortic and insufficiency murmur is heard. tricuspid and pulmonary valves are affected rarely [10]. in our study, the incidence of carditis was 57.9 % which is consistent with the literature [12]. we’ve seen isolated carditis in 25% (n=31) of the patients; together with arthritis in 38.3% (n=82) of the patients, with chorea in 4.7% (n=10) of them and with erythema marginatum in 0.5% (n=1) of the patients. while first symptom which patients with carditis presents with is shortness of breath and chest pain there was significant difference with those without carditis (p<0.05). while cardiomegaly was detected by tele during first presentation in 69.4% (n=86) of the patients with carditis heart failure was detected in 16.9% of the patients and it is seen more in those with carditis significantly (p<0.05). there was no significant difference in prolonged pr interval in patients with carditis compared to those without. arthralgia and positive aso incidence in patients with carditis was significantly much more than in those without (p<0.05). esr elevation was significantly much more in patients without carditis (p<0.05). in our study, most commonly seen valvular lesion in patients with carditis was isolated mitral insufficiency 62.2% (99). 5.7% (n=9) of the patients had isolated aortic insufficiency, %32 (n=51) of the patients had aortic and mitral insufficiencies together. valvular lesions, which were mild in the beginning, regress and disappear in time [13, 14]. meira et al. [14] detected 61% regression in 6 month to 7 years old surveys among mild and moderate valvular lesions. also in our study, recoveries in transitions from echo1 to echo2 and echo2 to echo3 in isolated aortic insufficiency were equal and 22%. recoveries were seen 21% in transition from echo1 to echo2 and 50% from echo1 to echo3 in isolated mitral insufficiency. recoveries were seen 50% in transition from echo1 to echo2 and 80% from echo1 to echo3 in patients with both mitral and aortic insufficiency. all recoveries but one was seen in aortic insufficiency, one was for both. recovery in patients with isolated mitral insufficiency was significantly more than those with isolated aortic insufficiency during approx. 12-40 months follow-up, however, recovery in isolated aortic insufficiency was more than those with both mitral and aortic insufficiency. chorea is a late sign of arf. we’ve seen chorea in 25 patients in our study of whom 4.7% (10 patients) had carditis. the incidence of chorea was significantly higher http://www.ajecr.org/ 155 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org in females than in males (18 female and 7 male) (p<0.05). erythema marginatum is a big macular lesion that is located in trunk and proximal portions of the limbs during the progress of arf. it’s seen in 5% of the patients [15]. we’ve seen erythema marginatum in 0.9% (n=2) of our patients. we haven’t come across any subcutaneous nodules. prednisolone is used in the treatment of carditis in arf. salicylates are added to the treatment while titrating down the steroids in the last week of treatment [16]. it’s been suggested that the incidence of heart disease has not changed after steroid and aspirin treatment. however, steroid treatments achieved faster acute phase responses and decline of numbers of cases undergone surgery [17, 18]. all patients were treated with benzathine penicillin and administered secondary prophylaxis. all the patients who had arthritis and mild carditis was started aspirin treatment, those with moderate or severe carditis who were treated with steroids were given aspirin during titration. six patients who had congestive heart failure were treated with diuretics and 2 patients with heart failure was treated with digoxin. haloperidol was used in patients who developed chorea. secondary prophylaxis is the only and the most important preventive intervention that affects the progression of the disease and has a proven effectiveness. the most effective way of prophylaxis is intramuscular injections of benzathine penicillin every three weeks. prophylaxis is the most effective preventive method today. in our study, 27 patients presented with recurrence, 2 (7%) of them had regular prophylaxis but 25 (%93) of them had irregular prophylaxis. as seen in our study, most important factor in recurrence of arf is irregular prophylaxis. since there is a correlation between irregular prophylaxis and surgical intervention, 5 patients who had surgery were cases with recurrences. four of them had irregular prophylaxis but 1 patient had recurrence despite of regular prophylaxis. among patients who had surgery, 4 of them had severe mitral insufficiency and 1 had severe mitral insufficiency and mild aortic insufficiency. mitral valves were repaired in all patients who had surgery. probability of having carditis during recurring attacks was higher among patients who had carditis during the initial arf attack [10]. attacks occurred most commonly in first 5 years of follow-ups. most important factors in preventing valve replacement are patient’s education and compliance of prophylactic treatment along with proper dosage and timing. in conclusion, arf is a cause of acquired and preventable heart disease and it can be reversed through right diagnosis and appropriate treatment. prognosis of arf is mostly defined by carditis. mitral and aortic valves are most commonly affected during carditis. most commonly seen valvular diseases are isolated mitral insufficiency, both mitral and aortic insufficiencies and isolated aortic sufficiency, respectively. mitral and aortic insufficiencies can be fixed by appropriate treatment and regular prophylaxis. especially, isolated mitral insufficiency instead of aortic and aortic component of both mitral and aortic insufficiencies can recover. patients who had carditis in their first episodes have significantly high risks of having a recurring episodes. most important factor preventing recurrence of arf carditis is regular prophylaxis. there is a significant association between irregular prophylaxis and surgical intervention. most commonly valve replacement is performed during surgery. therefore, the most important element in preventing valvular replacement is appropriate dosage and perfectlytimed prophylaxis accompanied by patient education. conflict of interest the authors declare no conflicts of interest and no financial support. references 1. williams rc: acute rheumatic fever. rudy s, harris ed, sledge cb (eds.) kelley’s textbook of rheumatology, sixth edition, wb saunders company, philedelpia, 2001; pp 1529-1540. 2. carapetis jr, mcdonald m, wilson nj. acute rheumatic fever. lancet 366:155-168, 2005. 3. olivier c. rheumatic fever-is it still problem? j antimicrob chemother, 45:13-21, 2000. 4. shiffman rn. guideline maintenance and revision. 50 years of the jones criteria for diagnosis of rheumatic fever. arch pediatr adolesc med 149:727-732, 1995. 5. vasan rs, shrivastava s, vijayakumar m, narang r, lister bc, narula j. echocardiographic evaluation of patients vvith acute rheumatic fever and rheumatic carditis. circulation 94:73-82, 1996. 6. maheu b, costes po, lionet p, kamblock j, papouin g, mansourati j. et al. contribution of doppler echocardiography to the diagnosis of the fırst attack of acute rheumatic fever. arch mal coeur vaiss 88:18331839, 1995. 7. otto cm. valvular regurgitation: diagnosis quantitation and clinical approach. text book of clinical echocardiography. second edition. saunders company, 2000, pp 265-300. 8. tani ly, veasy g, minich l, shaddy re. rheumatic fever in children younger than 5 years old. is the presentation different? pediatrics 112: 1065-1068, 2003. 9. aron am, freeman jm, carter s. the natural history of sydenham’s chorea. am j med 38:83-95, 1965. 10. ayoub em. acute rheumatic fever. in: moss and adams’ heart disease in infants, children and adolescents. allen hd, gutgesel hp, clark eb, driscoll dj (eds). 6th ed. philadelphia: lippincott williams and wilkins, 2001, pp1226-1241. 11. harlan ga, tani ly, byington cl. rheumatic fever presentings as monoarticular arthritis. pediatric infect dis j 25: 743-746, 2006. 12. narula j, chandrasekhar y, rahimtoola s. diagnosis of active rheumatic carditis. the echoes of change. circulation 100: 1576-1581, 1999. 13. karaaslan s, demirören s, oran b, baysal t, başpınar o, uçar c. criteria for judging the improvement in subclinical rheumatic carditis. cardiol young 13: 500505, 2003. http://www.ajecr.org/ 156 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org 14. meira zma, goulart ema, colosimo ea, mota ccc. long term follow up of rheumatic fever and predictors of severe rheumatic valvar disease in brezilian children and adolescents. heart 91:1019-1022, 2005. 15. guidelines for the diagnosis of rheumatic fever. jones criteria, 1992 update. special writing group of the committee on rheumatic fever, endocarditis, and kawasaki disease of the council on cardiovascular disease in the young of the american heart association. jama 268:2069-2073, 1992. 16. bland ef, jones td. rheumatic fever and rheumatic heart disease. a twentyyear report on 1000 patients followed since childhood. circulation. 4:836-843, 1961. 17. cilliers am, manyemba j, salojee h. antiinflammatory treatment for carditis in acute rheumatic fever. cochrane database syst rev 2:cd003176, 2003. 18. visnavathan k, manjarez rc, zabriskie jb. rheumatic fever. curr treat options cardiovasc med 1: 253-258, 1999. http://www.ajecr.org/ 38 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(3):38-46 original article stem cell therapy as one of temporary measures for management of heart failure and pulmonary hypertension in children aris lacis 1 *, inguna lubaua 2 , andis lacis 2 , andrejs erglis 3 1 department of pediatric cardiology and cardiac surgery, university children hospital, latvia 2 riga stradins university, latvia 3 the latvian institute of cardiology, latvia abstract. favourable results of experimental studies on animals and several conditions in adult population indicate that bone marrow derived progenitor stem cell (bmpsc) transplantation may play a crucial role. nevertheless, little is known about possible implementation of the bmpsc transplantation in children, dilated cardiomyopathy and pulmonary hypertension in particular. an increasing understanding of the nature and processes of idiopathic dilated cardiomyopathy and pulmonary arterial hypertension in children, as well as the limited treatment options have led our research towards the use of stem cell transplantation in the management of these patients. we performed intramyocardial bmpsc transplantation in 6 patients (4 months to 17 years) with dilated cardiomyopathy. all patients underwent complete detailed examination before and after the procedure. all patients demonstrated an increase in lvef and degree of shortening of the left ventricular diameter between end-diastole and end-systole after the procedure. a decreased concentration of natriuretic peptide or lvdd on 2d and 3d echocardiography was observed in 5 and 3 of the 6 patients respectively. intrapulmonary bmpsc transplantation was performed in two patients (9 and 15 years old) with severe pulmonary hypertension due to uncorrected large ventricular septal defects. both patients showed improvement in lungs’ vascularization. no serious periprocedural side effects were observed. if applied wisely, the stem cell therapy appears to be a safe and effective way for stabilization of critically ill patients with both severe pulmonary hypertension and idiopathic cardiomyopathy. keywords: stem cells, idiopathic dilated cardiomyopathy, pulmonary arterial hypertension, intramyocardial administration introduction dilated cardiomyopathy is a serious problem in pediatric cardiology field. despite the relatively low incidence of 0.57 to 2.6 per 100,000 children, the mortality rate is high. one third of patients die within the first year after diagnosis [1, 2]. up to 40% of these patients are defined as idiopathic dilated cardiomyopathy (idcm) characterized by ventricular dilatation and systolic dysfunction of unknown cause. the prognosis is poor in pediatric patients and survival appears to be related to the degree of systolic dysfunction. conventional medical therapy often provides a short term relief of symptoms but does not improve the outcome of the disease. however, recent clinical studies [3, 4] have suggested bone marrowderived autologous mononuclear cells or circulating progenitor cells [5, 6] as a promising therapy option for these patients. however, the most effective and safest way for delivery of these cells to the target area still needs to be established. pulmonary arterial hypertension (pah) is characterized by increased pulmonary vascular resistance resulting in extensive heart structural changes, limiting patients exercise capacity and eventually leading to right heart failure and death [7, 8]. a significant proportion of patients with congenital heart disease (chd), in particular those with systemic-topulmonary shunts, will develop the most advanced form of pah if left untreated. in these cases pah is characterized by obstruction of small pulmonary arteries leading to progressive increase in vascular resistance. this increases right ventricular afterload and consequently results in right ventricular failure. intima and media proliferation and its consequent pulmonary vascular obstruction are considered to be the key element in the pathogenesis of pah. vasoconstriction, vascular remodeling and thrombosis are factors that increase pulmonary vascular resistance in pah [9]. we hypothesize based on the data obtained from experimental and clinical studies, that intrapulmonary implantation of bone marrow derived progenitor stem cell (bmpsc) increases the vascular bed in the pulmonary circulation leading to alleviation of pah. locally implanted stem cells may trigger the neovascularization process in the lung, potentially leading to a decrease of the mean pulmonary artery pressure. justification for the use of intramyocardial and intrapulmonary stem cell implant ___________________________________________________________ * corresponding author: andis lacis, md (andis.lacis@rsu.lv). mailto:andis.lacis@rsu.lv 39 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org figure 1(a) schematic positioning of transcutaneous (1) and intra myocardial (2) punction. (b) injection of cell suspension into the apex cordis. tation was systematically discussed and planned by a team of cardiothoracic surgeons and cardiologists. materials and methods we used transdermal intramyocardial administration of bmpsc in critically ill patients with idiopathic dilated cardiomyopathy. all patients in this study gave informed consent and the study was approved by the central committee of medical ethics at the ministry of health. this innovative technique if combined with ultrasound monitoring offers the possibility for delivery of stem cells right into the target area in a safe and effective way. in patients with severe pulmonary hypertension we used intrapulmonary delivery of stem cells. for assessment of treatment results we used various visual diagnostics as well as functional and laboratory tests. preparation of bone marrow aspirate bone marrow (5 to 30 ml) was aspirated from iliac crest and bmcs were isolated ranging from 17 to 122 million bmcs. samples for flow cytometry were taken from 15 ml of mononuclear cell fraction and prepared for transplantation. preparation of the isolated cells was carried out using stem-kittm reagents (cat. nr. im3630; beckman coulter), that contained cd34-pe, cd45-fitcm isotype control, 7aad (viability dye) and stem-count fluorospheres. facs analysis was performed on fc-500 (beckman coulter) and analyzed using cxp software. each measurement contained at least 50,000 events. maximum number of events was 100,000. the numbers of cells/μl were calculated for a total number of cd34+ cells within the transplantation material. measurements with less than 50,000 events were excluded from the study. intramyocardial transplantation of stem cells the procedure was performed under general anesthesia in the operating theatre, positioning a patient horizontally on the back. we used the 0.95 mm × 220 mm optimed chiba needle. similar to the technique of pericardial puncture, we used the transcutaneuos subxiphoid approach, poking through the skin (dot (1) in fig. 1a) followed by gentle advancing toward the cardiac silhouette until a sligh figure 2 echocardiographical visualization of transcutaneous intramyocardial injection of stem cells. slight negative pressure was felt, searching for the apex cordis (dot (2) in fig. 1b). when the apex cordis was reached, under echocardiographical control (hewlettpackard sonos 4500) we performed an injection of 1 ml of cell suspension, subsequently followed by a second dose of 1 ml suspension with 1-2 min interval in between. a rationale to divide the injectable volume into two doses was based on concerns about a potential risk of barotrauma in myocardial tissues if the volume of a single dose would be too high. injection of cell suspension was controlled throughout the procedure via the us monitoring (fig. 2). the most challenging step of the procedure was simultaneous movement of the us probe and the needle towards the apex cordis. a special attention has to be paid to risks related to potential penetration of the ultra-thin wall of the left ventricle. shortly after injection of the cell suspension, we performed an additional us control to visualize the immediate results of the procedure. in some cases we also used radiographic visualization for further control for the short-term effects of the procedure. analgesic agent was administered as needed. figure 3 intravasal injection of the stem cells was performed by catheterization of both pulmonary arteries via superior vena cava (dots 1 and 4). intrapulmonary transplantation of stem cells intravasal injection of the stem cells was performed by 40 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org table 1 echocardiographic and heart failure parameters of the patients abbreviations: a: parameters before the procedure; p: parameters after the procedure; lvddleft ventricular diastolic dimension; fsfractional shortening; efejection fraction; lvdevleft ventricular end diastolic volume; mvr mitral valve regurgitation area; ntprobnpn-terminal of the prohormone brain natriuretic peptide catheterization of both pulmonary arteries via superior vena cava (dots 1 and 4, fig. 3). for delivery of stem cells right into the tissues of the upper and lower lobes (dots 2 and 3, fig. 5) of the right lung, we used a standard thoracocenthesis technique, followed by puncture (0.95 mm × 220 mm optimed chiba needle) of respective lung structures and injection of 1 ml of cell suspension, performed under chest radiological control. no periprocedural complications were observed. results we performed intramyocardial bmpsc transplantation in 6 patients (4 months to 17 years old) with dilated cardiomyopathy. all patients underwent quite detailed examination before and after the procedure. for monitoring the therapeutic response in patients with acute decompensated congestive heart failure we used clinical measures, including symptomatic relief of dyspnea, weight change, fluid balance, and resolution of s3 cardiac sounds, followed by other measurements to objectively follow up therapeutic responses, as illustrated in table 1. as shown in table 1, all 6 patients demonstrated an increase in lvef after the procedure. all patients after the procedure demonstrated an increase in the degree of shortening of the left ventricular diameter between enddiastole and end-systole. serial natriuretic peptide measurements revealed a decreased concentration after the procedure in 5 of the 6 patients. a decrease of lvdd on 2d and 3d echocardiography was observed in 3 of the 6 patients. we performed intrapulmonary bmpsc transplantation in two patients (9 and 15 years old) with severe pulmonary hypertension due to uncorrected large ventricular septal defects. both patients underwent radionuclide scintigraphy before the procedure, followed by re-examination at 6, 12, defects. both patients unde 24 and 36 months after that. latest test results showed improvement in lungs’ vascularization. no serious periprocedural side effects were observed. for monitoring the therapeutic response, we used a lung perfusion scintigraphy (lps) and a diagnostic imaging procedure that records the distribution of pulmonary arterial blood flow. for the lps, we used a radiopharmaceutical tc-99m-maa (macro-aggregated albumin) with siemensecam 2-head &-camera after i/v injection. spect was used to obtain a 3-dimensional evaluation of the perfusion. lung scans using a spect technique examination results in both patients appeared to be very similar therefore we decided to include in this report scan series just from one patient. scans revealed perfusion defects in the upper dorsal and lateral segments of the right lung. slightly lower perfusion was detected also in the upper segments of the left lung (fig. 4). the second study performed 6 months after the stem cell implantation procedure demonstrated remarkable improvement of perfusion in the upper segments of the right lung, as well as a small positive dynamics in the lateral segments of the right lung (fig. 5). further investigations carried out 18 and 32 months after the stem cell transplantation respectively (figs. 6 and 7) demonstrated a trend toward improving perfusion of the right lung. the right one in particular. none of the consecutive studies performed revealed considerable changes in perfusion of the left lung after the stem cell implantation. discussion despite a considerable number of studies in respective fields, the role of stem cell transplantation in restoration of myocardial and pulmonary tissues remains to be clarified. there is ample extensive research data available on factor patient 1 patient 2 patient 3 patient 4 patient 5 patient 6 a p a p a p a p a p a p lvdd (mm.) 47 41 40 42 55 55 45 41 33 29 44 46 fs (%) 5 22 21 22 18 28 7 29 21 40 21 26 lvef (%) 11 44 43 46 39 59 16 53 42 69 41 52 lvdev (ml) 63 47 87 93 177 141 85 78 47 39 87 41 mvr (cm 2 ) 2.7 0.4 0.56 0.5 1.0 0.5 0.7 0.6 nt-probnp (pg/ml) 1500 76 255 136 155 <20 4189 1469 1944 656 883 1784 41 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org figure 4 lung perfusion before the stem cell transplanttation. arrows (in red) indicate the areas with decreased lung perfusion. figure 5 lung perfusion 6 months after the stem cell transplantation. arrows (in red) indicate the areas with decreased lung perfusion. potential role of stem and progenitor cells in the management of ischemic heart disease. some investigators report the potential role of bone marrow cells to promote paracrine effects in the ischemic tissues (e.g. secretion of angiogenic factors), and suggest that paracrine signaling, rather than cell incorporation, promotes functional recovery [10, 11]. others conclude that progenitor cells have the capability to home myocardium in response to ischemia. cell adhesion markers, in particular integrins, play an important role in the trafficking of stem cells to myocardium [12]. in addition, damaged myocardium secretes several chemokines and growth factors that recruit these precursor cells to the heart. several studies have shown the role of different growth factors involved in the homing of stem cells to ischemic myocardium including stem cell factor [13], vascular endothelial growth factor [14], neural growth factor [15], granulocyte-colony stimulating factor [16], insulin-like growth factor [17], hepatocyte growth factor [18], and fibroblast growth factor [19]. several experimental studies have established a role of endothelial nitric oxide (enos) synthase as an essential component for neovascularization [20]. evidently, enos influences recruitment of stem and progenitor cells which may be considered contributing to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic nitric oxide bioactivity. erythropoietin has also been shown to improve cardiac function by inducing neovascularization. investigators suggest that this neovascularization appears to be related to increased mobilization and incorporation of bone marrow derived endothelial progenitor cells. erythropoietin has been shown to lead to preferential homing of endothelial progenitor cells to the ischemic border zone of myocardial infarcts resulting in improved microvascularization of ischemic cardiac tissue [21]. it still remains to be clarified which route of administration of stem cells is the safest and most effective. as reported by schächinger et al., in patients after myocardial infarction undergoing intracoronary infusion of 111 in-oxine-labeled proangiogenic progenitor cells, a substantial amount of radioactivity is detected for several days in the heart, indicating homing of progenitor cells to the myocardium. yet the amount of proangiogenic progenitor cells retained in the heart decreased progressively with time after the acute myocardial infarction. the study data suggest that proangiogenic progenitor cells preferentially home to extensive acute myocardial infarcts characterized by low viability and reduced coronary flow reserve [22]. schots et al. reported on rapidly diminishing concentration of intracoronaryinjected cd133+ peripheral blood progenitor cells in two patients, where detailed analysis showed 6.9% to 8.0% (after 2 hours) and 2.3% to 3.2% (after 12 hours) residual radioactivity at the heart [23]. this data may raise concerns whether intravascular injection of stem cells secures sufficient concentration in target tissues causing expected favorable effects. nevertheless it is equally true that there is no established relationship between exposure time and documented clinical efficacy of injected stem cells. another concern regarding intracoronary infusion of stem cells regards possible risks related to the procedure. de rosa et al., based on their study results concluded that intracoronary infusion of progenitor cells can be performed with adequate safety in patients with acute myocardial infarction or chronic heart failure, though cell-based based therapies require intracoronary or intracardiac instrumentation, potentially associated with periprocedural 42 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org figure 6 lung perfusion 18 months after the stem cell transplantation. arrows (in red) indicates the areas with decreased lung perfusion. ral risks such as vessel injury in the access site and coronary complications [24]. it is still not clear what do stem cells have to do with pulmonary hypertension but it is an area of great interest and much ongoing research. there are numerous publications showing remarkable benefits using various kinds of stem or progenitor cells in experimental models of pah. however, as humans are concerned, there is quite limited clinical experience with progenitor cell therapy in patients with pah, because very few definitive and rigorously designed trials have been performed so far. thus, it would be fair to say that the potential benefits and risks of progenitor cell therapy for pah are largely unknown. therefore, it has been widely recognized that this type of therapy may be offered only in the context of a research study that will help provide these answers. prior to the stem cell treatment it is necessary to assess possible advantages of available conventional treatment, evaluating expected benefits and risks, as well as to assess the possible ethical problems. although important advances in symptomatic treatments have occurred, many lung diseases including emphysema, pulmonary fibrosis, cystic fibrosis, and others have no cure [7]. a conventional therapy of pah consists of non-specific drugs including oral anticoagulation and diuretics as well as pah specific therapy, including prostanoids (epoprostenol, trepoprostenil, iloprost), endothelin receptor antagonists (bosentan, ambrisentan) and phosphodiesterase type 5 inhibitors (sildenafil, tadalafil) [8]. one of the most commonly used non-medical treatment strategies is atrioseptostomy, creating an artificial communication to decrease the right heart volume. the surgical creation of a right-left shunt decreases right auricular figure 7 lung perfusion 30 months after the stem cell transplantation. arrows (in red) indicate the areas with decreased lung perfusion. pressure and increases systemic blood flow, followed by reduction in right ventricular wall tension. however, atrioseptostomy has never been studied in controlled clinical trials and its role on long term survival has not been established. based on the data published by several investigators, it can be concluded that immediate mortality is high, reaching 14% during the first week, particularly in the case of severe desaturation and right heart failure [2527]. lung transplantation was historically the treatment of choice for severe pah and remains as such if medical treatments are insufficient [28]. however, this particularly heavy surgery can be proposed only to a minority of patients suffering from pah. moreover, long-term benefits remain disappointing, partly due to complications by acute and chronic rejection requiring lifelong immunosuppression with approximately 50% survival at 5 years, partly due to the donor graft induced antigenicity and extremely common infections, owing to the immunological and anatomical features of the lungs [29, 30]. besides, there is a critical shortage of donor lungs. all the above-mentioned points suggest that new approaches for lung diseases, including pah are desperately needed. there have been convincing results obtained from experimental studies with regeneration of damaged pulmonary vasculature by exogenously administered stem cells in dogs [30]. according to the researchers, neovascularization in the lung could increase the volume of the vascular bed in the pulmonary circulation and thus reduce the development of ph, presuming that epcs might be a potential cell source for neovascularization. the results obtained led the investigators suggest that epc transplantation into the lung is effective at preventing the progression of dehydromonocrotaline-induced ph in dogs, 43 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org posing a new therapeutic option for ph. similar results have been obtained from studies with rats. using a similar model of pah (monocrotaline-induced), the authors achieved restoration of the microvasculature structures and function of the artificially damaged lungs. the authors conclude that the regeneration of lung vascular endothelium by injection of progenitor cells may represent a novel treatment paradigm for patients with pah [31]. similar conclusions have been made based of results obtained from many other studies [32-34]. the latest data show that paracrine effects and secretion of growth factors are mainly responsible for stimulation of native angiogenesis, not just the epcs as was suggested by earlier studies. anyway, whatever mechanism(s) is behind these alterations, both anatomic and physiologic improvement in pulmonary hypertension has been clearly documented in animal models. it has been established that autologous implantation of bone marrow derived progenitor or stem cells leads to structural repair and regeneration of the lung. furthermore, currently very few studies on effects of autologous stem cell administration to ph in humans have been performed [35]. however, preliminary clinical trials using epc-based therapies in patients with pulmonary hypertension show benefit of this approach, thus revealing epcs as potential therapeutic targets [36-38]. in addition, information is available on two ongoing trials of cell therapy in pah patients, which are being conducted in china and canada, the results of which will be available in the coming years. therefore, many questions still have to be answered. for instance, the question about the best route of administration of the stem cells in patients with ph still remains controversial. it is not yet established whether these cells can engraft and acquire phenotype of structural lung cells following either systematic or intratracheal or local injection. it is also not clear which is the best cell source (bone marrow versus peripheral blood) and the best cell type (endothelial or haematopoietic progenitor cells or msc) to be used. it is still not established whether cell therapy is effective in idiopathic pah alone or in association with ph as well, and which is the best disease stage for cell therapy to be applied. data from experimental studies suggest that bone marrow cell transplantation reduces the development of ph by increasing vascular beds in pulmonary circulation [33, 38]. although the underlying mechanism is not yet determined, several factors are expected to contribute to cellular differentiation, transdifferentiation, paracrine and cell-cell effects [39]. these results led to human studies of pro-angiogenic cell transplantation, demonstrating significant clinical improvement in patients with pah [37, 38]. however, the trial design was limited by the fact that it included only a single 12-week follow-up, lack of sham treatment, and lack of blinding. these studies have now spurred a randomized controlled trial investigating enos transfected proangiogenic cells as a therapy for pulmonary hypertension (clinicaltrials.gov identifier: nct00469027). the above studies demonstrate a possible role for autologous angiogenic cell transplant in the treatment of pah and suggest that subsets of pro-angiogenic cells may have a protective application in pah. however, since proangiogenic hematopoietic cells do not permanently incorporate into the endothelium, the long-term effects of such treatment are unknown. cell therapies using various stem cells have been extensively evaluated. available research data indicate possible reparative roles of exogenously administered stem cells, demonstrating potential for structural repair for damaged lungs, notably repair of damaged lung vasculature through paracrine effects of circulating endothelial progenitor cells (epcs). these studies have also demonstrated potent immunomodulatory effects of adult bone marrow-derived mesenchymal stromal cells (mscs) in a variety of inflammatory and immune lung diseases. these initial observations have led to a cautious initial but growing exploration of epcs and mscs in clinical trials of pulmonary hypertension. mesenchymal stem cells (mscs) are the most extensively evaluated candidates for clinical cell-based therapy. many clinical trials using mscs have been registered and are ongoing. autologous mscs are easily isolated from the bone marrow and other tissues. mscs are expected to reduce inflammation and promote the repair process. these beneficial effects are thought to be based on the ability of mscs to modulate the immune system and their capacity to produce growth factors and cytokines, such as keratinocyte growth factor, hgf, and prostaglandin e2 [40]. since their discovery about a decade ago, epcs also have been subjected to intensive investigation [41]. however, as research opened deeper insights into epcs biology, the enthusiasm of the pioneer era has been damped in favor of a more critical view. recent studies are focused on three major questions: the fact that the number of epcs in peripheral blood is exceedingly low has consistently raised suspicion whether these cells can plausibly have an impact on physiological or pathophysiological processes. secondly, whereas the key role of epcs in tumourogenesis has been strongly emphasized by various groups in the past, recent publications are challenging this hypothesis. thirdly, the lack of consensus on epc-defining markers and standardized protocols for their detection has repeatedly led to problems related to comparability among papers. although there is sufficient evidence supporting the endobronchial [42] and intravenous [22, 23] administration routes, we suppose they are not optimal. as recognized by the investigators themselves, an assumption that endobronchial infusion of stem cells may lead to morphological alterations in the vascular bed of the lung is highly speculative and needs further exploration before to apply in critically ill patients suffering from chronic lung disorder [42]. we support an argument, that an acceptable safety profile is very important criteria when thinking about innovative treatment options. however, a safety profile should not be the main or only criteria to consider in case of rapidly deteriorating ph when available conventional treatment strategies have failed. 44 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org in fact, available scientific data from both experimental and clinical studies suggest that concentration of implanted stem cells in the target tissues rapidly decreases shortly after the intravascular injection. poor uptake of stem cells is explained by recirculation of the implanted cells by the blood flow [43, 44]. substantial experimental evidence points to the role of the circulating progenitor cells in vascular pathology, which characterizes chronic ph. however, it is widely recognized that more study is needed to determine the types of progenitor cells involved in vascular remodeling and their specific functions, once they take up residence in the vessel wall. much study is needed to determine the factors involved in their recruitment and retention [45-47]. it has been reported, that the hemodynamic alterations occuring with single-lung transplantation, that is, normalization of pressure and blood flow preferentially directed to the transplanted lung, may result in regression of the characteristic findings of ph in the native lung for primary ph patients [48]. we hypothesize that clinical and radiographical improvement of pah after intrapulmonary implanttation of stem cells can be caused by improvement of lung perfusion due to neovascularization of lung tisses. as demonstrated by the case presented in this article, intrapulmonary injections directly into the lung parenchyma may cause even better results if compared with injection into the lung arteries. these results may be explained by higher concentration of the injected stem cells in the target tissues. as the mechanism of action is still not clear, we also speculate that injected cells trigger a neovascularization process in the lung, affecting much larger areas than those directly exposed to the implanted cells. the results achieved might be explained by the upregulation of hypoxia-inducible factor in vascular cells, leading to the production of bone marrow-mobilizing factors that recruit pro-angiogenic progenitor cells to the pulmonary circulation where they contribute to angiogenic remodeling of the vessel wall in pah [49]. it still remains to be explored, if and why the effects of intrapulmonary injection of stem cells are not extended to the contralateral lung. in summary, it has to be recognized that the optimal route of administration of stem cells is not known. the optimal dosage regimen, including the lower effective doses, is also unclear. the true potential for different types of stem cells (mscs, escs) is not clear either. issues also remain regarding what group of ph patients are most likely to benefit from treatment, at what point in the disease is treatment most likely to be successful [50,51]. no long term benefits have been established as well. all the mentioned above suggest that more studies are necessary to answer at least the most important questions. until the prognosis for pah remains unsatisfactory, stem cell therapy poses a new promising therapeutic option, requiring deeper understanding of underlying processes and long term effects caused by it. conclusions if applied wisely, the stem cell therapy appears to be a safe and effective way for stabilization of critically ill patients with both severe pulmonary hypertension and idiopathic cardiomyopathy. this method provides additional opportunities for symptomatic treatment and serves as a bridge for potential heart or lung transplantation. potential benefits provided by the stem cell therapy should encourage multidisciplinary teams to think beyond the conventional and develop newer innovative strategies to obtain control of deteriorating clinical situations. conflicts of interest the authores declare no conflict of interest. references 1. alvarez j, wilkinson jd, lipshultz se. the pediatric cardiomiopathy registry study group. outcome preditors for pediatric dilated cardiomyopathy: a systemic review. prog pediatr cardiol 25-32, 2007. 2. arola a, tuominen j, ruuskanen o, jokinen e. idiopathic dilated cardiomyopathy in children: prognostic indicators and outcome. pediatrics 101:369-376, 1998. 3. rupp s, bauer j, tonn t, et al. intracoronary administration of autologous bone marrow-derived progenitor cells in a critically ill two-yr-old child with dilated cardiomyopathy. pediatr transplant 13:620-623, 2009. 4. limsuwan a, hongeng 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et al. essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. nat med 9:1370-1376, 2003. 21. westenbrink bd lipsic e, van der meer p, et al. erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization. eur heart j 28:20182027, 2007. 22. schächinger v, aicher a, döbert n, et al. pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium. circulation 118:1425-1432, 2008. 23. schots r, de keulenaer g, schoors d, et al. evidence that intracoronary-injected cd133+ peripheral blood progenitor cells home to the myocardium in chronic postinfarction heart failure. exp hematol 35:1884-1890, 2007. 24. de rosa s, seeger fh, honold j, et al. procedural safety and predictors of acute outcome of intracoronary administration of progenitor cells in 775 consecutive procedures performed for acute myocardial infarction or chronic heart failure. circ cardiovasc interv 6:44-51, 2013. 25. sandoval j, gaspar j, pulido t, et al. graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. a therapeutic alternative for patients nonresponsive to vasodilator treatment. j am coll cardiol 8:297-304, 1998. 26. rothman a, sklansky ms, lucas vw, et al. atrial septostomy as a bridge to lung transplantation in patients with severe pulmonary hypertension. am j cardiol 8:682686, 1999. 27. kurzyna m, dabrowski m, bielecki d, et al. atrial septostomy in treatment of end-stage right heart failure in patients with pulmonary hypertension. chest 8:977-983, 2007. 28. sitbon o, humbert m, simonneau g. primary pulmonary hypertension: current therapy. prog cardiovasc dis 8:115-128, 2002. 29. hosenpud jd, bennett le, keck bm, et al. the registry of the international society for heart and lung transplantation: eighteenth official report-2001. j heart lung transplant 8:805-815, 2001. 30. takahashi m, nakamura t, toba t, et al. transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs. tissue eng 10:771-779, 2004. 31. zhao yd, courtman dw, deng y, et al. rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and enos gene therapy in established disease. circ res 96:442-450, 2005. 32. lam cf, liu yc, hsu jk, et al. autologous transplantation of endothelial progenitor cells attenuates acute lung injury in rabbits. anesthesiology 108:392-401, 2008. 33. yoshida h, kitaichi t, urata m et al. syngeneic bone marrow mononuclear cells improve pulmonary arterial hypertension through vascular endothelial growth factor upregulation. ann thorac surg 88:418-424, 2009. 34. kanki-horimoto s, horimoto h, mieno s, et al. implantation of mesenchymal stem cells overexpressing endothelial nitric oxide synthase improves right ventricular impairments caused by pulmonary hypertension. circulation 114:181-185, 2006. 35. farkas l, kolb m. vascular repair and regeneration as a therapeutic target for pulmonary arterial hypertension. respiration 85:355-364, 2013. 36. fadini gp, avogaro a, ferraccioli g, agostini c. endothelial progenitors in pulmonary hypertension: new pathophysiology and therapeutic implications. eur respir j 35:418-425, 2010. 37. wang xx, zhang fr, shang yp, et al. transplantation of autologous endothelial progenitor cells may be beneficial in patients with idiopathic pulmonary arterial hypertension: a pilot randomized controlled trial. j am coll cardiol 49:1566-1571, 2007. 38. zhu jh, wang xx, zhang fr, et al. safety and efficacy of autologous endothelial progenitor cells transplantation in children with idiopathic pulmonary arterial hypertension: open-label pilot study. pediatr transplant 12:650-655, 2008. 39. hayes m, curley g, ansari b, laffey jg. clinical review: stem cell therapies for acute lung injury/acute respiratory distress syndrome hope or hype? crit care 16:205, 2012. 40. kubo h. concise review: clinical prospects for treating chronic obstructive pulmonary disease with regenerative approaches. stem cells transl med 1:627631, 2012. 46 am j exp clin res, vol. 1, no. 3, 2014 http://www.ajecr.org 41. resch t, pircher a, kahler cm, et al. endothelial progenitor cells: current issues on characterization and challenging clinical applications. stem cell rev 8:926939, 2012. 42. tzouvelekis a, paspaliaris v, koliakos g, et al. a prospective, non-randomized, no placebo-controlled, phase ib clinical trial to study the safety of the adipose derived stromal cells-stromal vascular fraction in idiopathic pulmonary fibrosis. j transl med 11: 171, 2013. 43. lacis a, lubaua i, erglis a, et al. management of idiopathic dilated cardiomyopathy with intramyocardial stem cell transplantation in children: a retrospective study of 7 patients. science j of clin med 2:129-133, 2013. 44. barbash im, chouraqui p, baron j, et al. systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. circulation 11:863-868, 2003. 45. peinado vi, ramirez j, roca j, et al. identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. am j respir cell mol biol 34:257-263, 2006. 46. firth al, yao w, ogawa a, et al. multipotent mes enchymal progenitor cells are present in endarterectomized tissues from patients with chronic thromboembolic pulmonary hypertension. am j physiol cell physiol 298:1217-1225, 2010. 47. yeager me, frid ma, stenmark kr. progenitor cells in pulmonary vascular remodeling. pulm circ 1:3-16, 2011. 48. levy nt, liapis h, eisenberg pr, et al. pathologic regression of primary pulmonary hypertension in left native lung following right single-lung transplantation. j heart lung transplant 20:382-384, 2001. 49. duong h, erzurum s, asosingh k. pro-angiogenic hematopoietic progenitor cells and endothelial colony forming cells in pathological angiogenesis of bronchial and pulmonary circulation. angiogenesis 14:411-422, 2011. 50. majka s, burnham e, stenmark kr. cell-based therapies in pulmonary hypertension: who, what, and when? am j physiol lung cell mol physiol 301: 9-11, 2011. 51. warburton d, perin l, defilippo r, et al. stem/progenitor cells in lung development, injury repair, and regeneration. proc am thorac soc 5:703-706, 2008. 341 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2019;6(2):341-345 case report evaluation of autologous adipose-derived mesenchymal stem cell therapy in a patient with acute ischemic cardiomyopathy masatatsu mishima1, xiaolan liu2 1miki medical corporation sun field clinic, time24 building, aomi, koto-ku, tokyo, japan 2world academy of anti-aging and regenerative medicine, imperial tower, tokyo, japan abstract. adipose-derived mesenchymal stem cells (admsc) is an innovative approach for the treatment of a range of diseases that are not responsive to standard therapies. their promising role in tissue engineering and ability to modulate the immune system are attractive. admsc can differentiate into endothelial cells, myocytes, chondrocytes and osteoblasts. our aim was to investigate safety and efficacy of intravenous autologous admsc in a patient with acute myocardial infarction. admsc treatment was associated with improved recovery of the left ventricular function (lvef), electrocardiographic (ecg) findings and serum bnp level in this patient. admsc may be considered to be one of future therapeutic agents for diseases that cannot be cured by conventional therapeutic methods. keywords: adipose-derived mesenchymal stem cells, autologous, myocardial infarction, cell therapy introduction it has been reported that bone marrow-derived hematopoietic and mesenchymal stem cells are administered to the left ventricular endocardial site of myocardial infarction patients to restore left ventricular ejection fraction (lvef) and myocardial infarction sites. [1-6]. it has also been shown that cardiac function has been improved by bone marrow-derived mesenchymal stem cells from hematopoietic cells administered via intracardial wall in myocardial infarction patients [7] or by intravenous administration [8]. this indicates that hematopoietic and mesenchymal stem cells promoted cardiac function improvement in myocardial infarction. on the other hand, it has been reported that cardiac function is restored even when adipose-derived mesenchymal stem cells (admsc) are administered to the intracardial inner wall of a myocardial infarction patient in addition to bone marrow origin [9]. there are few reports on the treatment of myocardial infarction using admsc, and cell therapy by intravenous administration is not well known. therefore, adipose tissues of patients with acute myocardial infarction were collected, stem cells were extracted and expanded, and intravenously administered to the same patient. as a result, the improvement of heart function was observed which is reported here. patient and treatment a 47-year-old male patient had chief complaint of sudden chest pain. he was admitted and hospitalized for treatment of acute myocardial infarction. the patient’s family history included his father with hypertension and heart disease, and nothing in particular from his mother. the patient had high blood pressure, dyslipidemia, obesity and alcohol consumption (3 cans of beer / week) but no smoking and diabetes mellitus. he was taking medicines, carvedilol and enalapril maleate. the patient received cardiac catheterization and stent therapy followed by thrombolytic therapy using antithrombotic drugs (clopidogrel sulfate, and baby aspirin) and radical scavenger. adipose-derived mesenchymal stem cells the procedure for isolation and propagation of admsc had been reported in details in our previous study [10]. after informed consent and approval of institutional review board, the adipose tissue was collected from abdominal (peri-umbilical) area of the same patient. stromal vascular fraction containing mesenchymal stem cells which were capable of differentiating into adipocytes, endothelial cells, bone cells or cardiomyocytes were prepared by treating the adipose tissue with collagenase and separation on concentrator (cytori manufactured by celution®). when this cell group is cultured in our proprietary culture medium, sf-dot (developed by biomimetics sympathies inc., tokyo, japan), cells other than mesenchymal stem cells die out, so that only admsc proliferate. this culture medium does not contain any animal-origin serum. ___________________________________________________________ * corresponding author: masatatsu mishima, md, phd (e-mail: m.mishima@sunfield-c.com). http://www.ajecr.org/ mailto:m.mishima@sunfield-c.com 342 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org figure 1. changes in bnp values of myocardial infarction patients. autologous admsc was administered at the time points indicated by red arrows. intravenously administered 54 million (2011/11/30), 36.8 million (2013/03/07) and 32.8 million (2013/04/04) cells. the values of bmp continued to decrease after admsc administrations. bnp (b-type natriuretic peptide) test b-type natriuretic peptide (bnp) is an excellent serum marker as a prognostic index for heart failure due to coronary artery injury. it can be easily measured in patient’s serum [11-17]. therefore, over 4 years after administration of admsc for treatment of myocardial infarction, the effect of admsc was examined mainly from the bnp concentration level. results after emergency hospitalization, the patient was diagnosed as having acute myocardial infarction and received cardiac catheterization and stent therapy followed by thrombolytic therapy using anti-thrombotic drugs. the patient’s serum bnp concentrations before and after admsc administrations at indicated time points are summarized in fig. 1. the patient received admscc at the time points indicated by red arrows. on september 12, 2011, the bnp value was 87.9 pg / ml. on november 30, 2011, 54 million admsc were administered intravenously. on september 12, 2012, cardiac function recovered to 98% as judged by electrocardiogram and echocardiography examinations. on march 7, 2013, 36.8 million admsc were intravenously administered again. finally, on april 4, 2013, 32.8 million admsc were administered intravenously. on march 10, 2015, lvef ejection fraction was 62% (echocardiogram examination), right heart load (electrocardiogram) and cardiac function had recovered to normal. on november 30, 2011, the bnp value recovered to 40.6 pg / ml after the first administration of admsc, but such a mild bnp concentration continued until january 8, 2013. after the third admsc administration on june 10, 2013, the bnp value was recovered to an average value of 19.8 pg / ml, but thereafter it was mildly high concentration (bnp 20.4 35.7 pg / ml) until september 30, 2014. the bnp value was 9.1-19.9 pg / ml since february 24, 2015 and the reference value was 18.4 pg / ml for more than 1 year. the lvef ejection rate was 62% (figure 2, table 1), right heart load (electrocardiogram) and other cardiac function test results were normal. figure 2. echocardiography diagnosis on 10th march 2015: left ventricle function: normal limit; right atrial load (-). the left ventricle lumen diameter is in the normal range, indicating that there is no left ventricular dysfunction maintenance and no right heart load discussion an increase in bnp concentration in the blood is seen from the early stage of myocardial infarction, it is proportional to an increase in left ventricular diastolic pressure and decrease in cardiac output and is considered to be a better predictive index than lvef [11-13]. the bnp value rises with the onset of myocardial infarction, peaks after about 20 hours and then declines [18]. however, there are reports that in severe cases bimodal rises again after 3 to 5 days [19]. in the reported patient, the bnp value was 87.9 pg / ml 4 days after onset, and the bnp value was 76.5 pg / ml even after 8 days. in the bnp high value group (bnp value of 80 pg / ml or more), the risk rate of myocardial infarction recurrence is reported to be 5 times as compared with the bnp low value group [14]. based on these findings, we considered the risk of myocardial infarction recurrence high in our patient, and performed cell therapy using admsc. admsc were administered intravenously 78 days after onset of myocardial infarction. this patient had a bnp value of 70 to 90 before administration and decreased to 40.6 pg / ml in 2 months after administration, but the bnp standard value 18.4 pg admsc were intravenously administered again because the condition of high concentration (33.3 to 34.7 pg / ml) continued for more than 1 year compared with / ml, and the cardiac function was not completely recovered. initial amsc administration, second and third cell therapies were made http://www.ajecr.org/ 343 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org table 1 measured values by echocardiography examination on march 10, 2015. in the 13 and 14 months after the first treatment and the bnp value 19.8 pg / ml was recovered to the average value at 2 months after the third amsc administration, but after 16 months it was mild. although bnp value was 9.1 to 19.9 pg / ml for more than one year, from about 2 years after the third amscs administration, the bnp concentration standard value of 18.4, a value correspondding to 29 pg / ml was shown. the lvef ejection fraction 62% (echocardiogram examination), right heart load (electrocardiogram) and cardiac function were also restored to normal. from these data, intravenous administration of amscs in myocardial infarction was shown to be an effective treatment. according to the results of this study, bnp concentration or lvef was not recovered to normal level by single administration of admsc. however, bnp concentration or lvef recovered to normal level by readministration of admsc. cell therapy of bone marrowderived or adipose tissue-derived mesenchymal stem cells is often based only on a single administration and it is thought that it will be a more effective treatment method by conducting re-administration of stem cells while looking for the bnp concentration to drop to normal limit. as an effect of mesenchymal stem cells administered to myocardial infarction patients, blood circulation improvement and infarct site reduction have been reported, and it is thought that admsc differentiated into vascular endothelial cells and cardiomyocytes [20-22]. however, in the experimental limb ischemia model, admsc are not found in the ischemic site, and there are reports that recovery of ischemia is due to endothelial cells and hepatocyte growth factor released from admsc. [23]. it is not clear whether improvement of cardiac function of this patient is due to differentiation of admsc administered intravenously into vascular endothelial cells or cardiomyocytes or by various growth factors from admsc. also, it took 1 to 2 months from administration of admsc for bnp concentration to decrease. it is considered this period to be the period necessary for the remodeling of blood vessels and myocardial walls of the ischemic part. improvement of blood circulation at the site of myocardial infarction is an important condition for recovery of cardiac function. it has been reported that the administration of admsc improved circulation of the ischemic part in the model of limb ischemia experiment in nude mice [24, 25]. this indicates that admsc are effective not only for myocardial infarction but also for treatment of patients with cerebral infarction. in fact, although not admsc, good therapeutic effects have been reported when bone marrow-derived mesenchymal stem cells are administered to patients with cerebral infarction [26]. cell therapy by stem cells is systemically administered by intravenous infusion or local injection of ischemic sites. the method used our patient was intravenous administration, and the stem cells were distributed to almost all tissues. myocardial infarction is a risk factor for hypertension, hyperlipidemia, diabetes or coronary artery disease. admsc used in our study have been reported to have a wide range of therapeutic effects such as diabetes, heart disease, osteoarthritis, repair of soft tissue injury or anti-inflammatory effect [27, 28]. in addition, admsc have paracrine function related to vascular endothelial cells and hepatocytes growth factors or anti-apoptotic factors [21]. from these facts, it is shown that admsc are not only involved in the regeneration of cardiomyocytes and vascular endothelial cells in the site of myocardial infarction, but also on risk factors for myocardial infarction. fat tissue collection has significantly less patient burden than tissue collection from bone marrow and can easily proliferate. besides, there is an advantage that 500 times more stem cells obtainable from adipose tissue than bone marrow [10, 29, 30]. as a treatment for myocardial infarction, coronary bypass surgery and thrombolytic therapy are known, but it is known that reperfusion injury (oxidative stress, inflammation, cardiomyocyte death occur [31, 32]. from the extensive therapeutic effects of admsc [21, 26, 27], it is also possible to protect myocardial walls from reperfusion injury. there are reports on intravenous administration of autologous and allogeneic bone marrow derived mesenchymal stem cells in myocardial infarction patients [7, 8]. from these reports, it is said that the effect of allogeneic stem cells promoted the reduction of infarct site and improvement of cardiac function like autologous stem cells. in addition, heterogeneous immune responses were not observed in patients receiving mesenchymal stem cells derived from allogeneic bone marrow [7, 8]. this indicates that stem cell therapy using allogeneic stem cells, not autologous, is possible. it is important to be able to use adipose tissue-derived stem cells of healthy persons for treatment instead of patients in severe conditions. stem cell therapy can be instantaneously administered to patients suffering from myocardial infarction or cerebral infarction or the like. this cell therapy is considered to be an effective treatment for serious patients with cardiovascular diseases. aortic diameter 32mm left atrium diameter 32mm interventricular septum thickness 10mm left ventricular posterior thickness 11mm left ventricular diastolic 55mm left ventricular end-systolic 37mm stroke volume1 91ml left ventricular fractional shortening 34% ejection fraction 62% ivc diameter 18/7mm measurement of values http://www.ajecr.org/ 344 am j exp clin res, vol. 6, no. 2, 2019 http://www.ajecr.org finally, cell therapy by mesenchymal stem cells has been reported to enhance immune competence and promote paracrine action of various growth factors or skin regeneration [27-29, 33, 34]. it is also involved in the cure and regeneration of arterial endothelial cell injury in diabetic lower limb ischemia [33]. these findings are thought to have caused "rejuvenation" by this cell therapy. mesenchymal stem cells including admsc are considered to be one of the future therapeutic agents for diseases that cannot be cured by conventional or standard medical or surgical treatment. conflict of interest the authors declare no conflict of interest. references 1. perin ec, hf, dohmann hf, borojevic r, et al. transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. circulation 107:2294-2302, 2003. 2. perin ec, silva gay, henry td, et al. a randomized study of transendocardial injection of autologous bone marrow mononuclear cells and cell function analysis in ischemic heart 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al. reactive oxygen species inhibit adhesion of mesenchymal stem cells implanted into ischemic myocardium via interference of focal adhesion complex stem cells. stem cells 28:555-563, 2012. 33. duscher d, barrera j, wong vw, et al. stem cells in wound healing: the future of regenerative medicine? a mini-review. gerontology. 2016; 62:216-225, 2016. 34. aso k, tsuruhara a, takagaki k, et al. adiposederived mesenchymal stem cells restore impaired mucosal immune responses in aged mice. plos one. 11:e0148185, 2016. http://www.ajecr.org/ https://www.ncbi.nlm.nih.gov/pubmed/30632499 https://www.ncbi.nlm.nih.gov/pubmed/30632499 https://www.ncbi.nlm.nih.gov/pubmed/30632499 https://www.ncbi.nlm.nih.gov/pubmed/30632499 424 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2021;8(1):424-428 review article a narrative review of transverse myelitis and multiple sclerosis: associations and threats masoud heydari1, hossein pourmontaseri2, fatemeh azizi3, ahad hasan syed hasani4, fatemeh noori5, maryam rahmannia4* 1faculty of nursing and midwifery, tehran university of medical sciences, tehran, iran 2student research committee, fasa university of medical sciences, fasa, iran 3faculty of nursing and midwifery, islamic azad university tehran medical sciences, tehran, iran 4student research committee, school of medicine, shahid beheshti university of medical sciences, tehran, iran 5faculty of paramedical, tehran university of medical science, tehran, iran abstract. the affiliation of multiple sclerosis (ms) with other immune system and inflammatory illnesses like transverse myelitis (tm) has been a theme of interest. even though, previous investigations tried to elucidate the relationship between these two illnesses, it remained unclear because of restricted and incompatible information in this subject. in this review, we researched the etiology, pathophysiology, epidemiology and treatments of ms and tm according to accessible articles in valid databases. we figured out multiple sclerosis (ms) can be associated with transverse myelitis (tm) in a number of aspects and longitudinal expanse of brain and spinal cord lesions found by mri, presence of drb1*1501 allele, and etc. however, further research is fundamental to perceive the remarkable relationship between multiple sclerosis and transverse myelitis in field of neurodegenerative diseases. an appraisal of research studies published in various bibliographic databases including pubmed, medscape, and google scholar were used. a total of 57 articles were sought and reviewed. results were classified into 6 specific subtitles including prevalence and incidence rates, complications, common cerebrospinal fluid (csf) agents and factors, prognosis and diagnosis, treatment, and finally risk factors. research articles were reviewed and analyzed for information regarding each subject. in this study, we aimed to review articles that detected links between tm and ms and those that described different aspects of these correlations. it was observed that in various aspects ms and tm had strong as well as weak correlations. on the other hand, we discussed that the lack of common appropriate treatment caused the inability to restrict symptoms on patients in either disease. further trials on candidate therapies may result in new reliable treatments with acceptable adverse effects. keywords: transverse myelitis, multiple sclerosis, neurodegenerative, risk factor, sssociation. introduction multiple sclerosis (ms) is a chronic central nervous system disease with cognitive and depressive disorders [1]. the last reports revealed that ms pathogenesis is commonly observed in women more than in men and usually occurs in young adults [2-5]. in the last decades, ms prevalence significantly increased in european and american countries (up to more than 120 per 100,000) [2-6]. the symptoms of ms would present in different levels (e.g., mild, moderated, severe progression) [7]. despite some studies suggested several hypothesizes, such as inflammatory mediated pathogenesis introduced by genetic deposition and environmental effects, the mechanisms of ms progression have remained unclear up to recent years [7-9]. moreover, some other studies showed that different cytokines, induced by autoreactive t cells, play the main role in cerebral inflammation leading to ms progression [5, 10]. therefore, it is still required to investigate new associations and pathogenesis mechanisms to provide more reliable treatments for ms. transverse myelitis (tm) is another neurological dysfunction that occurs in both brain hemispheres and would progress to the spinal cord as well [11]. several reports in earlier years revealed that more than 9 million people present different tm symptoms worldwide, but approximately around 90% of them experience mild symptoms [12, 13]. tm has shown the most association with connective tissues disorders, such as systemic lupus erythematosus and sjogren's syndrome [14]. moreover, recent studies demonstrated that acute partial transverse myelitis (aptm) would remain monophasic or change its pathogenesis to ms symptoms [15]. on the other hand, ___________________________________________________________ * corresponding author: maryam rahmannia, bs (mrahmannia77@gmail.com) http://www.ajecr.org/ 425 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org other studies on aptm showed that shifting to ms may occur very rarely in some cases, but with a higher symptomatic presentation [16, 17]. however, attempts to explore new findings of the relationship between ms and tm have continued in the last two decades [18]. in recent years, several studies have been carried out to find new associations and develop novel therapies for neurological disorders. therefore, in this study, we aim to review published articles to achieve links, promising treatments, and new common conditions for ms and tm patients. links between tm and ms 1. prevalence and incidence one of the first observations showed that around 3% of adult patients with tm completely convert to ms [19]. however, only 0.7% of patients with ms face acute tm, but the situation becomes different in the long term [20]. further clinical observations revealed that approximately 80% of patients with partial tm may present ms symptoms in 3 years [21]. a multi-central evaluation in new mexico showed that more than 20 percent of tm cases would be induced by ms mechanisms [22]. therefore, their remarkably observed correlation between tm and ms shows novel connections [23]. 2. complications pediatrics with tm or ms have higher levels of working memory, social life, and attention problems [24].tm and ms patients would present the same complications, such as depression, pain, and fatigue [25]. further studies showed that tm and ms patients have a higher risk for experiencing metabolic and cardiovascular disorders [26]. therefore, patients with these disorders may face several life-long mental and psychological problems require us to find novel management and control their complications. 3. common csf agents and factors some studies tried to introduce new common agents between tm and ms. the evaluation of cerebrospinal fluids (csf) in ms patients indicated that an increase in csf concentration of oligoclonal bands (tm-related agents) has a significant correlation with ms and would develop ms symptoms after 34 months [22, 27-29]. also, other trials showed that 14-3-3 proteins would elevate in patients with little recovered ms and acute tm [30, 31]. earlier studies revealed that il-17 would promote the level of il-6 in inflammation disorders [32]. in 2008, graber et al. demonstrated that il-17 induces astrocytes to resect more il-6 and develop tm and ms symptoms [33]. in contrast, other evaluations showed that ms agents found in csf specimens had no reliable correlation with the development of recurrent tm [34]. moreover, myelin oligodendrocyte glycoprotein immunoglobulin g is another diagnostic factor related to recurrent tm that provides ms-liked symptoms and caused multiple clinical misdiagnoses [35]. 4. prediction, diagnosis and prognosis in 1998, scott et al. introduced a novel prognosis for ms. in this study, they revealed that the correlation of neurological disorders with ms development is more significant in acute tm [36]. further investigations showed that symmetric sensory disorders have a higher prevalence in tm, while atm-related asymptomatic lesions are the most remarkable prognosis for ms [23, 36]. studies on adolescent patients with partial atm indicated that the presence of relapses and mri abnormalities in the brain predicts ms and results in higher complications [38]. on the other hand, in some situations, imaging would result in a misdiagnosis. in a recent study, asnafi et al. showed that longitudinal extensive tm would be mistaken with multiple ms. this misdiagnosis would be prepared with simple axial imaging in suspected cases [39]. moreover, imaging has low reliable efficiency in distinguishing between idiopathic atm and ms, as patients with idiopathic atm have a lower chance to show ms symptoms [29, 40]. therefore, we require further studies to figure out more criteria and provide diagnostic approaches with more efficiency [41]. 5. risk factors trials on antibody mutations indicated that the presence of immune system abnormalities is generally similar in both tm and ms. in a unique study, ligocki et al. introduced antibody gene signature (ags), as a novel risk factor for ms prediction. this study revealed that patients with a high concentration of ags had a serious risk of ms appearance, but these results would not provide reliable biomarkers to certainly diagnose ms [42]. another study on pediatric patients with ms and tm demonstrated that hladrb1*1501 allele, vitamin d abnormality, and viral infections, such as epstein-barr virus and cytomegalovirus, would increase the risk of ms and tm development [43]. in 2008, sellner et al. and revealed a complete list of ms risk factors. this observation indicated that partial atm with igg abnormalities, familial history of ms, serious complications at the beginning of symptoms, and brain msrelated lesions confirmed by mri, alongside oligoclonal bands would convert to ms more than the others [44]. moreover, further studies added gender (being female), smoking, and the appearance of hla-dr15 to the previous risk factors of ms incidence [26]. in contrast, saroufim et al. investigated that cardiovascular diseases, such as hypertension, would result in a higher risk of ms but have no relation with tm [45]. 6. treatments to date, several common treatments have been developed for tm and ms. new observations showed that medical marijuana would provide promising results to control the complications of tm and ms [46]. discussion and conclusion in this study, we aimed to review articles that detected links between tm and ms and described different aspects of these correlations. in 1998, paty et al. showed that the simultaneous incidence of tm and ms, and their conversion to each other in short term [47]; but, other long-term studies indicated http://www.ajecr.org/ 426 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org tight correlations between ms and tm [48]. the 3-year term had been introduced as a cut-off for co-appearance of these disorders [49]. however, we would require to design longer observations with bigger sample sizes to achieve more knowledge about the long-term clinical presentations. multiple studies reported that tm and ms patients may suffer from the same complications as well. while young cases would face some mental health disorders, such as social life, working memory, and attention problems, the older patients may present non-communicable diseases [50, 51]. thus, ms and tm patients may show different complications in various steps. in the past years, several csf biomarkers have been introduced to diagnose ms and tm. the presence of oligoclonal bands and il-17 had a significant association with higher incidence of tm and ms [52, 53]. despite these developments, we still have remarkable misdiagnoses in patients with different stages of tm or ms [54]. therefore, further studies are needed to investigate more reliable biomarkers with higher efficiency in prognoses. for the first observations, scott et al. indicated that neurological symptoms in acute tm and ms patients would occur in the same patterns [55]. based on further studies, mri has been introduced as a relatively reliable approach in diagnosing these neurological disorders [48, 56]. on the other hand, the other observations showed that diagnosis with mri would not be appropriate to detect all presentations. therefore, the specialists recently have serious doubts to rely on this procedure [57]. further studies would figure out novel promising methods to distinguish tm and ms in 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n=19) or open surgery (os; n=17). our laparoscopic surgical technique for sbo used an access port device and a small umbilical incision of 1.5 2.5 cm. the background information of the patients and the postoperative outcomes were collected and subjected to a statistical analysis. postoperative adhesions were the cause of the obstruction in 23 patients. incarcerated inguinal and internal hernias were identified in 6 and 4 cases, respectively, and a tumor, intussusception and volvulus of the small intestine were identified in one case each. three patients (15.8%) of the ls group were converted to laparotomy due to dense adhesion in one case and adhesion below the umbilicus in two cases. six patients required segmental resection through the access device at the umbilicus. no statistically significant differences were found between ls and os in the operative time (ls vs. os; 85.3 min vs. 88.2 min, p=0.8570), postoperative complication rate (5.26% vs. 5.88%, p=0.9355), or the postoperative hospital stay (10.3 days vs. 15.9 days, p= 0.3524). there was a significant difference in the mean timing of oral re-intake after surgery and the amount of intraoperative blood loss (2.5 days vs. 4.5 days p<0.05, 3.8 g vs. 92.9 g p<0.05, respectively). in conclusion, our results indicate that ls with an access port device was suitable for the treatment of sbo in most patients, and that it is useful both as a diagnostic tool and as a therapeutic surgical approach. keywords: small bowel obstruction, laparoscopy, acute abdomen introduction small bowel obstruction (sbo) is a relatively common disease in several conditions of acute abdomen and it results in emergency admission [1-4]. the clinical course is somewhat unfavorable, with a morbidity rate of about 30% if strangulation is present [5]. the most common causes of sbo are postoperative adhesions, which are present in 83.2% of all patients. this is not surprising given the marked increase in the number of elective surgeries for elective hernia repair to prevent incarcerated intestinal obstruction [6]. other causes of sbo include abdominal wall hernia and malignant tumors which account for 3.1% and 2.9% of patients, respectively [7-9]. open adhesiolysis is the conventional surgical approach for sbo patients who do not respond well to conservative therapy and in whom there are clinical findings of incarceration or perforation of intestine [10-12]. however, a potential problem when operating on patients with adhesion is that the new operation may cause even more adhesion. several clinical studies and most experimental studies have shown that adhesive formation is reduced in laparoscopic surgery (ls) in comparison to open surgery (os) [13]. on the other hand, surgeons consider the performance of adhesiolysis in ls to be unfavorable due to the risk of iatrogenic bowel injury, the difficulty of maintaining view field and their restricted ability to manipulate the instruments [7]. in 1991, bastug et al. [14] presented the first report on laparoscopic surgery for sbo. from that time, numerous reports have shown the feasibility and utility of the laparoscopic approach for the management of sbo [13, 1517]. in addition, with increased experience and technical advances in laparoscopic techniques and instrumentation, morbid obesity and previous abdominal surgery are ___________________________________________________________ * corresponding author: ryosuke nakata, md (s00-056@nms.ac.jp). http://www.ajecr.org/ mailto:s00-056@nms.ac.jp 379 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org table 1 etiology of small bowel obstruction table 2 etiology of conversion (overall conversion rate, 15.8%; n=3) table 3 demographic characteristics asa: american society of anesthesiologists; bmi: body mass index. table 4 clinical data preoperatively no longer considered to be contraindications for laparoscopy [15]. we herein present our experiences with ls for sbo of various etiologies to assess the feasibility, efficacy, and clinical outcomes associated with this therapeutic method and present our methodology. materials and methods we respectively reviewed the clinical records of 36 patients who were hospitalized in our department from april 2014 to march 2015 for sbo and who underwent surgical treatment due to clinical findings of intestinal ischemia or the failure of conservative management. the diagnosis of sbo was performed based on the patient’s complete medical history and a physical examination combined with laboratory blood tests and imaging studies. abdominal computed tomography (ct) was a particularly useful modality for diagnosing and detecting the location of obstructions. patient management included nasogastric intubation, the placement of a long intestine suction tube, and vigorous resuscitation with the administration of intravenous fluid and electrolytes. patients with virgin abdomens usually underwent immediate laparoscopic exploration. in addition, the presence of signs of peritoneal irritation or an elevated and rising white blood cell count prompted urgent surgery. when elective surgery was indicated, conservative measures were continued 5-7 days. if the obstruction did not resolve with this period, surgical intervention was performed. the surgical method (ls or os) was chosen by the operating surgeon based on the clinical findings of massive abdominal distension or if there was a risk of dense broad adhesion. the administration of antibiotics was initiated in accordance with the indications of each patient. the preoperative patient demographic characteristics, including sex, age, body mass index, preoperative intubation with a long intestinal tube, and previous abdominal surgery were included in the analysis. the patients were evaluated for perioperative risks based on the statuses of the american society of anesthesiologists (asa). clinical preoperative data, including the white blood cell count (wbc), and the c-reactive protein (crp), creatine phosphorus kinase (cpk), lactate dehydrogenase (ldh), and total bilirubin (t-bil) levels were also retrospectively compared between the ls group and the os group. the operative data, including the operative time, amount of blood loss, the performance of bowel resection, the timing of the restarting of oral-intake and postoperative hospital stay were compared between the ls group, os group and the conversion laparotomy group. surgical procedures all operations were performed by experienced laparoscopic surgeons or by residents under supervision. detailed informed consent for laparoscopy and possible conversion was obtained from all patients. patient positioning for laparoscopic surgery for adhesiolysis is similar to that of other laparoscopic procedures such as appendectomy. all patients underwent general anesthesia and were placed in the supine position with one or both arms alongside of the patient’s body. the placement of the operator and monitor was determined by the findings of the etiology n laparoscopic surgery open surery conversion adhesion 23 (63.9%) 7 13 3 incarcerated inguinal hernia 6 (16.7%) 2 4 0 incarcerated internal herunia 4 (11.1%) 4 0 0 intussusception 1 (2.8%) 1 0 0 tumor obstruction 1 (2.8%) 1 0 0 volvulus of the small intestine 1 (2.8%) 1 0 0 total 36 16 (44.4%) 17 (47.2%) 3 (8.3%) etiology no. of patients (n=3) % dense adhesion 1 33.3 adhesion below the umbilicus 2 66.6 laparoscopic surgery (n=19) open surgery (n=17) p age 61.5 (35-85) 70.9 (45-89) <0.05 sex (women/men) 6 / 13 8 / 9 0.4955 asa classification 0.0782 i 8 (42.1%) 3(17.7%) ii 11 (57.9%) 11 (64.7%) iii 0 3(17.7%) iv 0 0 bmi 20.7 (16.5-25.2) 20.7 (14.0-24.8) 0.9313 long intestinal tube 7 (36.8%) 5 (29.4%) 0.7317 previous abdominal surgery 11 (57.9%) 15 (88.2%) 0.0652 laparoscopic surgery (n=19) open surgery (n=17) p white blood cell count (×10 3 /μl) 12.0 (5.5-20.8) 8.5 (4.1-15.0) <0.05 c-reactive protein (mg/dl) 1.3 (0.02-5.5) 2.5 (0.03-25.5) 0.4337 creatine phosphorus kinase (iu/l) 85.5 (29-211) 88.8 (13-210) 0.8429 lactate dehydrogenase (iu/l) 215.5 (135-372) 225.1 (145-370) 0.7634 total bilirubin (mg/dl) 0.87 (0.4-1.6) 0.68 (0.2-1.2) 0.1809 http://www.ajecr.org/ 380 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org table 5 operative data ls: laparoscopic surgery; os: open surgery. figure 1 the setting of the ea-access at the umbilicus and a 5 mm trocar at the lower abdomen. obstructive location in preoperative abdominal ct. the surgical operator stood opposite to the monitor, which was placed on the same side as the obstruction. the camera operator stood next to the surgeon. a skin incision of 2-3 cm was made in the umbilical region. we used an ez-access with a lap-protector (hakko medical inc., chikuma, japan) as the umbilical access device. two 5 mm trocars were placed through the ezaccess device for a 5 mm laparoscope and a 5 mm instrument. after attaching the ez access device to the lapprotector, pneumoperitoneum was established and maintained at 10 mmhg using co2. a 5 mm trocar was placed under laparoscopic guidance into the lower abdomen (figure 1). two types of laparoscope, a flexible or a 30º side-view 5-mm telescope, were used. the other essential instruments included atraumatic graspers, scissors with cautery capability, and an ultrasonic dissector. after sufficient instruments were prepared in the abdomen, the procedure began with detecting and approaching the point of obstruction. the surgeon should be mindful that the dilated and edematous bowel is easily injured by small instruments. to the extent that was possible, the small bowel was run with atraumatic graspers in a retrograde manner from the cecum to detect the point of obstruction. in addition, the grasping of the mesentery to manipulate the bowel also decreases the incidence of bowel perforation. for cases of simple adhesive intestinal obstruction, a combination of a sharp dissection with the sensible use of electrocautery or an ultrasonic scalpel was applied to treat adhesiolysis. in such cases, it is necessary to consider that electrical intestinal injury may not be recognized immediately and may present as a delayed perforation. after adhesiolysis, the state of the lysed intestinal serosa can be assessed through the umbilical lap-protector. the indications for repair and resection are evaluated based on the direct visual findings and palpation. for cases of abdominal wall hernia, obstruction is improved by the laparoscopic reduction of the incarcerated hernia. in cases where reduction was possible, we performed a laparoscopic evaluation of the viability of the incarcerated bowel segment based on the color, the presence of peristalsis, and venous congestion. in cases where bowel resection was not required, tapp was performed in the usual manner. on the other hand, when obviously necrotic intestine was detected, immediate bowel resection and anastomosis was performed extracorporeally through an umbilical lap-protector. the same treatment was used when any tumors were identified as the cause of obstruction. the nasogastric tube was left in place, and the patient was maintained on intravenous fluids, with nothing by mouth until the return of bowel function was heralded by bowel sounds or the passage of flatus. subsequently, the nasogastric tube was removed, and the diet was advanced as tolerated. statistical analysis statistical analyses were performed to compare the characteristics and results of the ls and os groups. all statistical analyses were performed using the jmp ® 11.0.0 software program (sas institute japan, tokyo, japan). patient characteristic and perioperative variables were compared in a univariate analysis to determine the association of each variable with the outcome. fisher’s exact test and the mann–whitney u test were performed. a p value of <0.05 was considered to be statistically significant. results the etiologies of sbo are outlined in table 1. postoperative adhesion was identified in 63.9% of patients (n=23), as well as incarcerated inguinal hernia (n=6), incarcerated internal hernia (n=4), intussusception (n=1), tumor (n=1), and volvulus of the small intestine (n=1). laparoscopy was initially attempted in 10 patients with adhesive ileus. the other 13 patients who had a medical history of repeated ileus underwent open surgery due to suspected dense adhesion. the conversion rate from laparoscopy to laparotomy was 15.8% (n=3). the reasons for conversion, which are shown in table 2, were dense adhesion (n=1), and adhesion below the umbilicus (n=2). in the umbilical adhesion patients, exploratory laparoscopy with an access port device was performed to confirm that no other obstructions were present after adhesiolysis. variable laparoscopic surgery (n=16) open surgery (n=17) conversion (n=3) p (ls vs. os) operative time (min) 85.3 (39-168) 88.2 (24-190) 127.3(47-240) 0.7321 amount of blood loss (g) 3.8 (0-830) 92.9 (0-60) 46.7 <0.05 bowel resection 6 (37.5%) 10 (58.8%) 2(66.7%) 0.3028 postoperative complication 1 (5.3%) 1 (5.9%) 0.9335 oral re-intake (day) 2.5 4.5 2 <0.05 postoperative hospital stay (day) 10.3 15.9 8.3 0.2891 http://www.ajecr.org/ 381 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org figure 2 the bowel status after adhesions is was ascertained through the umbilical access port device. figure 3 an operative scar 2 weeks after surgery. the patient characteristics and preoperative clinical data are listed in tables 3 and 4. the patients in the ls group were younger than the patients in the os group. there was no significant differences in the other characteristics of the patients in the two groups. regarding the laboratory findings, the white blood cell count was higher in the ls group than os group. the perioperative data are shown in table 5. the amount of intraoperative blood loss was significantly lower in the ls group than in the os group (3.8 g vs. 92.9 g, p=0.0017). oral re-intake occurred more quickly in the ls group than in the os group (2.5 days vs. 4.5 days, p<0.05). each of the two groups had one case of postoperative complication that was not lethal but which delayed re-oral intake. there were no significant differences between the two groups with regard to the mean operative time, the number of cases in which bowel resection was performed or the duration of postoperative hospital stay (10.3 days vs. 15.9 days, p= 0.3524). discussion in this retrospective study, we evaluated the feasibility, efficacy and clinical outcome of the laparoscopic surgery for sbo in comparison to open surgery and presented our methodology. the main cause of sbo was adhesion (63.9% of patients). in the ls group, the overall conversion rate was 15.8%. the rate of postoperative complications did not differ significantly between the ls and os groups (5.3% vs. 5.9% p=0.9335). all of the patients survived the surgery. there was a significant difference in the timing of postoperative oral re-intake; the ls group achieved oral reintake more quickly than the os group (2.5 days vs. 4.5 days, p=0.0374). this indicates that bowel function recovered faster in the ls group, which is in line with a previous report [18]. however, we did not find any significant difference in the postoperative hospital stay. the earlier oral re-intake of the ls group suggests the possibility that the hospital stay may be shortened. laparotomy has traditionally been the standard surgical intervention for sbo when conservative therapies fail. on the other hand, the laparoscopic approach for sbo is associated with a potential problem in that it may cause further adhesion. a large number of clinical and experimental studies have shown a reduction in adhesion formation after laparoscopic surgery in comparison to open surgery [13]. in addition, laparoscopic surgery for bowel operations has been shown to significantly reduce the incidence of sbo in comparison to open surgery [16]. these data show that ls is an attractive and reasonable therapeutic option for sbo. beck et al. reported in a postoperative trial that in 63% of laparotomy incisions, the length of the incision was involved in the formation of adhesion in the intraperitoneal region [19]. furthermore, the incidence of ventral hernia after laparotomy is reported to be from 11% to 20%, whereas that in laparoscopic surgery is reported to be 2.5% [16,20,21]. the additional benefits of laparoscopic approach are reported to be a decreased incidence of wound infection and postoperative pneumonia, a more rapid return of bowel function, and a shorter hospital stay [18]. all suspected cases of intestinal obstruction can initially be approached by laparoscopy. in experienced hands, the laparoscopic management of sbo is successful; 66% of cases are performed without conversion [7]. as a controversial point, there are measures that may be implemented to prevent intestinal perforation at the time of the first trocar insertion [22-26]. we conducted the preoperative ultrasound mapping of the adhesions as a way of ascertaining the presence of dense adhesion at the umbilicus. in addition, in our methodology, the first trocar was set with ez-access after the insertion of a lap-protector into the umbilical incision. because the first trocar was inserted under direct vision, none of the patients in our series suffered an intestinal injury. another safety-related issue concerning laparoscopic surgery in sbo patients with bowel distension is the instrumentation and handling of the bowel, due to the increased risk of perforation. wullstein et al. reported a 26.9% rate of bowel injury in patients who underwent ls, http://www.ajecr.org/ 382 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org in comparison to a 13.5% rate in patients who underwent laparotomy [27]. although missed enterotomy can occur in association with laparotomy, the incidence is higher with laparoscopic surgery. however, the risk of bowel injury can be diminished with good surgical practices, which include avoiding the use of electrocautery, minimizing the grasping of the dilated bowel, manipulating the bowel using atraumatic graspers and by handling the mesentery whenever possible. this indicates that during laparoscopic surgery for sbo, the surgeon needs to be careful in the handling of bowel distension and in ascertaining the presence of bowel injury after adhesiolysis. we should pay attention to the possibility of thermal injury to the bowel due to the use of a monopolar electrocautery or a harmonic scalpel. in our methodology, it is easy to ascertain the presence of bowel injury through the umbilical access device. if the injury is detected or suspected, the bowel repair or resection can be performed concurrently. for the 8 patients of the ls group, some procedures were conducted through the umbilical access port device. six patients required bowel resection and anastomosis for enterotomy with adhesiolysis, necrotic change for ischemia or an intestinal tumor. in two patients, the bowel status was ascertained with direct vision after the releasing strangulation. it was not hard to move the bowel through the small umbilical incision (approximately 2 cm) after the reduction. in our study, three patients were converted to laparotomy before reduction. the reasons for conversion were dense adhesion in one patient, and adhesion below the umbilicus in two patients. in the umbilical adhesion patients, adhesiolysis was performed at the time of the insertion of the lap-protector. exploratory laparoscopy was performed with access port devices after adhesiolysis to confirm that there were no other obstructions. exploratory laparoscopy can explore the intraperitoneal region more widely than open adhesiolysis or mini-laparotomy. it is considered that this procedure can lead to a reduction in the number of cases of reoperation for multiple obstructions. in a recently published review, ghosheh et al. reported that 356 of 1,061 patients who underwent laparoscopy for acute sbo required conversion to open laparotomy (conversion rate, 33.5%) [7]. conversion was most frequently due to dense adhesions (27.7%), followed by the need for bowel resection (23.1%) as a result of injury, ischemia, gangrene, and other causes. in our procedure, another 5 mm skin incision was positioned on the lower abdomen to assist the surgeon. the skin incision became less noticeable after 2 weeks. setting a 5 mm trocar upon the lower abdomen allowed the operator to maintain eye-hand and hand-hand coordination. in addition, the basic principle of triangulation of instrumentation is also maintained and it avoids instrument interference. when drain placement is required, it can be inserted into the skin incision on the lower abdomen. a sufficient preoperative examination, such as abdominal ct or ultrasound mapping, is important in ls for sbo. several studies have demonstrated the value of ct in confirming the diagnosis and revealing the cause of sbo [28,29]. moreover, jerome et al. reported that mpr can increase both the accuracy and confidence in the location of the transition zone in ct of sbo; the authors reported that the accuracy of transition zone location was increased from 90% to 93% [30]. the preoperative detection of the transition zone is useful for detecting the origin of sbo with laparoscopy. with an adequate preoperative examination, it is possible to provide sufficient therapy for each patient. the present study is associated with several limitations. given that the assignment to the laparoscopic group was nonrandom, there is concern that overall healthier individuals were more likely to be selected for laparoscopic surgery than their sicker counterparts. we noted that patients undergoing laparoscopic adhesiolysis were more likely to be younger than the patients who underwent open surgery (61.5 vs. 70.9, p=0.0439). despite these limitations, this retrospective study shows the feasibility of 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adhesive small bowel obstruction. br j surg 90:1147-1151, 2003. 28. boudiaf m, soyer p, terem c, pelage jp, maissiat e, rymer r. ct evaluation of sbo. radiographics 21:613624, 2001. 29. frager d, medwid sw, baer jw, mollinelli b, friedman m. ct of sbo: value in establishing the diagnosis and determining the degree and cause. ajr am j roentgenol 162:37-41, 1994. 30. hodel j, zins m, desmottes l, boulay-coletta i, jullès mc, nakache jp, rodallec m. location of the transition zone in ct of small-bowel obstruction: added value of multiplanar reformations. abdom imaging 34:3541, 2009. http://www.ajecr.org/ 229 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(3):229-231 case report seconder paroxysmal hemicrania caused by mucocele burc esra sahin 1 *, ece ozdemir oktem 2 1 department of neurology, faculty of medicine, ahi evran university, kirsehir, turkey 2 department of neurology, faculty of medicine, manavgat state hospital, turkey abstract. paroxysmal hemicrania (ph) is classified as a subgroup of trigeminal autonomic headaches among primary headaches. the pain is unilateral, located frequently at the orbital, supraorbital and/or temporal regions. it is sharp, severe, and these headaches tend to occur at least 5 times a day, which last for 2 – 30 minutes, accompanied by autonomic findings such as conjunctival injection, lacrimation, nasal congestion, nasal discharge, ptosis and orbital edema. secondary ph is associated with tumor or vascular pathologies. most frequently hypophyseal tumors were detected. in our case, there was a mucocele originating from the left ethmoidal sinus and compressing on the medial rectus muscle in the cranial mr examination of a patient admitted with clinical features of ph. in this case, it was seen that neuroimaging is mandatory in patients with tob, in order to exclude secondary causes. also, nociceptive impulses originating from surrounding tissues with the compression effect of the mucocele induce trigeminal autonomic reflex, supporting the hypothesis that autonomic symptoms and pain occur with hypothalamic and trigeminal connections. keywords: trigeminal autonomic cephalalgy, paroxysmal hemicrania, mucocele introduction paroxysmal hemicrania is classified among trigeminal autonomic cephalgies subgroup among primary headaches [1]. tos is a kind of headache characterized by autonomic symptoms at trigeminal nerve somatic distribution [2]. the pain is frequently seen at the ophthalmic region of the trigeminal nerve. ihss-3 have remarked that ph pain may be seen as orbital, supraorbital, temporal or any combination of the trigeminal nerve [1]. ph is characterized by very severe pain accompanied by sharp, short unilateral autonomic symptoms [3]. the pain is typically described as severe, throbbing, and squeezing type of sensation [3, 4, 5, 6]. pain is intermittent and starts suddenly. it frequently lasts for 10-30 minutes, with a range duration between 2-45 minutes. more than 60% of patients experience restlessness and pain between episodes [7]. ihss-3 beta criteria requires accompaniment of at least one autonomic symptom such as ipsilateral conjunctival injection, lacrimation, nasal congestion, rinorrhea, sweating of forehead and face, myosis, ptosis and/or eye edema [1]. the prevalence of ph is not as low as it may be thought. either the cases were not recorded or the diagnosis was not considered. case series have described both typical and atypical cases, and 22% of cases show an atypical presentation [8]. secondary ph is typically associated with tumors and vascular pathologies. most frequent causes of ph and other tos include pituitary gland pathologies, and for this reason cranial mr imaging is mandatory [8]. in our case, there was a mucocele which originated from the left ethmoidal sinus and compressed the medial rectus muscle at cranial mr imaging in a patient admitted with clinical features of ph. case report a 52-year-old female patient was admitted at our outpatient clinics with complaints of headache which had started 3 months ago, and showed an increase in frequency and severity in the last 15 days. the pain was experienced around the left eye, lasted for 5-10 minutes, and had occurred for at least 10-20 episodes per day. the patient described the pain as very severe in intensity and stinging/stabbing. there was injection and ptosis at the ipsilateral eye during an episode. treatment was initiated with indomethacin 75 mg/day and complete remission was obtained on the second day. a lesion originating from the left anterior ethmoid sinus and compressing the left medial rectus muscle was detected in cranial mri (figure 1). the patient had a surgical intervention at the ent department, and histopathological examination revealed a mucocele. the patient did not experience pain during oneyear of follow-up. discussion ph is a rarely encountered type of headache, which ___________________________________________________________ * corresponding author: burc esra sahin, md (besras11@yahoo.com). http://www.ajecr.org/ mailto:besras11@yahoo.com 230 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org figure 1. cranial mr a) t1, b) t2, c) t2 flair imaging: mucocele originating from the left anterior ethmoid sinus and compressing the left medial rectus muscle discussion ph is a rarely encountered type of headache, which may be due to missing this diagnosis or not recording it. there may be a group of patients, which may not exactly fulfill the ihss-3 diagnostic criteria [8]. there is overlap in clinical findings and treatment of tacs, which may be caused by a common physiopathology of tacs [9, 10]. in a pet study in patients with ph, activation was observed in the contralateral posterior hypothalamus, ventral midbrain, red nucleus and substantia nigra [11]. trigeminal autonomic reflex (tar) was held responsible for the symptoms of trigeminal autonomic pain syndromes. tar activation is responsible for the acute attacks of tacs [12]. this reflex is probably triggered due to activation of hypothalamus and structures closely related with it [12]. ph and other autonomic findings of tacs probably develop with central dysinhibition of tar by direct hypothalamic – trigeminal connections of the hypothalamus [13]. pituitary gland lesions constitute the most frequent cause of secondary ph and tac. for this reason, cranial mri should be part of our imaging studies in ph and tacs [8]. in conclusion, a mucocele originating from the ethmoidal sinus and compressing the rectus muscle induces tar with nociceptive impulses that originate from the surrounding tissues. autonomic symptoms and pain occur as a result of hypothalamic – trigeminal connections. neuroimaging should be done in order to exclude secondary causes in all patients with a diagnosis of ph. conflict of interest the authors declare no conflicts of interest. references 1. headache classification subcommittee of the international headache society. the international classification of headache disorders, 3rd ed. cephalalgia 33:629-808, 2013. 2. indomethacin-responsive headaches. vanderpluym j. curr neurol neurosci rep 15:516, 2015. 3. lambru g, matharu ms. trigeminal autonomic cephalalgias: a review of recent diagnostic, therapeutic and pathophysiological developments. ann indian acad neurol 15:51-61, 2012. 4. fuad f, jones ns. paroxysmal hemicrania and cluster headache: two discrete entities or is there an overlap? clin otolaryngol allied sci. 27:472-479, 2002. 5. prakash s, belani p, susvirkar a, trivedi a, ahuja s, patel a. paroxysmal hemicrania: a retrospective study of a consecutive series of 22 patients and a critical analysis of the diagnostic criteria. j headache pain 14:26, 2013. 6. boes cj, dodick dw. refining the clinical spectrum of chronic paroxysmal hemicrania: a review of 74 patients. headache. 42:699-708, 2002. 7. cittadini e, matharu ms, goadsby pj. paroxysmal hemicrania: a prospective clinical study of 31 cases. brain 131:1142-1155, 2008. http://www.ajecr.org/ 231 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org 8. sanjay prakash, rushad patell. paroxysmal hemicrania: an update curr pain headache rep 18:407, 2014. 9. goadsy pj, lipton rb, a review of paroxismal hemicranias, sunct syndrome and other shortlasting headaches with autonomic feature, including new cases. brain 120:193-209, 1997. 10. prakash s, hansen jm (2011) mechanisms of cluster headache and other trigeminal autonomic cephalalgias. in: martelletti p, timothy j, steiner tj (eds) handbook of headache: practical management, 1st ed. springer verlag, pp 330–340. 11. matharu ms, cohen as, frackowiak rsj, goadsby pj. posterior hypothalamic activation in paroxysmal hemicrania. ann neurol 59:535-545, 2006. 12. may a. cluster headache: pathogenesis, diagnosis, and management. lancet 366:843-855, 2005. 13. malick a, strassman rm, burstein r. trigemino hypothalamic and reticulohypothalamic tract neurons in the upper cervical spinal cord and caudal medulla of the rat. j neurophysiol 84:2078-2112, 2000. http://www.ajecr.org/ 321 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2019;6(1):321-325 original article a united states pharmacopeia chapter 800-centered process improvement proposal for compounding antineoplastic medications in an academic medical center pharmacy veronica bonderski1, mary clay1*, raoof abdellatif2 1college of pharmacy, purdue university, stadium mall drive, west lafayette, indiana, usa 2department of pharmacy, northwestern memorial hospital, east huron street, chicago, illinois, usa abstract. it is well described that handling antineoplastic medications without proper personal and environmental protections leads to malignancy, reproductive toxicity, and organ damage. in december 2019, the united states pharmacopeia chapter 800 (usp<800>), which addresses the proper handling of hazardous drugs, will be enforceable for any facility handling these agents. northwestern memorial hospital in chicago, illinois has developed a process improvement for the central pharmacy clean room to increase compliance to usp<800> standards. this plan specifically focuses on adjusting the workflow of four antineoplastic agents that are compounded in their central pharmacy clean room. keywords: united states pharmacopeia chapter 800, pharmacy, antineoplastic, national institute for occupational safety and health, compliance introduction the standards described in united states pharmacopeia general chapter <800> hazardous drugs handling in healthcare settings (usp<800>) [1], a detailed addendum to the brief hazardous drugs (hds) section of general chapter <797> pharmaceutical compounding – sterile preparations (usp<797>), will be officially enforceable across all institutions that handle hds as of december 2019. prior to the publication of usp <800>, minimal standards existed regarding the preparation, administration, and transportation of hds; however, this chapter now provides practical standards that must be implemented in order to increase the protection of healthcare workers and patients from unnecessary contact with hds. usp<800> was developed in response to publications and alerts from safety organizations and healthcare facilities, such as the national institute for occupational safety and health (niosh). notably in 2004, niosh released their alert entitled “preventing occupational exposure to antineoplastic and other hazardous drugs in health care settings”[2]. it presented compiled data and reports regarding hds as far back as the 1960s which documented negative outcomes for healthcare workers exposed to hds. adverse events included reports of malignancies, reproductive toxicity in both men and women, and overall organ damage. thus, it was clear that an enforceable standard was needed to protect the safety of those involved with the preparation of hds. at northwestern memorial hospital, a 900 bed academic medical center in chicago, illinois, the pharmacy staff are working to update the facility’s practices and procedures in order to become fully compliant with usp <800>. like many other hospitals throughout the country, several barriers to achieving full compliance exist ranging from financial barriers, changes to workflow, or staff education. additionally, improvements must ideally be implemented without affecting the current standard of patient care. little has been published to date detailing hospitals’ efforts toward increasing usp<800> compliance and the barriers involved in doing so. in order to move forward with the changes needed to improve northwestern memorial’s compliance to usp<800>, a task force including pharmacists, pharmacy technicians, and pharmacy students was created to amend and improve current practices. more specifically, the task force identified that the manipulation of several niosh class i hazardous drugs, or antineoplastic drugs, was being performed in conditions that do not meet the specifications for safe handling of these dangerous compounds [3]. because of this, northwestern memorial has developed a process improvement proposal which addresses these issues. the overall objectives of the task ___________________________________________________________ * corresponding author: dr. mary clay (claym@purdue.edu). http://www.ajecr.org/ mailto:claym@purdue.edu 322 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org force charged with this initiative are to improve the safety of hospital employees and patients as they come into contact with hds throughout the entire medication delivery process and to increase compliance to usp<800> while maintaining efficient pharmacy workflow. usp<800> overview usp<800> outlines the standards for the handling and preparation of hds so that pharmacies and other facilities are able to maximize patient, employee, and environmental safety. these standards range from maintaining engineering controls, proper drug storage, disposal protocols, documentation, and other exposure control measures. as the date approaches when usp<800> will be enforceable by pharmacy regulatory agencies, hospitals are working to implement these standards. in general, facilities that compound or prepare hds are required to have engineering controls that ensure containment of the hd throughout its normal course of manipulation or in the case of a leak, spill, or error. this includes, but is not limited to, a negative pressure iso class 7 buffer room and containment primary engineering controls (c-pec) such as biological safety cabinets (bsc) or compounding aseptic containment isolators (caci) that are externally vented. as for the storage of hds, antineoplastic agents that are not in their final dosage forms (i.e. they will be repackaged or manipulated in some way) must be stored separately from non-hds. additionally the containment secondary engineering controls (c-sec) for these drugs involve storage in a negative pressure, externally vented room, with at least twelve air changes per hour. if refrigeration is required, there must be a dedicated hazardous drug refrigerator, which should be stored in a negative pressure environment with twelve air changes per hour. negative pressure spaces with a refrigerator should have an exhaust near the compressor and the back of the refrigerator. disposal of these drugs further complicates the implementation of usp<800>. hds and contaminated materials must be disposed of according to federal, state, and local regulations. because of this, those involved in the general disposal processes for the pharmacy must also be trained on how to dispose of hds. many facilities also employ separate disposal mechanisms for bulk waste and trace waste, such as personal protective equipment (ppe), further adding to the need for trained personnel. because ppe worn during hd manipulation is subject to higher risk in the event of contamination, there are additional requirements for handling it in usp<800>, as compared to usp<797>. those compounding hds are required to wear chemotherapy gloves that meet the american society for testing and materials standard d6978, hair and beard covers, gowns that are proven not to be permeable to hds, two pairs of shoe covers, eye protection, and respiratory protection beyond a surgical mask. while these ppe requirements are in place to help protect those involved from exposure to the hds during manipulation or if accidents like spills occur, there are additional standards for the management and cleaning of hd spills. firstly, facility-mandated standard operating procedures for these situations must be maintained at all times. whether the spill occurs in a drug storage area or in the direct compounding area, they should be cleaned immediately by trained personnel. spill kits should be on hand at all times, and those trained to use them must be present any time hds are being handled. at the time of the spill and at other regularly scheduled times, cleaning of the usp <800> environment is required. the cleaning process is far more extensive than that required by usp <797>. the sequential cleaning process begins with decontamination. decontamination is defined by “inactivating, neutralizing, or physically removing hazardous drug residue from non-disposable surfaces and transferring it to absorbent, disposable materials…”. next, the cleaning phase begins which involves removing contaminants from objects and surfaces using specific detergents or other chemicals. the last step of cleaning is disinfection which is the process of destroying microorganisms from the compounding areas or supplies. together, these steps ensure a safe space for the manipulation of hds [1]. assessment of current practice northwestern memorial hospital has several pharmacy locations distributed throughout the different wings of the facility. of most importance to this initiative are the central pharmacy which houses a large, usp<797> compliant, non-hazardous drug clean room, an inpatient oncology pharmacy with a designated hazardous drug compounding room, and a smaller general satellite pharmacy with a bsc. early on, it was recognized by pharmacy staff that these facilities had the equipment needed to redistribute the workflow of hds to optimize employee safety without affecting the quality of patient care. in order to begin identifying specific areas for improvement for usp<800> compliance, fourth year pharmacy students began reviewing usp<800>, completing simplifi797ⓡ critical point training modules, and observing procedures in the pharmacy locations described above [4]. by doing this, they were able to create a list of potential areas for improvement. next, the students met with clean room technicians and pharmacists to hear and evaluate their specific questions and concerns about hd handling in the pharmacies. it was very apparent that these employees were most concerned about their exposure to antineoplastic drugs in the central pharmacy clean room, a compounding space not designed to handle the preparation of hds. seeing as this was an issue identified by the both the pharmacy students and pharmacy employees, it was decided that this was the best area of focus. from there, the pharmacy students and a clean room pharmacist met to review the niosh list of antineoplastic and other hazardous drugs in healthcare settings [3]. from the list of niosh class 1 drugs, they identified the agents which are handled and manipulated in the general clean room. the drugs that raised the most concern were mitomycin, hydroxyurea, the bacillus calmette-guerin (bcg) vaccine, and methotrexate. http://www.ajecr.org/ 323 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org mitomycin usp<800> standard and manufacturer guidance in the package insert for mitomycin, an alkylating agent, there were vague guidelines for the handling and disposal of this hazardous drug. therefore, we must turn to the guidance of usp<800> for recommendations, keeping in mind that usp<800> resulted from data and case reports that were made public after mitomycin and other hds first came to market. section 5.3 of usp<800> describes in detail the requirements outlined previously pertaining to the compounding environments in which hds should be handled. these controls can help drastically mitigate the risk of inhalation, contact to mucous membranes, or ingestion of mitomycin [1, 5, 6]. current practice mitomycin is compounded in the central pharmacy clean room upon request by a surgeon performing an oncologic procedure for bladder irrigation. the drug is reconstituted, drawn up into a syringe, and the final product is placed in an amber bag. a nurse or technician comes to pick up the drug or deliver it to the operating room, respectively. because the delivery of this medication is extremely time sensitive, it is currently prepared in the central pharmacy to reduce transit time to the operating rooms which are housed in the same wing of the hospital. proposed solution the inpatient oncology pharmacy operates from 0730 to 2300 during the weekdays. this location has a negative pressure, externally vented hd clean room with two caci. typically, only one caci is used at a time when the scheduled chemotherapy compounding begins in the late morning. additionally, mitomycin is usually requested starting from 0830 and throughout the day. therefore, mitomycin can be compounded in the mornings after opening of the inpatient oncology pharmacy, leaving adequate time to clean the caci at the beginning of the shift. the second caci may be used if it is open and an additional technician is available to avoid disrupting current workflow. from there, a pharmacy technician not working in the hd clean room would deliver the mitomycin dose to the appropriate operating room. hydroxyurea usp<800> standard and manufacturer guidance hydroxyurea is an antimetabolite drug used as chemotherapy. northwestern memorial often uses this medication as a compounded oral suspension, which is non-sterile. thus, section 5.3.1 of usp <800> was also considered which details the non-sterile hd compounding standards. however, because there is a risk of aerosolization of the hydroxyurea drug particles as it is compounded into a liquid, c-pec, c-sec, and ppe used in sterile hd compounding should still be used [1, 7, 8]. current practice hydroxyurea oral suspension is batched in the central pharmacy cleanroom. compounding of the suspension involves opening hydroxyurea capsules. risk for exposure to this drug drastically increases when the capsules are opened and drug powder is mixed with liquid. the suspension is stored with non-hd oral liquids in the main pharmacy area. scheduled patient specific doses are drawn up into oral syringes each day for the afternoon delivery for administration overnight or in the morning. the patient-specific label states that chemotherapy precautions should be taken, but technicians generally just wear gloves and sometimes a surgical mask while drawing up the dose. proposed solution since hydroxyurea is batched for multi-use purposes and is not time-sensitive, the transition of its initial compounding from the central pharmacy to the inpatient oncology pharmacy is logistically viable. the oncology pharmacy usually has rush periods when they make their scheduled inpatient chemotherapy doses beginning in the late morning. outside of these hours, hydroxyurea can be batched by the technicians handling hds since it only needs to be done on a weekly basis. the batched oral liquid can continue to be stored in the central pharmacy where it is to be kept in a bag designated for chemotherapy drugs to prevent unnecessary exposure and as an alert to all staff that handle it. when working on the patient specific oral syringe doses each day, the technicians will first complete all nonhd orders. they will then don gloves and masks before continuing with the hd oral liquid orders and finish the process by thoroughly cleaning the directly exposed surfaces with 70% isopropyl alcohol wipes. additionally, pharmacists will wear gloves and a mask when verifying all doses and place each patient-specific set of syringes in a chemotherapy bag as described previously. these steps will reduce personnel exposure to the drugs and also prevent potential, unnecessary contamination of other medications. an important factor for the implementation of this proposed solution involves further education of all pharmacy staff involved with handling hydroxyurea. information relevant to the potential hazards presented by contact with hds and instructions for procedural changes should be highlighted to enhance the safety surrounding the handling of this hd. overall, this is only a temporary solution for the compounding of hydroxyurea; the full enforcement of usp<800> will require not only the batching but also the patient specific doses to be drawn up under a negative pressure environment. further evaluations and alterations of the process will be necessary to reach total compliance in addition to the alterations proposed above. bacille calmette-guerin vaccine usp<800> standard and manufacturer guidance the bcg (tice) vaccine is a live vaccine that can be used for tuberculosis prophylaxis and the prevention or treatment of some bladder cancers. currently, it is believed that the bcg vaccine produces an inflammatory response http://www.ajecr.org/ 324 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org that stimulates macrophages to have anti-tumor effects. according to the manufacturer of ticeⓡ, the preparation of the vaccine suspension should be done using aseptic technique and in a separate, designated area. preferably, the preparation should occur in a c-pec in order to minimize the risk for inhalation, which could result in an outbreak of tuberculosis. these recommendations align with section 5.3 of usp<800> [1, 9, 10]. current practice the bcg ticeⓡ vaccine suspension is currently prepared in the central pharmacy cleanroom for patients requiring bladder irrigation. pharmacy staff know when these patients are coming several weeks in advance. the vaccine is reconstituted into a suspension for irrigation, dispensed in a 60 ml toomey catheter-tip syringe, and wrapped in foil. a nurse picks up the scheduled dose from the pharmacy window. in addition to preparation, there are existing, informal hospital protocol for cleaning of the direct compounding area after bcg preparation. this process takes approximately 30 minutes. deactivation, decontamination, and cleaning are performed with 2% sodium hypochlorite (bleach). the sodium hypochlorite is removed with sodium thiosulfate or followed by use of a germicidal detergent in sterile water. finally, the hood is disinfected with sterile water and 70% alcohol. after this process, the hood must remain unused for 5 minutes to allow the alcohol to dry. proposed solution since the timeline for the preparation and administration is known so far in advance, the bcg vaccine can be moved to the inpatient oncology pharmacy for preparation and inserted into the scheduled chemotherapy workflow. because this is a live vaccine, there is concern for contamination of other products, especially those for transplant patients. therefore, the existing hospital protocol described above for bcg preparation must be followed and caci downtime should be added into the workflow. nursing staff will pick up the dose after preparation at the pharmacy. methotrexate usp<800> standard and manufacturer guidance methotrexate is a folate synthesis inhibitor used for a variety of inflammatory conditions and malignancies. the central pharmacy clean room specifically prepares doses for intravitreal injection or for the treatment of ectopic pregnancy. like the manufacturer guidance for handling of mitomycin, usp<800> section 5.3 contains the best standards for the conditions in which methotrexate should be handled. the risks for contact to mucous membranes, inhalation, and ingestion to pharmacy employees is similar to that of mitomycin [1, 11, 12]. current practice methotrexate is compounded in the central pharmacy cleanroom for ectopic pregnancy and intravitreal injections. the stock methotrexate vials are stored in the central pharmacy talystⓡ machines. for ectopic pregnancy, the drug is simply drawn from the stock vial into a syringe. the intravitreal injections are prepared by appropriately diluting the stock methotrexate solution and drawing them up into a syringe. proposed solution both methotrexate products can be moved to the inpatient oncology pharmacy. in fact, this pharmacy is located in the same wing of the hospital as the women’s pavilion, actually improving the logistics of delivery of the medication for ectopic pregnancy. however, this becomes an issue if an order for methotrexate for ectopic pregnancy is placed overnight. fortunately, a remedy for this problem stems from the smaller satellite pharmacy with the bsc. if there is an order overnight, a pharmacy technician can retrieve the drug vials from the inpatient oncology pharmacy and bring them to the small satellite pharmacy for preparation and pharmacist verification since it is open 24 hours a day and is conveniently located in the same wing as the oncology pharmacy. as for the intravitreal injections, they are used during normal business hours, and nurses are already accustomed to picking up these doses from the pharmacy. the oncology pharmacy can insert these into their normal work flow if the ordering physicians are aware that they can expect an hour order time on the medication. the second caci may also be utilized if it is open to reduce the wait time on the medication. discussion the proposed solutions outlined above are just the beginning of a longer journey to usp<800> compliance and optimal employee and patient safety. an ongoing discussion between pharmacy staff and hospital administration will be necessary to continue implementing changes. in this initiative, changes to the workflow were only made regarding niosh class i hazardous drugs, as they posed the greatest, most immediate threat to pharmacy staff exposed to these drugs. further plans to improve usp<800> compliance should involve a comprehensive address for drugs in all other niosh classes to ensure they are being handled and prepared according to the manufacturer's instructions. additionally, discussion surrounding the remodeling of the central clean room pharmacy to include a usp<800> compliant compounding space should be initiated. this plan would include significant financial and logistical barriers, but as usp<800> becomes enforceable, as the joint commission begins to review compliance to the chapter, and as the introduction of new, hazardous drugs to the market continues to rise, remodeling the central pharmacy will be a necessary, long-term solution. acknowledgements: the authors acknowledge gail santucci, pharmd and basil hussein, b.s.pharm, cpht for help in preparation of the manuscript. http://www.ajecr.org/ 325 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org conflict of interest the authors declare no conflicts of interest. references: 1. hazardous drugs—handling in healthcare settings (general information chapter 800). in: the united states pharmacopeia, 39th rev., and the national formulary, 34th ed. rockville, md: united states pharmacopeial convention; 2016:7721-7739. 2. united states department of human services. preventing occupational exposures to antineoplastic and other hazardous drugs in health care settings. washington, dc: national institute for occupational safety and health, 2004. 3. united states department of human services. niosh list of antineoplastic and other hazardous drugs in healthcare settings. washington, dc: national institute for occupational safety and health, 2016. 4. wolters kluwer. simplifi797. available at: http://www.pharmacyonesource.com/products/simplifi797/. accessed may 23, 2018. 5. mitomycin. micromedex solutions. truven health analytics, inc. ann arbor, mi. available at: http://www. micromedexsolutions.com. accessed may 23, 2018. 6. mitomycin injection, powder, lyophilized, for solution [package insert]. canonsburg, pa: mylan institutional llc; 2017. 7. hydroxyurea. micromedex solutions. truven health analytics, inc. ann arbor, mi. available at: http://www. micromedexsolutions.com. accessed may 23, 2018. 8. hydroxyurea [package insert]. gurnee, il: teva pharmaceuticals usa, inc; 2016. 9. bacillus of calmette and guerin vaccine, live. micromedex solutions. truven health analytics, inc. ann arbor, mi. available at: http://www.micromedexsolutions. com. accessed may 23, 2018. 10. tice bcgbacillus calmette-guerin powder, for suspension [package insert]. roseland, nj: organon usa inc; 2016. 11. methotrexate sodium. micromedex solutions. truven health analytics, inc. ann arbor, mi. available at: http://www.micromedexsolutions.com. accessed may 23, 2018. 12. methotrexate injection [package insert]. canonsburg, pa: mylan institutional llc; 2014. http://www.ajecr.org/ 175 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(3):175-179 original article analysis of carbamazepine side effects associated with bone metabolism, folate and vitamin b12 serum levels in isfahan epileptic patients mohammad-reza najafi1, zahra tolou-ghamari2* 1department of neurology, isfahan neurosciences research center, faculty of medicine, isfahan university of medical sciences, isfahan, iran 2department of urology, isfahan urology and renal transplantation research center, isfahan university of medical sciences, isfahan, iran abstract. according to previous publications, chronic uses of aeds reduce bone health, increase homocysteine levels, alkaline phosphates and many other biochemical changes. the aim of this study was to investigate carbamazepine (cbz) effects on bone density, serum levels of folate, vitamin b12 and other biochemical variables. twenty-one adult epileptic patients who received cbz for treatment of epileptic attacks were enrolled. bone mineral density (bmd) was evaluated by dual-energy x-ray absorptiometry method. biochemical indices of bone metabolism, including serum calcium, phosphorus, alkaline phosphatase contents and also serum levels of folate, vitamin b12, homocysteine were measured. all clinical, laboratory and pharmacological data were recorded in d-base and analyzed using spss (version 16) for windows. bmd z-scores, and 25-dihydroxyvitamin d3 concentrations was not differ significantly, but it seems that, the female patients had diminished bmd at the femoral neck (p≤ 0.05). folate or vitamin b12 levels tend toward lower values. this seems to be connected to a tendency toward higher mean corpuscular volume (mcv) with a mean of 84.9 fl (ranged from 59-120 fl). there was no correlation between cbzc0 and homocysteine levels (p≤ 0.63). in compare to normal value the serum calcium content was lower with a mean of 7.8 mg/dl (ranged; 7.0-8.9 mg/dl; p ≤ 0.04). with a mean of 213 u/l serums alkaline phosphatase was significantly higher (p ≤ 0.01) than normal values. due to the need for chronic prescription strategy in order to control convulsion attack, pharmacotherapy with aeds may consequence to changes in serum contents of calcium, phosphorus, alkaline phosphatase in addition to decrease in bmd and increase in homocysteine levels. further studies related to efficacy and side-effects of cbz as the most prescribed aeds in iranian epileptic population seem to be valuable. keywords: carbamazepine, homocysteine, bone metabolism, folate introduction epilepsy is one of the most common chronic neurological disorders, and its significances encompass future beyond the event of seizures. antiepileptic drugs (aeds) are widely used as a chronic treatment strategy to control seizure attack and carbamazepine (cbz) is commonly used aeds. there is evidence for the presence of bone disease in epileptic patients. the special effects of aeds on bone metabolism and the endocrine system are not completely recognized [1-5]. according to previous publications, cbz affect bone metabolism, vitamin b12 and folic acid levels by induction or inhibition of cyochrom-p450 linked to metabolic events [4-17]. bone is a metabolically dynamic tissue which experiences constant formation, maintenance and resorption that termed as osteoblasts, osteocytes and osteoclasts. under regular situations, bone osteoclasts and osteoblasts are strongly joined to each other, so that the amount of bone detached is continuously identical to the quantity of recently bone formed. this equilibrium is accomplished and controlled over the exploit of several systemic hormones and local mediators. [1] adult epileptic patients are in abundant danger for bone damages. this could be associated to underling disease, imbalance and the effects of aeds on bone strength. biochemical abnormalities of bone uptake comprising hypocalcaemia, hypophosphatemia, vitamin d deficiency and augmented alkaline phosphatase have been described previously. chronic treatment with particular drugs deliberates a higher risk of osteomalacia that is accredited to vitamin d deficit. several studies have shown that enzyme-inducing aeds are associated with reduced levels of 25hydroxyvitamin d [7-17]. bone mineral density (bmd) is a differentiation that could be made between osteopenia versus osteoporosis (t score; −1 to ___________________________________________________________ * corresponding author: prof zahra tolou ghamari (toloeghamari@pharm.mui.ac,.ir). http://www.ajecr.org/ mailto:toloeghamari@pharm.mui.ac,.ir 176 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org −2.5 vs < −2.5 sd) respectively. therefore, normal value is a t-score of -1 0r higher. in 1994, the world health organization (who) established a classification of bone mineral density (bmd) according to the standard deviation (sd) differences between a patients' bmd and that of a young-adult reference population. this value is how commonly expressed as a "t-score". a t-score that is equal to or less than -2.5 is consistent with a diagnosis of osteoprosis; a t-score between -1.0 and – 2.5 is classified as low bone mass (osteopenia); and a t-score of -1.0 or higher is normal [1]. many studies have demonstrated that low bmd is associated with an increased risk of fracture [1, 12-17]. another side-effects related to cbz-therapy in epileptic patients could be mentioned as its' effect on metabolic events related to homocysteine levels that could be results as demethylation of methionine. to be metabolized, it needs cofactors folate, vitamin b6 and vitamin b12 which are needed in homocysteine metabolic pathway either through re-methylation or trans-sulfuration. impaired homocysteine metabolic pathways results to hyperhomocysteinemia (hhcy). hhcy is a risk factor for decreased mental function in epileptic patients. it seems that aeds may change metabolic pathways of homocysteine, leading to an alteration of plasma homocysteine levels. hyperhomocysteinemia has been reported for some aeds in epileptic patients after chronic treatment including with cbz and sodium valproate. according to previous publications subsequent to aeds serum biochemical such as homocysteine, folate, and vitamin b12 could be changed in epileptic patients. treatment with most of the frequently used aeds is linked with decreased folate or vitamin b12 serum concentrations and could be a risk factor for hyperhomocysteinemia. earlier studies described that cbz provoke the production of an extensive variety of monooxygenase and conjugating enzymes. these agents are well recognized to decrease the level and activity of many lipidand nonlipid-soluble drugs. as a result enzyme-inducing aeds such as cbz may deliver to the progress of a number of comorbidities, including osteoporosis, sexual dysfunction, vascular disease and cognitive function [17-24]. it is still not completely clear whether that long term treatment with cbz, could be an important risk factor for metabolisms of bone and homocysteine [21]. as prescriptions of cbz in iranian epileptic patients increasing [2-5], therefore study of biochemical changes and bone turnover was of interest that investigated. materials and methods a cross-sectional study in patients under treatment with cbz monotheray visited isfahan epileptic clinics was carried out between the years 2012 to 2013. only patients who received cbz with no history or laboratory results expressive of liver or bone disease were nominated. patients with cbz monotherapy for a period of at least six months were entered in this study. the study was conducted at isfahan neurosciences research centre (inrc) and isfahan urology and kidney transplantation centre and was approved by the institute research ethics committee (irec; grant no; 291158). for each subject bmd referred to be measured by dual x-ray absorptiometry called dxa technology. dxa measured bone mineral content (bmc in grams) and bone area (ba, in square centimeters), then calculated "area" bmd in g/cm2 by divided bmc by ba. t-score, the value used for diagnosis osteoporosis, is the mean bmd of a young-adult reference population from the patients' bmd divide by the standard deviation (sd) of young-adult population. z-score, used to compare the patients' bmd to a population of peers, calculates by subtracting the mean bmd of an age, ethnicity and sex-matched reference population from the patients' bmd and divide by the sd of the reference population. fasting morning blood was obtained for measurements of albumin, total calcium and cbz. sample for homocysteine, was collected in edta vial and was measured using enzyme immunoassay (eia). serum calcium concentrations were measured by standard autoanalzyer technique (normal, 8.2–10.6 mg/dl). serum 1, 25-dihydroxyvitamin d (3) (normal range, 20–74 pg/ml) was measured by radioimmunoassay. serum alkaline phosphatase (normal range, 4-40 u/l) was measured by competitive enzyme immunoassay. trough level of cbz (normal range, 7–14 mg/l) was measured by enzyme immunoassay for each patient. levels of calcium, phosphate, alkaline phosphatase, demographic, clinical, hematological-biochemical and pharmacological data were recorded in d-base. statistical analysis statistical analyses were done using the spss for windows, version 16 (spss inc., chicago, il). descriptive statistics such as means, median and range was calculated for variable of interest and correlation between variables was defined by multiple logistic regression analysis (step-wise). a p value of ≤0.05 was considered as significant. results twenty-one patients (comprised of 10 females and 11 males) with a mean age of 27 years as ranged from 18 to 50 years were studied. the range for serum total alkaline phosphatase in patients received aeds polytherapy was 87-559 iu/1, for those with cbz monotherapy, the value ranged from 87 to 553 with a mean of 213 iu/1, seems to be significantly higher than normal values (60-360 iu/1, p ≤ 0.01). with a mean homocysteine level of 9.6±1.7 (normal values; 5-15 µmol/ l), there was no correlation between cbzc0 and homocysteine levels (pearson’s correlation r= 0.083, p ≤ 0.63). folate (normal range; 2.7 17 ng/ml) or vitamin b12 (normal range; 200-800 pg/ml) levels tended toward the lower side of the reference ranges with a mean value of 3.4 ng/ml and 250 with disparities among individuals. this seems to be connected to a tendency toward higher mean corpuscular volume (mcv). as the mean normal value related to mcv ranged from 79 to 96 fl, in the small number of studied patients in here was with a mean of 84.9 fl that ranged from 59-120 fl. the mean level of serum calcium content was 7.8 mg/dl (ranged; 7.0-8.9 mg/dl) seems to be significantly (p≤0.04) lower when compared to normal values, ranged from 8.2 to http://www.ajecr.org/ 177 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org 10.6 mg/dl. in compare to normal value related to serum phosphorus levels (2.5-4.5 mg/dl) the mean value in population studied was 2.3 mg/dl as ranged from 2.3 to 4.0 mg/dl in population studied. bmd z-scores and 25 figure 1 variability of cbz concentrations among twenty one epileptic patients. dihydroxyvitamin d3 concentrations did not differ significantly. the mean 25-dihydroxyvitamin d3 seems to lie in lower parts of recommended normal values or 20-60 pg/ml. the mean value in population studied was 27.99 pg/ml with a range of 20 to 40.2 ng/ml. bmd was 0.809±0.017 g/cm2 (hip) and z-score was −0.4±0.2. in the small number of females it seems that bmd at the femoral neck decreased. in the 8 out of 21 patients the minimum concentration for cbz ranged from 4.8 to 8 mg/ml. as shown in figure 1, cbz c0 was associated with marked irregularity and large discrepancy with a mean value of 6.8 ng/ml ranged from 0. 5 to 11 ng/ml (normal range; 4-12 ng/ml). discussion for management of epileptic patients, considering of many factors behind a good approach to control convulsion attack seems to be necessary. within this population morbidity and mortality related to hip fractures and cardiovascular disease have been reported to be widespread. therefore, attention to metabolism related to bone and homocysteine could be categorized as one of the most important issue. as a result, in this study we aimed to investigate the effect of cbz as a higher prescribed or best seller aeds on metabolism of bone and biochemical markers such as homocysteine, folate, vitamin b12 and alkaline phosphatase levels. in spite of the side-effects of some aeds including carbamazepine as an enzyme inducer and valproate sodium as an enzyme inhibitor of cytochrome p450, there are limited data in our epileptic patients regarding the monitoring and treatment of bone health and bone disease in this population. many studies recommend that patients with epilepsy under treatment with aeds have an increased risk of fracture, low bmd, and variations in bone metabolism and showed an association between uses of anticonvulsant medications, reduced bone mineral density, and increased fracture risk. [12-15] fall during convulsion attack, instability, immobility and a hereditary tendency to low bmd could be mentioned as several issues that expected to contribute related to the increased risk. [15] fracture risks in patients with cbz have been reported by many authors as 1.88% (95% ci;1.33–2.65), 1.31% (95%ci; 1.14–1.51). [7] in agreement with previous studies, serum alkaline phosphatase (alp) levels were higher in patients. [12-17] previous evidence suggests that cbz affects bone metabolism by changing or reducing vitamin d concentrations. [17-24] in patients with epilepsy vitamin d receptor polymorphism is linked to low bmd, that could be arbitrated throughout vitamin d-parathormone mechanism [16]. according to many previous publications b6, b12 and folate are necessary for the metabolism of homocysteine to methionine [17-23]. an increase in serum levels of homocysteine could be a cause for hyperhomocysteinemia,[25] however due to inter and intra individual variability further studies on epileptic patients are needed to confirm this correlation with cbzmontherapy. there is data indicating that homocysteine might be a risk factor for stroke and dementia. it has been shown that some enzyme inducer aeds through cytochrome p450 might cause disparities in cholesterol, lipoprotein and homocysteine. raised serum levels of homocysteine are a tough and self-directed prognosticator for augmented danger of atherosclerosis development in a dose-dependent consensus. this situation could lead to an increased level of the latent threat production for heart disease and endothelial dysfunction, asymmetric dimethylarginine or the regulator of nitric oxide. asymmetric dimethylarginine is a product of methylation of l-arginine and endogenous nitric oxide synthase inhibitor. nitric oxide plays a role in the seizure attack [21]. finally, due to small size of patients in this study, further clinical investigations are desirable to monitor the prescription of cbz in terms of therapeutic drug monitoring (tdm) among iranian epileptic population and its' association to bone, other biochemical variables and cardiovascular disease. acknowledgement this study was supported by grant no. 291158 from isfahan university of medical sciences. conflict of interest the authors declare no conflicts of interest. references 1. meier c, kraenzlin, me. antiepileptics and bone health. ther adv musculoskelet dis 3: 235-243, 2011. 2. tolou ghamari z. antiepileptic drugs (aeds) polypharmacy could lead to buried pharmacokinetic interactions 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http://www.ncbi.nlm.nih.gov/pubmed?term=misra%20a%5bauthor%5d&cauthor=true&cauthor_uid=20933174 http://www.ncbi.nlm.nih.gov/pubmed?term=aggarwal%20a%5bauthor%5d&cauthor=true&cauthor_uid=20933174 http://www.ncbi.nlm.nih.gov/pubmed?term=singh%20o%5bauthor%5d&cauthor=true&cauthor_uid=20933174 http://www.ncbi.nlm.nih.gov/pubmed?term=sharma%20s%5bauthor%5d&cauthor=true&cauthor_uid=20933174 http://www.ncbi.nlm.nih.gov/pubmed?term=nakken%20ko%5bauthor%5d&cauthor=true&cauthor_uid=20201711 http://www.ncbi.nlm.nih.gov/pubmed?term=taub%c3%b8ll%20e%5bauthor%5d&cauthor=true&cauthor_uid=20201711 459 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2022;9(1):459-464 original article survival and clinicopathologic characteristics of meningioma mehrdad roozbeh1, shokouh taghipour zahir2, mahrooz roozbeh3, farzan safidahaj4* 1department of neurology, brain mapping research center, shahid beheshti university of medical sciences, tehran, iran 2surgical and clinical pathology, shahid sadoughi university of medical sciences, yazd, iran 3cognitive neuroscience, institute for cognitive science studies, tehran, iran 4general medicine, shahid sadoughi university of medical sciences, yazd, iran abstract. with the prevalence of one-third of intracranial brain tumors, meningioma prognosis is affected by the size, location, ability to access the tumor through surgery, and the degree of histological malignancy. we intended to investigate patients' prognosis and clinical features with central nervous system meningioma. records of all patients with central nervous system and spinal cord meningiomas between 2006-2016 in yazd shahid sadoughi, shahid rahnemoun, mortaz, goodarz, and mojibian hospitals were studied. using spss version 17, survival, age, type of treatment, grade, size, and tumor location were investigated in this retrospective cross-sectional study. of the 175 patients (66 (37.7%) male, 109 (62.3%) female) with an average survival time of 78.5 months, tumors recurred in 12 (10.7%) patients. among them, 63 (47%) were between 18-54, and 72 (53%) were between 55-87 years. syncytial was the most common histological subtype, and a significant relationship between age and tumor subtype was found (p-value = 0.03). primary symptoms of headache and nausea/vomiting were in most of them (51, 57%). the most and the most minor expected location was the brain lobes (14 cases) and the spinal cord (2 patients). there was no significant relationship between meningioma subtype-symptoms, survival-gender/ tumor subtype, recurrence-gender/ age, or tumor subtype. also, a significant relationship between age and survival was found (p-value = 0.05). an increase in the prevalence of meningioma in recent years has been due to improvements in imaging techniques and the population's aging. overall, there was a significant relationship between age and meningioma prevalence. keywords: meningioma, brain tumor, survival, recurrence introduction meningiomas are predominantly benign adult tumors, accounting for a third of all intracranial brain tumors [1]. these tumors are often attached to the dura matter and originate from arachnoid meningothelial cells. meningiomas may develop along any surface outside the brain and inside the ventricular system. they usually present with non-localized and vague symptoms or have local findings related to lower brain tissue compression. although most meningiomas are separated from the lower brain tissue, some tumors invade the brain, increasing tumor recurrence. the general prognosis of meningiomas is affected by the size, location, ability to access the tumor surgically, and degree of histological malignancy. very different histological patterns are found in meningiomas, including syncytial (spiral clusters of compact cells in clusters with no visible cell membranes), fibroblastic (with long cells with abundant collagen between them), transitional (characterized by having syncytial and fibroblastic features), psammomatous (abundant psammoma bodies), secretory (with eosinophilic drops and pas-positive). nf2 (neurofibromatosis gene) genetic mutations are more common in tumors with a specific growth pattern (fibroblastic, transitional, and psammomatous) [2]. regarding who, meningiomas are classified into four grades, i to iv. so, grade i is benign and contains the most significant amount of total meningiomas, while only 5% of meningiomas are atypical and anaplastic. unlike grade i meningiomas, which are more common in women, anaplastic and atypical meningiomas are more common in men. grades ii and iii are associated with a higher risk of recurrence. atypical meningiomas are characterized by histological features of prominent nucleoli, increased cellularity, growth without a definite pattern, and a higher mitosis rate, categorized in grades ii / iv. lesions with higher recurrence rates and local growth are more aggressive. anaplastic meningiomas are highly invasive tumors that resemble sarcomas or carcinomas with a high degree of malignancy. however, there is usually some histological evidence that they originated from meningothelial cells. these are tumors in grades iii / iv [35]. so far, studies have been performed on meningiomas of the nervous system, which have examined these tumors in terms of epidemiology and clinical features. most of these ___________________________________________________________ *corresponding author: dr. farzan safidahaj (safi.farzan@gmail.com) http://www.ajecr.org/ mailto:safi.farzan@gmail.com 460 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org table 1 frequency distribution of tumor subtype by age, gender, and status of radiotherapy p meningioma types variable total angiomatous fibroblastic atypical psammomatous transitional syncytial 0.82 49 1(2%) 5(10.2%) 6(12.2%) 5(10.2%) 14(28.6%) 18(36.7%) male gender 91 2(2.2%) 9(9.9%) 6(6.6%) 15(16.5%) 28(30.8%) 31(34.1%) female 140 3(2.1%) 14(10%) 12(8.6%) 20(14.3%) 42(30%) 49(35%) total 0.03 63 1(1.6%) 7(11.1%) 4(6.3%) 8(12.7%) 28(44.4%) 15(23.8%) 18-54 age 72 2(2.8%) 6(8.3%) 8(11.1%) 10(13.9%) 14(19.4%) 32(44.4%) 55-87 135 3(2.2%) 13(9.6%) 12(8.9%) 18(13.3%) 42(31.1%) 47(34.8%) total 129 3(2.3%) 13(10.1%) 11(8.5%) 19(14.7%) 37(28.7%) 46(35.7%) no radiotherapy 4 0 0 1(25%) 0 2(50%) 1(25%) yes 133 3(2.3%) 13(9.8%) 12(9%) 19(14.3%) 39(29.3%) 47(35.3%) total studies have been conducted in the united states and europe. these studies have been able to study its prevalence and prognosis in different populations and the pathways involved in the pathogenesis of the disease, factors affecting the course of the disease, and the survival of patients with meningiomas to pathologists [2, 6–12]. in iran, in some cases, studies have been performed on meningioma and its epidemiological and histopathological factors. however, no reflection on this subject has been published in yazd so far, and on the other hand, in some of the published articles, the time of publication has been related to the past years [1319]. therefore, we decided to study the characteristics of patients with central nervous system meningioma in yazd from 2006 to 2016 to review the epidemiological, clinical, and pathological findings and prognosis of patients so that in the future and with additional studies, measures can be taken in the control of possible risk factors, diagnosis and treatment steps, and more accurate prognosis of patients. materials and methods study design the present study is a retrospective descriptiveanalytical study. the study population included 175 patients with central nervous system, and spinal cord meningioma referred to hospitals in yazd who underwent neurosurgery from the beginning of 2006 to the end of 2016. the required data were extracted from the archived files of these patients. in the present study, the required data were collected by referring to the pathology department of all hospitals in yazd, including shahid rahnemoun, shahid sadoughi, mortaz, goodarz, and mojibian hospitals. according to the pre-prepared checklist, the required data, including age, sex, clinical symptoms, pathological findings, tumor size, tumor grade, type of treatment performed, and the percentage of recurrence after surgery, were extracted by month and entered into the checklist. the recurrence rate was obtained by referring to patients' files and, if not available, by telephone contact. statistical analysis the obtained data is entered into the software spss v. 17 (spss, inc., chicago, il, usa). the relationship between the variables was tested using chi-square and log rank statistical tests. the results were recorded in tables and graphs. the significance level was also considered 0.05. ethical considerations according to the helsinki convention, patients' information is confidential and was not used anywhere other than the dissertation for research purposes. permission to view patient records and collect the required data has been obtained from the medical centers. a trained medical student called the patient's family for survival information. results as a cross-sectional analytical study and survival analysis for ten years, this study was performed from 2006 to 2016 in shahid sadoughi, shahid rahnemoun, mortaz, goodarz, and mojibian hospitals and on 175 patients with meningioma of the central nervous system. of the 175 patients studied, 66 (37.7%) were male, and 109 (62.3%) were female; the tumor subtype was unknown in 35 patients. of patients whose recurrence status was known, 12 with meningioma (10.7%) recurred. six patients with known adjuvant treatment status (3.7 %) underwent radiotherapy. of the 159 patients whose current life condition was known, five died. among 140 patients with known tumor subtypes, 49 (35 %) were male, and 91 (65%) were female. the most common subtypes in both men and women were syncytial (18 men (36.7%) and 31 women (34.1%)). there was no significant difference between meningioma subtypes by gender (p-value = 0.82) (table 1). among 135 patients with known tumor subtypes and age, 63 cases (47 %) were in the age range of 18-54 years, and 72 patients (53%) were in the age range of 55-87 years. the highest frequency among the group of 18-54 years was related to the transitional subtype with 28 cases (44.4%), and then the syncytial with 15 cases (23.8%) had the highest frequency. these findings indicate a significant relationship between the age of meningioma patients and tumor subtypes (p-value = 0.03) (table 1). in this study, among 113 patients whose clinical http://www.ajecr.org/ 461 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org figure 1 survival kaplan-meier survival curve by the age group in patients with meningioma (log-rank test: 0.05). symptoms were evaluated, the highest prevalence of symptoms was related to 65 patients (37.1%) who presented with early symptoms of headache and vomiting/ nausea and were diagnosed with meningioma by imaging. next are 30 patients (17.1%) randomly diagnosed with meningioma in imaging without clinical symptoms. this study has seven cases (4%) with seizures and paralysis. the lowest number of patients was 4 cases (2.3%), belonging to the group of patients with other symptoms such as urinary incontinence. among 90 patients whose tumor subtype and clinical manifestations were known, 51 (57 %) with headache and nausea, and vomiting, 25 (27 %) were asymptomatic, 4 (4 %) with seizures, 7 (8) % had presented with paralysis, and three patients (3%) had other symptoms. among patients with headaches, nausea, and vomiting, the most reported histological subtypes were transitional, syncytial, psammoma, atypical, fibroblastic, and angiomatous. the most common asymptomatic patients were transient, syncytial, psammoma, fibroblastic, and atypical. in patients with seizures, the highest frequency was related to the syncytial form, followed by transitional and fibroblastic. in patients with the initial manifestation of paralysis, syncytial and transitional were the most common. the most common type of tumor diagnosed in patients with miscellaneous symptoms was syncytial. there was no significant relationship between the meningioma subtype and patients' clinical symptoms (p-value> 0.05). in this study, among 34 patients whose meningioma location was known, most cases were located in the brain lobes (14 cases), and the minor cases were related to the spinal cord (2 cases). it should also be noted that 13 cases (7.4%) had meningioma at the base of the brain, and 5 cases (2.9%) had parasagittal and midline meningioma. among 133 patients with known subtype and radiotherapy status, 4 (3%) underwent radiotherapy, of which one was atypical, two were transitional, and one was syncytial subtype (table 1). among the 114 patients who were followed up, the mean survival time in the study was 78.5, with a 95% confidence interval in the range of [76.19-80.90]. the number of patient deaths was 4 cases. of the 114 patients who were followed up, 46 were male, and 68 were female. the average survival time of the men was 76.34 months with a 95% confidence interval [71.25 81.43], and the average survival of women was 80 months with a 95% confidence interval [78.5-81.94]. these findings did not show a significant relationship between patients' survival and gender. of the 111 patients whose living conditions were known, four patients died, all in the 55-87 age group. the mean survival time in the 55-87 was 79.21 months, while in the group of 18-54 was 80 months. the findings showed that survival time decreased statistically significantly with age (figure 1). out of 92 patients whose meningioma subtype and survival time was identified, three deaths (9.4%) occurred in the meningioma group with the syncytial subtype. the average survival in this group is 79.19 months. death did not appear in the other groups. there was no statistically significant relationship between the meningioma subtype and survival time. out of 108 patients with available information about tumor recurrence, 10 (9.3%) experienced recurrence with a mean recurrence time of 74.55 months with a 95% confidence interval in the range of [70.75-78.35]. out of 108 patients with recurrence information, 46 were male, and 62 were female, of which 10 were four males, and six were female. also, the average recurrence time in the group of men was 74.90 months with a 95% confidence interval [69.21-80.59], and in the group of women, 74.34 months with a 95% confidence interval in the range [69.2879.39]. no statistically significant relationship was found between recurrence time and patients' gender (p-value = 0.89) of the 105 patients whose recurrence and age data were available, 51 were in the age range of 18-54, and 54 were in the age range of 55-87. four cases of recurrence were in the age group of 18-54 and 6 cases in the age group of 55-87. in the group of 18-54 years old, the average recurrence time was 75.42 months with a 95% confidence interval of [70.1980.66], and in the 55-87 age group, the average recurrence time was 73.43 with a 95% confidence interval in the range [67.75-79.12]. thus, there was no statistically significant relationship between recurrence time and the age of patients (p-value = 0.61). among 86 patients whose recurrence information and histological subtype of tumors were known, in the syncytial group, 2 cases (6.9%) with a mean recurrence time of 76.38 months, and in the transitional group, one patient (4.3%) with a mean recurrence time of 77.83 months, in the atypical group 2 cases (18%) with a mean recurrence time of 47.94 months, and in the fibroblastic group 2 cases (22%) with a mean recurrence time of 67 months had experienced a recurrence. no recurrence was seen in the psammomatous and angiomatosis groups. overall, recurrence occurred earlier in the atypical group than in other groups, possibly due to the malignant nature of this pathological subtype. these findings showed no association between the meningioma subtype and tumor recurrence (p-value = 0.42). http://www.ajecr.org/ 462 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org discussion meningiomas are benign tumors found in all parts of the central nervous system, including the brain, spinal cord, and orbit. by who definition, meningiomas originate in the arachnoid cells of the leptomeninges. these arachnoid cells have a dual origin of embryonic mesenchyme and the anterior portion of the neural crest. according to this definition, meningiomas are classified into 15 subtypes in 3 grades. the most common category of grade i account for 80-90 % of cases and mainly includes meningothelial, fibroblastic, transitional, angiomatous, and psammomatous subtypes. grade ii comprises 5-15 % of all patients and atypical subtypes, clear cell and choroid. malignant cases in grade iii comprise 1-3 % of all cases. this category includes anaplastic, papillary, and rhabdoid subtypes [20–28]. according to studies in the united states, meningioma is the most common intracranial tumor in adults and accounts for one-third of primary central system tumors. the annual incidence of meningioma in the united states is reported to be 8.3 per 100,000 population. this rate increases with age, so in people over 70, the risk is 3.5 times higher over 70 [23, 29, 30]. criteria for patients entering databases differ in different studies. so, histological confirmation was not required in the cbtrus database, unlike the databases used in the extensive survey of xavi et al. in france. hence, the age group involved in the french study was 55-64 years, while in the cbtrus databasebased studies, the prevalence of meningioma increases with age [31,32]. recent studies have shown an increase in the incidence of meningioma in different communities, which may be due to improved quality of imaging techniques or increasing age of communities because this tumor has slow growth and is more common among older age groups [33]. in our study, more meningioma cases were found in older age groups. in addition, there was a significant difference in the frequency of meningioma subtypes in the age groups of 18-54 years and 55-87 years. the incidence of meningioma in women is 56% higher than in men [31]. although in the present study, no significant difference was found between meningioma subtypes and gender, 37.7% of patients were men, and 62.3% were women, which is consistent with the findings of previous studies. in the most extensive study of 13038 meningioma patients in france from 2006 to 2010, the most common clinical manifestation was a headache, seen in one-third of patients, followed by motor and sensory disturbances with 29% and seizures with 24.6% in the following categories. other manifestations included mental disorders, increased intracranial pressure, and miscellaneous manifestations. of patients, 9% did not show any symptoms at the time of surgery [32]. these findings are similar to the data from our study in which the most common manifestations were headache, nausea, and vomiting (57.7%), except those asymptomatic cases (26.5%) were the second most common clinical manifestation of patients and seizures, paralysis. miscellaneous symptoms were in the following categories. in the study of kallio et al., the most common sites of meningioma were the parasagittal regions and cerebrum falx, sphenoid ridge, the middle cavity, and cerebral lobes, respectively, and the lowest frequency was related to the posterior cavity [34]. in our study, the highest frequency of meningioma was observed in the cerebral lobes (41.2%), cranial base (382%), midline, parasagittal (14.7%), and spinal cord (5.9%), respectively. patterson et al., following a 25 -year follow-up of patients with parasagittal meningioma as one of the most common areas of meningioma, showed that the tumor recurrence rate after 25 years was 47%, which was higher compared with tumors in other areas of the nervous system. this finding may be due to the specific location of the tumor and the ease of growth of tumor tissue in the area. therefore, they suggested longterm follow-up to evaluate the recurrence rate of tumors in this area. on the other hand, the special location of tumors in this area, due to its proximity to venous sinuses and cerebral veins, deprives the surgeon of the possibility of complete resection of the tumor, so the rate of tumor recurrence is higher than tumors in other places [35]. factors influencing meningioma recurrence include the extent of tumor resection (based on the simpson scale), tumor grade, older age, and female gender [29,36,37]. although treatment protocols for meningioma treatment include surgery, radiotherapy, and chemo-therapy, tumor resection is essential in treating and reducing recurrence. although complete resection of the tumor is curable to treat cases of benign meningioma, there will still be a small risk of local recurrence, with rates of up to 60% reported over 15 years [38]. however, in studies such as the mccarthy et al. study, which had a shorter follow-up period, a 5 -year recurrence rate of 19.2% was reported for benign meningiomas and 32.4% for malignancies, regardless of the type of treatment [39]. in another 10 -year study by rogers et al., the recurrence rate was 20-39 % [38]. these differences indicate the need for long-term follow-up to assess the recurrence rate of meningioma in patients. in our study, the overall recurrence rate was 10.7%. in 2016, a study by fonkem et al. on 376 meningioma patients showed that the prevalence of meningioma increased with age over 45. despite the higher prevalence of meningioma in blacks, its majority from 1976 to 2013 is on the rise among whites. in this study, it was shown that there is a significant relationship between age and the frequency of meningioma. still, due to the lack of a classification of patients with race characteristics or skin color, this relationship was not measured [24]. a study by das et al. in singapore found that the most common tumor of the central nervous system was a meningioma, which also showed dysfunction of p53 and apoptotic disorder has not been effective in the pathogenesis of meningiomas [40]. while in a study by taghipour zahir et al. conducted in yazd province from 2006 to 2013, the highest prevalence of central nervous system tumors is related to astrocytoma [25]. tumor pathogenesis was not investigated in our study. a 2015 study by harrison et al. investigated the effect of gender on the incidence and prognosis of 1709 cases of spinal cord meningioma, showing that spinal cord meningiomas accounted for a high percentage of spinal cord tumors. although the prevalence of spinal meningioma is higher in women, the mortality of this tumor is higher in http://www.ajecr.org/ 463 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org men [31]. in this study, we had two cases of spinal cord meningioma that did not cause mortality. in 2007, rogers et al. studied radiotherapy's role in treating intracranial meningiomas. they concluded that small, asymptomatic, slow-growing meningiomas could be followed up by serial examination and imaging. if indicated for surgery, subtotal resection with radiotherapy helps local control of the tumor and improves survival. for meningiomas that were close to the visual pathways or had a high risk of cerebral edema, the technique fractioned external beam radiotherapy was suggested [41]. in this study, six patients underwent radiotherapy after surgery, but surgery and radiotherapy techniques were unavailable. regarding pathological subtype, one case was atypical, 2 cases were transitional, and one was syncytial. a study by ikawa et al., conducted in 2017 in japan on surgery for the elderly with meningioma, found that postoperative mortality rates increased with age (especially over 65 years). this doubles the importance of deciding on surgical indications for the elderly [42]. a systematic review and meta-analysis by poon et al. found that elderly patients undergoing meningioma surgery were found to have an overall risk of postoperative complications of 20% for these patients. before surgery, the risk of complications for the patient was calculated so that a better decision could be made about the patient's surgical indication [43]. in the study of kuratsu et al., the risk of complications after meningioma surgery was 23.3% in people over 70 years and 3.5% in young people [22]. in our study, there were five deaths, all in old age. a long-term follow-up study conducted by rochat et al. in denmark from 1935 to 1984 followed children who underwent meningioma. they concluded that their prognosis was worse than expected. this was partly due to the lack of advanced imaging techniques in those years and the resulting large tumor size at diagnosis. pediatric meningiomas can also be associated with mutations in the neurofibromatosis two genes, which makes tumor resection difficult [44]. in our study, the minimum age was 18 years and did not include pediatric meningiomas, which may be due to referrals of pediatric brain tumors to more wellequipped surgical centers. it is suggested that a database with complete details of patients and comorbidities at the time of surgery be prepared. that information about patient visits after surgery is added to the database. also, if the patient dies, the cause and its connection with meningioma should be determined as much as possible. long-term follow-up studies will help find more accurate survival and surgical complications. conclusion the increasing prevalence of meningioma in recent years might be due to improved imaging quality and increasing population age. in previous studies, the majority of meningioma in women was significantly higher. still, in this study, despite a large number of women with meningioma, no significant difference was found between gender and meningioma subtype. the prevalence of meningioma was statistically significant with age. the findings suggest the need for a longer follow-up study. acknowledgments thanks to our dear friend, dr. aryanfar, for offering pearls of insight to us throughout this research and for passionate support. i'd want to thank the pathology departments of shahid rahnemoun, shahid sadoughi, mortaz, goodarz, and mojibian hospitals and their authorities for their assistance in providing me with permission to access data collection. also, special gratitude to the patients` families for providing us with survival information. conflict of interest the authors declare no conflicts of interest references 1. alruwaili aa, de jesus o. meningioma. [updated 2021 sep 15]. in: statpearls. treasure island (fl): statpearls publishing; 2022. available from: https://www. ncbi.nlm.nih.gov/books/nbk560538/. 2. kamenova m, guzman r, soleman j. demogra-phics and outcome of histologically confirmed intracranial meningiomas. clin transl neurosci 3:2514183x19894945, 2019. 3. huntoon k, toland ams, dahiya s. meningioma: a review of clinicopathological and molecular aspects. front oncol 10:579599, 2020. 4. riemenschneider mj, perry a, reifenberger g. histological classification and molecular genetics of meningiomas. lancet neurol 2006;5(12):1045–54. available from: http://www.ncbi.nlm.nih.gov/pubmed/ 17110285. 5. aster jc, abbas ak. robbins basic pathology. philadelphia, pa: elsevier saunders; 2013. 6. wanis ha, møller h, ashkan k, et al. the incidence of major subtypes of primary brain tumors in adults in england 1995-2017. neuro oncol 23:1371-1382, 2021. 7. ostrom qt, francis ss, barnholtz-sloan js. epidemiology of brain and other cns tumors. curr neurol neurosci rep 21:68, 2021. 8. miller kd, ostrom qt, kruchko c, et al. brain and other central nervous system tumor statistics. ca cancer j clin 71:381-406, 2021. 9. garzon-muvdi t, yang w, lim m, et al. atypical and anaplastic meningioma: outcomes in a population-based study. j neurooncol 133:321-330, 2017. 10. fonkem e, dandashi ja, stroberg e, et al. a retrospective analysis of meningioma in central texas. j epidemiol glob health 6:87-93, 2016. 11. chebil c, boumediene f, cicero ce, et al. epidemiology of primary brain tumors in the province of catania during the 2003-2016 period. neuroepidemiology 55:473-483, 2021. 12. barresi v, caffo m, tuccari g. classification of human meningiomas: lights, shadows, and future perspectives. j neurosci res 94:1604-1612, 2016. 13. mehrazin m, rahmat h, yavari p. epidemiology of primary intracranial tumors in iran, 1978-2003. asian pac j cancer prev 7:283, 2006. 14. bitaraf ma, azar m, miri sm, et al. radiosurgery for skull base meningiomas: a study on 230 cases in iranian http://www.ajecr.org/ 464 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org gamma knife center. tehran univ med j 68:162-167, 2010. (in persian). 15. faraji m, birjandi a. pediatric meningioma. ofoghe-danesh 2004;10(1):14-17, 2004. (in persian). 16. samadi n, ahmadi sa. meningioma: a clinicopathological evaluation. malays j med sci 14:46-52, 2007. 17. shiravi khozani m, moradi a, solat yekani s f, et al. pathologic study of ki-67 and p53 protein expression in patients with meningioma referred to shohadayetajrish hospital. res in med 37:102-106, 2013. (in persian). 18. taghipour m, razmkon a, bakhtazad a. high prevalence of intracranial meningioma in jewish population in shiraz, southern iran. neurosurg q 20:68-70, 2010. 19. zakarian b, tonkaboni js. meningioma in a dog in iran: a case report, together with a review of the literature and special reference to the aetiology. j small anim pract 12:37-43, 1971. 20. mashayekhi f, saberi a, mashayekhi s. serum timp1 and timp2 concentration in patients with different grades of meningioma. clin neurol neurosurg 170:84-87, 2018. 21. da broi m, borrelli p, meling tr. predictors of survival in atypical meningiomas. cancers (basel) 13:1970, 2021. 22. ogasawara c, philbrick bd, adamson dc. meningioma: a review of epidemiology, pathology, diagnosis, treatment, and future directions. biomedicines 2021;9(3):319. 23. farokhi mr, ansari z. recurrence of intracranial meningioma and its contributive factors; a 20year study. tehran univ med j 65:91-96, 2007. 24. fonkem e, dandashi ja, stroberg e, et al. a retrospective analysis of meningioma in central texas. j epidemiol glob health 6:87-93, 2016. 25. zahir st, vakili m, navabii h. clinicopathological findings and five-year survival rates for patients with central nervous system tumors in yazd, iran 15:103191023, 2014. 26. komori t. the 2016 who classification of tumours of the central nervous system: the major points of revision. neurol med chir (tokyo) 57:301-311, 2017. 27. perry a, stafford sl, scheithauer bw, et al. meningioma grading: an analysis of histologic parameters. am j surg pathol 21:1455-1465, 1997. 28. bhat ar, wani ma, kirmani ar, ramzan au. histological-subtypes and anatomical location correlated in meningeal brain tumors (meningiomas). j neurosci rural pract 5:244-249, 2014. 29. jordan jt, plotkin sr. benign intracranial tumors. neurol clin 36:501-516, 2018. 30. kuratsu j, ushio y. epidemiological study of primary intracranial tumours in elderly people. j neurol neurosurg psychiatry 63:116-118, 1997. 31. kshettry vr, hsieh jk, ostrom qt, et al. descriptive epidemiology of spinal meningiomas in the united states. spine (phila pa 1976) 40:e886-889, 2015. 32. zouaoui s, darlix a, rigau v, et al. épidémiologie descriptive de 13 038 cas de méningiomes opérés en france entre 2006 et 2010. neurochirurgie 64:15-21, 2018. 33. park bj, kim hk, sade b, et al. epidemiology. in: lee jh, editor. meningiomas: diagnosis, treatment, and outcome. london: springer-verlag; p 11. 2009. 34. kallio m, sankila r, hakulinen t, et al. factors affecting operative and excess long-term mortality in 935 patients with intracranial meningioma. neurosurgery 31:212, 1992. 35. pettersson-segerlind j, orrego a, lönn s, et al. long-term 25-year follow-up of surgically treated parasagittal meningiomas. world neurosurg 76:564-571, 2011. 36. louis dn, perry a, reifenberger g, et al. the 2016 world health organization classification of tumors of the central nervous system: a summary. acta neuropathol 131:803-820, 2016. 37. simpson d. the recurrence of intracranial meningiomas after surgical treatment. j neurol neurosurg psychiatry 20:22-39, 1957. 38. rogers l, barani i, chamberlain m, et al. meningiomas: knowledge base, treatment outcomes, and uncertainties. a rano review. j neurosurg 122:4-23, 2015. 39. mccarthy bj, davis fg, freels s, et al. factors associated with survival in patients with meningioma. j neurosurg 88:831-839, 1998. 40. das a, tang wy, smith dr. meningiomas in singapore: demographic and biological characteristics. j neurooncol 47:153-160, 2000. 41. therapy gr, city sl. role of radiation therapy in treating intracranial meningiomas. neurosurg focus 23:e4, 2007. 42. ikawa f, kinoshita y, takeda m, et al. review of current evidence regarding surgery in elderly patients with meningioma. neurol med chir (tokyo) 57:521-533, 2017. 43. poon mt-c, fung lh-k, pu jk-s, et al. outcome of elderly patients undergoing intracranial meningioma resection – a systematic review and meta-analysis. br j neurosurg 28:303-309, 2014. 44. rochat p, johannesen hh, gjerris f. long-term follow up of children with meningiomas in denmark: 1935 to 1984. j neurosurg 100(2 suppl pediatrics):179-182, 2004. http://www.ajecr.org/ 184 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(4):184-186 original article the effect of delayed diagnosis on mortality in doudenal injuries hassan calis*, nuraydin ozlem 1department of general surgery, ahi evran university training and research hospital, kirsehir, turkey abstract. duodenal injuries can occur after penetrant or blunt trauma. they are mostly seen after penetrant trauma. delayed diagnoses of duodenal injuries are often accompanied by morbidity and mortality. in this study, follow-up and examination data from cases with duodenal injuries were evaluated retrospectively. hospital records of 18 patients with duodenal injury who were followed-up were evaluated. four patients with missing information related to follow-up and treatment processes were excluded from the study. thus, 14 patients were included in the study. of these, 6 patients were injured after penetrant and 8 patients after blunt trauma. one patient with mural hematoma on the first part of duodenum after a blunt trauma was medically followed-up. sepsis-related late stage mortality was observed in 5 patients. sepsis-related mortality was observed in 3 patients with delayed diagnosis and examination after getting stabbed. mortality related to sepsis occurring after suture deficiency on the repair line was observed in one patient with duodenal injury after blunt trauma. mortality related to sepsis after polytetraflouroethylene graft primary repairment was observed in one patient with iatrogenic duodenal injury. following the stabilization and resuscitation of the patients after duodenal injuries, if the specialists are not skeptical in the first examination, there is usually a delay in the diagnosis. early period vital sign and examination findings and radiological evaluations of the patients may not give exact results after trauma. in such a case, repeated examination and radiological evaluations may help. early stage mortality is usually related to large vessel injuries, however late-stage mortality is related to delayed diagnosis and treatment, sepsis, duodenal fistula, pancreatic and choledochal injuries. keywords: duodenum, injury, trauma, delayed diagnosis, mortality introduction duodenal injuries, most of which are caused by penetrant traumas, are generally seen around the age of 30 years old. these injuries may lead to serious complications and death if there is a delay in the diagnosis. in this study we have retrospectively evaluated the effect of diagnosis and treatment of duodenal injury on mortality in the examined cases. materials and methods in this study, 18 patients who were followed up because of duodenal injury during 1990-2015 were retrospectively examined. four patients with missing information related to follow-up and treatment processes were excluded. thus, 14 patients were included in the study. the records of the patients related to demographical characteristics, type of injury, duration until the surgical intervention, intra-operative data and the procedures were obtained. results of the patients, 9 were female and 5 were male. the average age was 35 (range: 12 58). 6 patients were injured after penetrant and 8 patients after blunt trauma (table 1). in one case with injury after blunt trauma; the patient with mural hematoma in the first part of duodenum was medically followed up and discharged. whipple procedure was applied to a patient who had grade 4 avulsion on duodenum and head of the pancreas. one patient had grade 5 duodenal injury and the patient was treated with roux-en-y diverticulization, cholecystectomy and bile duct drainage. another patient had grade 4 spleen injury and grade 2 duodenum laceration and he was treated with primary suture repairment, splenectomy, cholecystectomy and bile duct drainage. two of the patients had motor vehicle accident and as a result they had duodenum injury on the second part and they both received primary repairment. one patient had go-kart accident and as a result he had duodenal injury on the second part and he received laparoscopic primary repair. one patient had grade 4 duodenum injury and was treated with primary repairment, but the patient had suture failure and died because of sepsis on the 23rd post-operative day. four patients had penetrant trauma injury and two had iatrogenic trauma after being stabbed. one patient had fullthickness injury on the liver, right kidney and the intersection of the duodenum parts 3-4 after being stabbed and the patient received primary repairment. three patients ___________________________________________________________ * corresponding author: hassan calis, md (drhasancalis@hotmail.com). http://www.ajecr.org/ mailto:drhasancalis@hotmail.com 185 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org were injured after being stabbed and they had exploratory surgery and no duodenal injury was observed. a new operation was planned since the abdominal examination findings of the patients were abnormal (on account of the fact that abdominal ultrasonography and abdominal ct scan were not commonly used in 1990s, re-laparotomy was exercised). the patient who received re-laparotomy on the fourth post-operative day had injury on the 3rd part of the posterolateral inferior of the duodenum and he was treated with primary suture repairment, cholecystectomy, bile duct drainage, tube duodenostomy and feeding jejunostomy. the patient who had suture failure on the primary repairment line did not accept reoperation and died. the patient who received re-laparotomy on the fifth postoperative day was treated with lateral duodenostomy, feeding jejunostomy and tube gastrostomy. the general condition of the patient kept deteriorating and the patient died on the 20th post-operative day because of sepsis. the patient who received re-laparotomy on the seventh postoperative day had injury on the lateral first part of the duodenum and he received primary suture repairment and pyloric exclusion but he died on the 17th post-operative day because of sepsis. in one of the patients who had iatrogenic duodenum injury, sponge was forgotten in foramen winslow after conventional cholecystectomy and then the patient was re-operated on the fifth post-operative month, he received gastrotomy and sponge extraction. the other patient developed injury on the second part of the duodenum dependent on cautery injury during laparoscopic cholecystectomy and he was treated with primary repairment using polytetrafluoroethylene graft. the patient died on the 12th post-operative day because of sepsis. discussion duodenal injuries are observed in %4.3 (% 3.7% 5) of the patients who have abdominal injuries. duodenal injuries are observed five times more in man and %70 of the cases are at ages 16-32 [1]. the frequency of the duodenum injury dependent on blunt trauma is %11-26 [2] and in our study this figure is %57 higher. since duodenum is neighbor to vital organs, sole injury of the duodenum is rare. liver is accompanied the most in injuries (%17). %15 vascular injuries, %13 colon, %12 pancreas, %11 small intestine, %9 stomach follows the liver in injuries respectively [1]. since duodenum is neighbor to vital organs the diagnosis and treatment is also vital. in our cases; liver, pancreas, spleen and right kidney were the organs injured other than duodenum. in duodenum injuries 2nd part was the most frequently injured area with %36, 3rd part was %18, 4th part was %15 and 1st part was %13 in the frequency of injuries. duodenum multiple area injury is observed %18 [1]. though it has low sensitivity, measurement of serum amylase may be useful in duodenal injuries [3]. some authors argue that %50 of the cases with gastrointestinal and duodenal injuries have an increase in the level of serum amylase [4]. though it is not specific, amylase measurement in peritoneal lavage liquid may be useful in the cases with duodenal injuries [5]. however, some authors argue that measurement of serum amylase should not be used as an indicator for exploratory surgery [1]. subdiaphragmatic free air in abdominal graph, retroperitoneal air, absence of the psoas shadow and lumbar spine scoliosis may give clue about injuries during pre-operative radiological evaluation [6]. ultrasonography (focus assesment for the sonographic evaluation; fast) provides little benefit since it is not efficient enough in the evaluation of retroperitoneal structures of the duodenum. opaque ct scan imaging is the most useful way especially in the evaluation of retroperitoneal structures of the duodenum [5]. occasionally negative results may be yielded in contrasted ct scan imaging in case it is performed in early stage and if there is an abnormal intestine image related to paraduodenal hematoma or inexplicable low level of liquid [7]. if the radiological evaluation does not help and the physician suspects of duodenal injury for a case, another means of diagnosis may be laparoscopy or laporotomy [6]. since radiological evaluation was not sufficient enough, three of our patients received diagnostic laparotomy and one patient received diagnostic laparoscopy. following the stabilization and resusitation of the patients after duodenal injuries, if the specialists are not skeptical in the first examination, there is usually a delay in the diagnosis. a thorough examination must be performed in cases of injury mechanism (acceleration and deceleration), upper abdominal rigidity and tachycardia, vomiting and fever. vital and examination findings, radiological evaluations of the patient may not give exact results in the early post-traumatic stage. in such a case, repeated examination and radiological evaluation may help. the first evaluations and exploratory laparotomy findings of the three patients with duodenal injury caused by being stabbed were considered as normal. since the abdominal examination findings of the patients were abnormal, and on account of the fact that abdominal ultrasonography and abdominal ct scan were not variable no. male/female 5/9 blunt t rauma 8 follow-up 1 whipple procedure 1 roux-en-y divert iculizat ion, 1 cholecyst ect omy and bile duct drainage p rimary sut ure repairment , splenect omy, 1 cholecyst ect omy and bile duct drainage p rimary repairment 3 laparoscopic primary repairment 1 p enet rant t rauma 6 p rimary repairment 1 p rimary repairment , cholecyst ect omy, 1 bile duct drainage, t ube duodenost omy and feeding jejunost omy lat eral duodenost omy, feeding jejunost omy 1 and t ube gast rost omy p rimary sut ure repairment and 1 pyloric exclusion gast rot omy and sponge ext ract ion 1 p rimary repairment using 1 polyt et rafluoroet hylene (p t fe) graft t able 1 t reat ment modalit ies in p at ient s http://www.ajecr.org/ 186 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org commonly used in 1990s, re-laparotomy was exercised to these patients. if surgery is planned for the patients with suspicious duodenal injury and if these patients have conditions such as duodenal subcutaneous emphysema, bile in the duodenal wall, free bile, retroperitoneal hematoma around the duodenum or perirenal hematoma, the physician should mobilize the duodenum and evaluate each of the four parts. even if perforation is detected on the anterior wall during the operation, posterior wall should also be evaluated with mobilization. duodenal mobilization should be performed even for the cases without post-traumatic injury [8]. full-thickness injury of one patient on the posterior wall of the intersection of the duodenum parts 3-4 after being stabbed could only be realized after the complete mobilization of the duodenum. duodenal injuries can be treated with simple methods such as primary repairment (duodenoraphy) or it can be treated with complicated methods such as resection, anastomose, duodenal diverticulation, pyloric exclusion, pancreatic duodenectomy. no single method is successful in preventing duodenal fistula [9]. most authors prefer using single or double layer primary repairment or resection and anastomose for duodenal injuries. it is important to keep the lumen width while performing primary repairment. if the patient has sepsis in the late stage, these options are limited [10]. even if we performed some surgical methods such as primary suture repair plus cholecystectomy plus bile duct drainage plus tube duodenostomy plus feeding jejunostomy, lateral duodenostomy plus feeding jejunostomy plus tube gastrostomy, primary suture repair and pyloric exclusion, and primary repair with ptfe punch graft, the result did not change in our patients who developed mortality caused by sepsis. mortality rates in duodenal injuries may change around 5-30%. early stage mortality is usually related to large vessel injuries, however late stage mortality is related to delayed diagnosis and treatment, sepsis, duodenal fistula, pancreatic and choledochal injuries. sepsis related late stage mortality was observed in 5 patients. three patients, who got stabbed, died because of sepsis after delayed diagnosis and examination. one patient with iatrogenic duodenum injury died because of sepsis after polytetrafluoroethylene graft primary repair was performed. after blunt trauma, suture failure developed on the repair line in one patient with duodenal injury, and the patient died because of sepsis. delay in diagnosis and treatment results in high levels of mortality and morbidity [1]. mortality was observed in three patients because of delayed diagnosis and treatment; limited radiological evaluation also had an impact on their mortality. the relationship between an increase in radiological evaluation possibilities and lower levels of mortality is a remarkable detail. most surgeons may not be suspicious of a duodenal injury because especially the ones following blunt traumas are rare. repeated examinations and auxiliary imaging methods are important for diagnosis. additionally, intra-operative skepticism is also important. in cases of duodenal injuries, it is important to mobilize the duodenum and evaluate each of the four parts. it should be kept in mind that a delay in diagnosis and treatment results in high levels of mortality and morbidity. conflict of interest the authors declare no conflicts of interest. references 1. esther garcia santos, ana soto sanchez, juan m. verde, corrado p. marini, juan a. asensio, patrizio petrone. duodenal injuries due to trauma: review of the literature. cir esp 93:68-74, 2015. 2. velmahos gc, constantinou c, kasotakis g. safety of repair for severe duodenal injuries. worl j surg 32:7-12, 2008. 3. fang jf, chen rj, lin bc. surgical treatment and outcome after delayed diagnosis of blunt duodenal injury. eur j surg 165:133-139, 1999. 4. asensio ja, demetriades d, hanpeter de, gambaro e, chahwan s. management of pancreatic injuries. curr probl surg 36:325-419, 1999. 5. rotondo mf, newell ma. pancreatic and duodenal injuries. in: current surgical therapy (cameron jl, ed). 9 th edition. cv mosby, missouri, pp 992-996, 2008. 6. jayaraman mv, mayo-smith ww, movson js, dupuy de, wallach mt. ct of the duodenum: an overlooked segment gets its due. radiographics 21:147160, 2001. 7. osuka a, idoguchi k, muguruma t, ishikawa k, mizushima y, matsuoka t. duodenal disruption diagnosed 5 days after blunt trauma in a 2-year-old child: report a case. surg today 37:984-898, 2007. 8. hemanga k bhattacharjee, mahesh c misra, subodh kumar, virinder k bansal. duodenal perforation following blunt abdominal trauma. j emerg trauma shock 4:514-517, 2011. 9. degiannis e, boffard k. duodenal injuries. br j surg 87:1473-1479, 2000. 10. rickard mj, brohi k, bautz pc. pancreatic and duodenal injuries: keep it simple. anz j surg 75:581-586, 2005. http://www.ajecr.org/ 434 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2021;8(1):434-439 original article the effect of therapeutic use of music on quality of life in fibromyalgia patients özdemir ç1*, aypak c2, özdemir i3, akbıyık di4, görpelioğlu s2 1department of family medicine, kütahya provincial health directorate, kütahya, turkey 2department of family medicine, ankara dışkapı yıldırım beyazıt training and research hospital, ankara, turkey 3department of physical medicine and rehabilitation, yoncalı physical therapy and rehabilitation center, kütahya, turkey 4department of family medicine, hitit university, çorum, turkey abstract. this study’s aim was to determine the effect of music therapy on symptoms and functions in patients with fibromyalgia syndrome (fms). women diagnosed with fms for at least 6 months included in the study. 16 patients were in the music treatment group and 51 patients were in the control group. patients in music treatment group were given a music cd which includes nature sounds recommended by the turkish psychological association. the patients were evaluated with fibromyalgia impact questionnaire (fiq) before and after the treatment. the fiq-function (p<0.001), fiq-overall impact (p=0.001) and fiq-symptoms (p<0.001) subscales improved after music treatment. all fiq scores were similar in controls before and after treatment. fiq total score (p=0.001), symptoms score (p=0.006), overall impact score (p=0.008) and function score (p=0.010) were lower in the music group after treatment. in conclusion, this study supports a potential benefit of music in the treatment of fms and fms-associated symptoms. keywords: fibromyalgia, music therapy, fibromyalgia impact questionnaire. introduction fibromyalgia syndrome (fms) is a chronic pain syndrome accompanied by fatigue, sleep disorders, mood changes, cognitive and memory disorders, and also sensory processes can be affected [1-4]. it has been suggested that the central and autonomic nervous system, neurotransmitters, hormonal or immune system dysfunctions, or psychiatric factors are effective on fms pathogenesis [5]. however, fms pathophysiology has not been fully elucidated due to the absence of a diagnostic laboratory and imaging method available for the diagnosis of fms. its prevalence has been reported to range between 1.7 % and 5.4 % [6]. fibromyalgia has not considered as a disease by the world health organization until 1992 [7]. fms is a challenging and non-curable disease because of the complicated and incomprehensible pain observed in patients experiencing a restriction in intentional movements and functional mobility due to generalized pain [8]. the most preferred treatments in the treatment of fms include anti-convulsant, serotonin-noradrenaline reuptake inhibitors, exercise-based treatments, psychology-based treatments, and complementary/alternative treatments. there is increasing evidence that non-pharmacological treatments are effective as well as pharmacological treatments [9]. based on current data concerning non-pharmacological treatments acupuncture, biofeedback, chiropractic, cognitive-behavioral therapies, aerobic exercise, hydrotherapy/spa therapy, hypnotherapy, massage, meditation, awareness / mind-body therapy, homeopathy can be effective [10]. music, which is a common consensus between cultures, is taken into consideration as a general relaxant and a hobby. today, therapies are available in different concepts, with varying duration and repetitions, applied with the help of music therapists or musical instruments. with the increasing number of evidence-based studies, the effect of music therapy, when added to the treatment of some diseases, has been proved. it has been suggested that the analgesic effect of music is exerted by central mechanisms [11]. considering mood disorders and pain observed in fms patients, it is thought that music can be used in the treatment of fms. in a limited number of studies, the effects of different types and complicated music treatments used for fms patients were examined. therefore, in this study, unlike other studies in fms patients, the aim was to show the effect of music therapy composed of sounds of nature on disease-related ___________________________________________________________ * corresponding author: çağla özdemir, md (cagla_gocen06@yahoo.com.tr) http://www.ajecr.org/ 435 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org figure 1. study design. symptoms and functions in randomized controlled study design. materials and methods this study was planned between 01/06/2017 and 01/12/2017 in a prospective randomized controlled study design. between these dates, 101 fms patients who met the inclusion and exclusion criteria of the study were reached. for fms, female patients over the age of 18, who met the 2010 american college of rheumatology (acr) criteria and diagnosed with fms at least six months previously, were included in the study. people with hearing problems, pregnancy, another accompanying central sensitization syndrome or schizophrenia, etc.; those with a psychiatric illness, and patients whose pharmacological treatment for fms changed within the last three months, those without music listening devices, and not sufficient socio-cultural level to understand the questionnaire or scale items to be responded by them were excluded from the study. the patients were randomized to both groups in a 1: 1 ratio. accordingly, 50 patients in the intervention and 51 patients in the control group were enrolled in the study. however, 34 of the 50 patients left the study because they declined music therapy, moved out, or lost –to follow-up. the study was performed with 16 fm patients in the intervention group and 51 fm patients in the control group. with g * power 3.1.9.2 program, when an alpha error of 0.05, it was calculated that the power of the study was 87% in the number of 16/51 patients. figure 1 represents the study design. a total of 101 patients with fibromyalgia were included for the study. the patients were randomized to both groups in a 1: 1 ratio. 23 patients were excluded from the study because of move out or lost to follow up.11 patients were drew from the study because of non-adherence to treatment. analysis did not include these 34 patients. the study was completed with 16 patients). data concerning age, education level, occupation and comorbid disease of all patients were recorded at baseline. no changes were made to the conventional treatments that patients were receiving in both treatment groups. the intervention group was asked to listen to a 30-minute music concert consisting of sounds of nature. they were asked to listen to music in a comfortable and quiet environment. they were requested not to divide, stop, change the speed until the music ended. the piece of music was created under the supervision of an experienced music therapist. music pieces were chosen from sounds of nature because they are universal, easily accessible, and had been previously shown to be effectively used for therapeutic purposes [12]. music therapy lasted 30 minutes every day for a total of 20 days. patients were called by phone and their compliance with music therapy applied every other day, and side effects associated with music therapy were questioned. before treatment, all patients were evaluated using the fibromyalgia impact questionnaire (fiq) scoring system, and fiq scores of patients were re-evaluated on the 20th day after treatment. intraand intergroup changes in fiq scores were analyzed. the fibromyalgia impact questionnaire developed in 1991 by burckhardt et al. [13] was used in the study. this scale was revised in 1997 [14]. ediz et al. [15] conducted the turkish validity and reliability study of the revised fibromyalgia questionnaire (fiq) in 2011. the revised fiq scale is a scale that evaluates the limitations and functional disability in fm patients with 20 questions in three sections: function, overall impact, and symptoms. the higher the score obtained from the survey, the disability of fibromyalgia worsens. compliance with ethical standards this study was approved by the clinical research ethics committee of dışkapı yıldırım beyazıt training and research hospital (date 16.01.2017, approval no: 34/13). this study was carried out in accordance with the helsinki declaration. informed consent was obtained from all individual participants included in the study. the study was registered at clinicaltrials.gov id: nct04823117. statistical analysis statistical analyzes were made using spss version 20.0 (ibm®, chicago, usa) package program. the suitability of variables to normal distribution was examined using visual (histogram and probability graphs) and analytical methods (shapiro-wilk test). in normally distributed numerical data descriptive statistics age, fiq function before and after treatment, symptom, and total score) were expressed as mean and standard deviation. for non-normally distributed numerical data they were given as median and min-max value, and for nominal data as numbers and percentages. normally distributed numerical variables were analyzed between the two groups by using the t-test in independent groups and within the group with matched-pairs t-test. spearman and pearson correlation tests were used in the correlation analysis. numerical variables not normally distributed were compared between the two groups with the mann -whitney u test and within the group with the wilcoxon signed-rank test. nominal data were evaluated between two groups using the chi-square test. in statistical analyzes used in the study, p-value below 0.05 was considered statistically significant. http://www.ajecr.org/ 436 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org table 1 analysis of sociodemographic and clinical features * t-test; **chi-square test. table 2 distribution of fiq scores before treatment * t-test; **mann whitney u test; fiq= fibromyalgia impact questionnaire. table 3 distribution of fiq scores after treatment * -test; **mann whitney u test; fiq= fibromyalgia impact questionnaire. results sociodemographic and clinical features the mean age of the conventional treatment group was 43.1 ± 10.2 years, and the average age of the music therapy group was 41.8 ± 8.9 years. there was no age difference between the groups (p = 0.640), and the majority of the patients were primary school graduates in both groups (68.6 % vs. 68.8 %, p = 0.927). very few patients were actively working in both the conventional treatment group and the music therapy group (21.6 %, 18.8 %, p = 0.809). thirty-nine (58.2 %) patients had comorbid diseases. the groups were similar in terms of the frequency of comorbid diseases (cardiovascular system, gastrointestinal system endocrine system, psychiatric and musculoskeletal diseases) (p = 0.054). the sociodemographic and clinical features of the patients are summarized in table 1. intergroup analyzes before treatment, fiq-function (p = 0.440), fiq-overall impact (p = 0.767), fiq-symptom (p = 0.666) and fiq-total score (p = 0.806) did not differ between the groups (table 2). after treatment, fiq -function (p = 0.010), fes-overall impact (p = 0.008), fiq-symptom (p = 0.006) and fiq-total scores (p = 0.001) in the music therapy group were significantly lower than the conventional group. in the music therapy group, there was a decrease of 30.8 % in fiqfunction scores, 40.6 % in fiq-overall impact score, 28.3 % in fiq-symptom scores, and 32.6 % in fiq-total scores (table 3). intragroup analyzes before the treatment, mean values for fiq-function 3.9 ± 2.3, fiq-overall impact 10.0 (0-20.0), fiq-symptom 48.0 ± 13.0, fiq-total 62.3 ± 16.3 scores in the control group were as indicated. after their current treatments, mean values for fiq-function 3.9 ± 2.2, fiq-overall impact 10.0 (0-20.0), fiq-symptom 47.0 ± 14.4, fiq-total 63.2 ± 17.5 scores were as indicated. as expected in the control group, no changes were observed in fiq-function (p = 0.827), fiqoverall impact (p = 0.601), fiq-symptom (p = 0.304) and fiq-total scores (p = 0.237) after their current treatments. before the treatment, in the music therapy group, mean values for fiq -function 3.4 ± 1.2, fiq-overall impact 10.0 (0-20.0), fiq-symptom 49.6 ± 12.1, fiq-total 63.3 ± 12,4 scores were as indicated. after treatment, mean values for fiq-function 2.7 ± 1.1, fiq-overall impact 4.2 (0-15.7), fiq-symptom 33.7 ± 15.5, fiq-total 42.6 ± 16.9 scores were as indicated. in the music therapy group, fiq-function (p < 0.001), fiq-overall impact (p = 0.001), fiq-symptom (p < 0.001) and fiq-total scores (p < 0.001) decreased significantly after treatment (table 4). fiq scores and related factors there was no correlation between patients’ ages and fiq-function (p = 0.663), fiq-overall impact (p = 0.431), fiq-symptom (p = 0.710) and fiq-total scores (p = 0.651) before treatment. there was no correlation between patients’ ages and fiq-function (p = 0.586), fiq-overall impact (p = 0.738), fiq-symptom (p = 0.953), and fiq-total scores (p = 0.971) after treatment. fiq-function, fiq-overall impact, fiq-symptom, and fiq -total scores did not change both before and after treatment according to the education, occupational status, presence of comorbid disease of the patients (all p > 0.05). discussion fms treatment is among the challenging diseases due to its unclear etiology and pathophysiological mechanisms. preferred evidence-based pharmacological treatments include antidepressants (amitriptyline, duloxetine, milnacipran), anti-epileptics (pregabalin) and other drugs (tramadol, pramipexole, memantine) [16]. however, the effect of pharmacological treatments alone is limited. on the other hand, the effect spectrum of non-pharmacological treatments has been reported to be higher than the effect spectrum observed in drug treatments [17]. a variety of methods have been preferred for non-drug treatments. these p value age (mean ± sd) 0.640* education 0.927** primary school 35 68.6 11 68.8 high school/university 16 31.4 5 31.2 profession 0.809** housewife/unemployed 40 78.4 13 81.2 employed 11 21.6 3 18.8 comorbidity (+) 33 64.7 6 37.5 0.054** musculoskletal (+) 7 13.7 3 18.8 0.623** variable conventional therapy n=51 (%) music therapy n=16 (%) 43.1 ± 10.2 41.8 ± 8.9 conventional therapy music therapy (n=51) (n=16) function (mean±sd) 3.9 ± 2.3 3.4 ± 1.2 0.440* median overall impact (range) 10.0 (0-20.0) 10.0 (0-20.0) 0.767** symptom (mean±sd) 48.0 ± 13.0 49.6 ± 12.1 0.666* total (mean±sd) 62.3 ± 16.3 63.3 ± 12.4 0.806* fiq p value conventional therapy music therapy (n=51) (n=16) function (mean±sd) 3.9±2.2 2.7±1.1 0.010* median overall impact (range) 10.0 (0-20) 4.2 (0-15.7) 0.008** symptom (mean±sd) 47.0±14.4 33.7±15.5 0.006* total (mean±sd) 61.0±17.5 42.6±16.9 0.001* fiq p value http://www.ajecr.org/ 437 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org table 4 distribution of fiq scores before/after treatment * t-test; **wilcoxon signed ranks testi; fiq= fibromyalgia impact questionnaire. methods include exercise therapy, hot or cold application, spa treatment, cognitive behavioral therapy, magnetotherapy, biofeedback, and music therapy [18]. it was previously stated that music therapy is effective in patients with fms, in reducing pain and it also provides increased functionality associated with a decrease in pain [19]. however, data in the field of music therapy are limited. on the other hand, the fact that the available data, music concerts and culture differ among geographic regions reduces the generalizability of the results of the studies. this study aimed to evaluate the effect of more universal-themed music therapy on the loss of function and symptoms caused by fms and to compare it with conventional therapy. the first remarkable finding of this study was the increase in the functions evaluated with fiq scale with music therapy, and the decrease in the overall effect level and symptom severity due to fms. the developments observed were more pronounced compared to the conventional treatment group. our findings indicated that music therapy was effective in fms patients. the effectiveness of music therapy has been confirmed in a small number of studies performed on patients with fms [19-25]. in spite of these findings, it can be said that music therapy in fms patients should be among the treatment methods of fms patients despite the distinct methodological differences between studies. the analgesic effect of music on pain has been linked to cognitive and emotional effects originating from listening to music. these effects include distraction of pain, feeling of pleasure-happiness, stimulated memory, and relaxation [2628]. besides, listening to music, have been associated with the secretion of dopamine from the caudate nucleus and nucleus accumbens [26]. dopamine is known to play a role in central analgesia [29]. it has been suggested that musicbased analgesia is central-type analgesia, and that analgesic effect is exerted in the brainstem and by functions of the emotional cortex through central neurotransmitters rather than peripheral receptors [21]. despite significant methodological differences, it has been reported in previous studies that music therapy has an impact on the functional status and / or symptoms of fms patients. in a study performed by garza-villarreal et al. [21] in the year 2014, 11 fms patients listened relaxant music pieces selected by the patients; while in another group, 11 patients listened “pink noise” consisting of environmental noise. in the music therapy group, improvement in pain and functional mobility was noted as assessed by the pain and “timed up & go” (tug) test. in the single-blind randomized study conducted by espi-lopez et al. [19] in 2016, the fms patients were divided into therapeutic exercise and music therapy (n = 13), therapeutic exercise (n = 13) and the control groups (n = 9), and it has been shown that therapeutic exercise is effective in the relief of discomfort and depression, but when combined with music therapy, the therapeutic exercise was found to be more effective. in their study in 2014, picard et al. [22] analyzed the change in fiq and jenkin sleep scale scores after music therapy in 20 patients. in this study, it was stated that it is a music with mostly 2 hz binaural beats promoted sleep. all participants were provided with the same mp3 device and headphones, and it was reported that music should be preferred at bedtime. how often and how well the patients complied with the treatment was also recorded. significant improvement in fiq total score and jenkin sleep scale scores were observed in the study. naghdi et al. (2015) investigated the effect of low-frequency sound stimulation in 19 fms patients. there was a marked improvement in the fiq score, jenkins sleep scale, and pain scores. in addition, it was stated that painless sitting and standing time prolonged, and range of motion of the cervical joint increased. no side effects were observed during low frequency sound stimulation. however, there was no control group in this study. in a randomized controlled study conducted in 2016 in our country by alparslan et al. [20], the effect of music therapy on pain was evaluated in 21 intervention patients and 16 control subjects who were followed up in the rheumatology clinic. it was stated that the severity of pain assessed at the 1st, 7th and 14th days in the music group decreased significantly, but not in the control group. in the study conducted by demirbağ et al. [25] in 2012, the effectiveness of music therapy was evaluated in 162 female fms patients. preferred music included at least four classical turkish music maqams, ie. housseini, saba, rast, and ussaq. it was stated in the study that depression levels were more markedly decreased with music therapy, besides, non-restful sleep, headache, fatigue, and intestinal problems were also reduced. in a randomized controlled study conducted by weber et al. [30], 120 fms patients were divided into four groups: group 1 listened to bach classical music, vibratory stimuli were applied on acupuncture points in group 2. in group 3, patients listened to bach, and also vibratory stimuli were applied at acupuncture points while group 4 was the control group. the patients were evaluated with fiq and “health assessment questionnaire” (haq). while improvement in fiq and haq scores was observed in all intervention groups, the best results were obtained in group 3 that received music and vibratory stimulus therapy. fiq before treatment after treatment p value function (mean±sd) 3.9 ± 2.3 3.9 ± 2.2 0.827* median overall impact (range) 10,0 (0-20) 10.0 (0-20.0) 0.601** symptom (mean±sd) 48.0 ± 13.0 47.0 ± 14.4 0.304* total (mean±sd) 62.3 ± 16.3 63.2 ± 17.5 0.237* fiq before treatment after treatment p value function (mean±sd) 3.4 ± 1.2 2.7 ± 1.1 <0.001* median overall impact (range) 10.0 (0-20.0) 4.2 (0-15.7) 0.001** symptom (mean±sd) 49.6 ± 12.1 33.7 ± 15.5 <0.001* total (mean±sd) 63.3 ± 12.4 42.6 ± 16.9 <0.001* music therapy conventional therapy http://www.ajecr.org/ 438 am j exp clin res, vol. 8, no. 1, 2021 http://www.ajecr.org although music therapy has been reported to reduce symptoms of fms in most studies, there were important methodological differences between studies in terms of the duration, application method, repetition, and type of music therapy. the most important reason for choosing the sounds of nature in this study was that they were accessible to everyone. it has been previously shown that the sounds of nature can be used for the treatment of different diseases. it was previously reported that music therapy is useful in the treatment of anxiety [12] in reducing psychological stress [31] and in reducing pain in cardiac surgery patients [32]. it was stated by the turkish psychological association that the sounds of nature provide relaxation [20]. on the other hand, in this study, it has been shown that music therapy is a method that can be applied by patients because it does not require additional costs and cause complications. also, it can be added to the routine treatments of patients. in this study, the home treatment method was preferred due to the fact that the treatment can be done in the home environment, and does not cause additional health expenses, besides health professionals do not need to spare time for home treatment. in this way, hospitals and hospital staff are not needed for music therapy. on the other hand, it should be noted that patient compliance will be lower when music therapy is provided in the home environment. the study had some limitations. firstly, since treatment is recommended in this study using music cds or similar methods, it can be thought that patients listen to music with different sound quality and different sound levels. secondly, patients who did not apply music therapy for the desired time period and frequency were excluded from the study. therefore, it may be thought that more voluntary patients comply with the treatment. in addition, patients were evaluated after a relatively short treatment in this study. we could not determine the longevity of the effectiveness of music therapy in this study. in conclusion, in this randomized controlled study, it was found that music therapy consisting of sounds of nature showed improvement in the overall effects, symptoms, and functions as shown by the fiq scale scores in female fms patients, and the success rates achieved were significant compared to the control group. it can be said that music therapy, which does not require additional cost, and complication is not observed can be added to the routine treatments of the patients. music therapy can be preferred in the treatment of patients who do not get the desired response with pharmacological treatment. although it is stated in the literature that many different music forms may be effective, music therapy consisting of sounds of nature may be more 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hemoptysis treated using bronchial artery embolization hamidreza varastehravan, aryan naghedi* department of cardiology shahid sadoughi hospital, shahid sadoughi university of medical sciences, yazd, iran abstract. massive hemoptysis is an important medical emergency which needs immediate managements. there are different managements offered for this event but there is no universal accordance on management of massive hemoptysis. available database is not adequate in this field. here we aimed to present a series of iranian patients with massive hemoptysis managed in yazd. we entered 33 referred patients into our study with their own informed consent. the patients had already been diagnosed with their pathology. except 5 patients, they all underwent embolization intervention. tuberculosis and bronchiectasis were the most common causes of hemoptysis detected in 11 (39%) and 10 (35%) patients respectively. other causes of hemoptysis in studied patients were primary pulmonary hypertension, eisenmenger’s syndrome, lung tumor and cystic fibrosis. all patients underwent angiography and were treated using bronchial artery embolization technique. five patients involved recurrent hemoptysis, accounting for 17% of the patients. two patients involved unwanted complications of embolization therapy. unfortunately, we recorded two related mortalities in our follow up. our study showed that embolization technique is followed by a low rate of mortality and recurrence and remains as one of the most effective procedures for management of hemoptysis. keywords: hemoptysis, embolization, angiography, intervention introduction hemoptysis is defined as expectoration of blood originated from lung parenchyma or lower respiratory airways. in this definition upper respiratory tract bleedings are not considered as hemoptysis [1]. hemoptysis is categorized into massive and non-massive groups [2]. massive hemoptysis stands a low portion of total hemoptysis but if happens it will lead to high rate of mortality [3, 4]. despite high risk of mortality still there are not adequate researches performed about management of hemoptysis. the number of case series and their follow ups and outcome reports are also limited [5]. etiologies leading to hemoptysis have changed during time. recent statistics indicate bronchiectasis, tb, necrotizing pneumonia and bronchogenic carcinoma as the most common etiologies of massive hemoptysis [6]. hemoptysis can be managed using medical, surgical or embolization approaches but up to dated investigations consider embolization technique as the most effective treatment with better outcomes and lower risk of mortality [3, 7]. available database about embolization management in patients with massive hemoptysis is limited either in the world or in our region, iran. embolization and angiographic intervention techniques are well developed in iran and are being used with similar outcomes as other countries. in this paper we present a series of patients with hemoptysis managed via transcatheteral bronchial artery embolization in yazd, iran. patients and methods we are about to present a series of 33 patients referred to shahid sadoughi hospital during 6 months since february 2013 till august 2013 with massive hemoptysis. all referred patients had already been diagnosed and bleeding sites were determined using bronchoscopy, chest x-ray (cxr) or computed tomography (ct-scan). all referred patients who entered our study were informed about all details while signing their informed consent and our study was carried out with institutional review board approval. they first underwent bronchial angiography via 5f cobra catheters for primary imaging and evaluation. of the patients, 28 were treated via embolization method. immediate success rate was 100% and hemoptysis stopped in all patients after embolization. all procedures were done by one of shahid sadoughi staffs personally, this provided equal operator depended factors for all patients. as suggested by m.c o’dell et al., we followed our patients to check persistent hemoptysis, non-target embolization and unstable hemodynamics for 2 years [8]. five cases that ___________________________________________________________ * corresponding author: dr. aryan naghedi (a.naghedi@yahoo.com). http://www.ajecr.org/ mailto:a.naghedi@yahoo.com 327 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org table 1 summed up results tb: tuberclosis, pph: primary pulmonary hypertension, es: eisenmenger’s syndrome, cf: cystic fibrosis. were not candidate for embolization treatment were as below: 1. patient with evidences of a large pulmonary artery aneurysm as the cause of hemoptysis. 2. young patient diagnosed as bronchiolitis obliterans organizing pneumonia (boop) syndrome. 3. high risk case of lung tumor for embolization which was suggested for conservative treatment. 4. known bronchiectasis case that was candidate for other treatment options. 5. a case which was referred back for a more comprehensive diagnostic work up. other patients were treated using embolization via “merit medical” polyvinyl alcohol (pva) 355-500 or 500-710 micron particles. the average hospitalization ranged from 1 to 3 days. five patients involved recurrence among those who were treated using embolization technique (17.85%): three of them were treated using repeated embolization. one was observed in hospital without angiographic intervention and was discharged with stable hemodynamics. the last one was a known case of tb that her second hemoptysis led to death. two patients developed treatment complications including self-limiting thoracic aorta dissection and inevitable dysphagia. here is the list of recurrent hemoptysis cases after first time embolization: 1. a 53 years old man with history of bronchiectasis treated with embolization technique and discharged with stable hemodynamics. he was re-admitted 3 months later with complaint of massive hemoptysis, respiratory arrest and unstable hemodynamics. the patient was intubated and treated using common bronchial artery embolization. there was no more complaint of hemoptysis during our follow up. 2. this case was a 27 years old man and known case of es with hemoptysis and anemia. he was treated using right inter costobronchial artery embolization. he was readmitted after 1 month with evidences of bleeding from major aortopulmonary collateral artery (mapca) and was successfully treated using mapca embolization. 3. the next case was a 65 years old woman with history of tb and hemoptysis. she was treated with right bronchial artery embolization for the first time but she was readmitted with pervious symptoms. during investigations a non-bronchial artery branched from intercostal arteries was claimed to be responsible f or hemoptysis. patient’s symptoms disappeared after second embolization of nonbronchial artery. this non-bronchial artery was also one of the esophagus supply sources thus she faced unavoidable dysphagia after embolization. dysphagia recovered completely after 2 or 3 days. 4. the other case was a 64 years old man diagnosed with pph and poor unstable hemodynamic conditions so was too risky for a second embolization therapy. he was transferred to intensive care unit (icu) for conservative care. after one month his hemodynamic condition became stable and his hemoptysis stopped without any interventional therapy. 5. the last case was a 73 years old woman with hemoptysis due to tb infection which was treated successfully for the first time. unfortunately, she passed away at home due to second massive hemoptysis. two mortality cases are also recorded. one of them was the fifth case mentioned above who passed away at home due to massive hemoptysis. the other one was a patient with complicated heart disease who passed away before cardiac surgery because of two vessel disease (2vd) and severe mitral regurgitation (mr). results the average age of massive hemoptysis cases was 51 years varied from 20 to 86 years old. twenty cases were female and only 8 of them were male. majority of patients were known cases of tb (39.28%) which active tb infection was detected in one of them. other cases included bronchiectasis (35.71%), primary pulmonary hypertension (pph) (7.14%), lung tumor (7.14%), eisenmenger’s syndrome (es) (7.14%) and cystic fibrosis (cf) (3.57%) respectively. tb patients were the ones with highest rate of recurrence and complication. they accounted for 40% of recurrence cases and 100% of complicated patients. discussion hemoptysis is not a rare clinical manifestation. it is defined as coughing up blood that is originated from lung parenchyma or lower airways, trachea, pharynx and bronchi [1]. hemoptysis incidence is 1 out of each 1000 outpatients per year. it makes 6.8% of outpatient pulmonary clinic visits so each first line physician will encounter hemoptysis cases 4 or 5 times a year [2, 6]. although hemoptysis can be self-limiting in many cases but it may need icu admittance and can even lead to death [9]. hemoptysis is categorized into 2 broad groups traditionally, massive or severe and non-massive or so called mild or moderate. massive hemoptysis has an incidence of less than 5% of total hemoptysis [3]. it is low, but its mortality rate is more than 50% which is much higher than non-massive hemoptysis. for this reason, ibrahim et al. suggested that “life threatening hemoptysis” expression should be used instead of massive hemoptysis [10]. based on this it is mandatory for any medical doctor to be able to manage patients with chief compliant of massive or non-massive hemoptysis [2]. massive hemoptysis is defined by bleeding amount of 200 to 1000 milliliter (ml) during 24 hours but the most frequent embolization recurrence complication mortality tb 11 2 2 1 bronchiectasis 10 1 pph 2 1 1 es 2 1 lung tumor 2 cf 1 total no. 28 5 2 2 no. of patients condition http://www.ajecr.org/ 328 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org amount used in literature is ranged between 300 and 600 ml during 24 hours. other terms are also seen to be used for describing amount and severity of hemoptysis such as major (>200 ml/24 hours), severe (>150 ml/12 hours) and exsanguinating (>1000 ml total) [11]. an amount of 400 ml of blood within the lung parenchyma is enough for respiratory failure and may be followed by asphyxia and exsanguination as the most common causes of death due to hemoptysis [1]. massive hemoptysis in pediatrics means bleeding more than 8ml/kg during 24 hours or 200ml during 24 hours [12]. the main etiologies for hemoptysis are categorized under 3 main groups: airway diseases, parenchymal diseases and vascular diseases [1]. hemoptysis etiologies have changed during time and vary in different geographic zones. at the 1960s, common etiologies were mostly tb, bronchiectasis and lung abscess. other etiologies including bronchiectasis, lung cancer and pneumonia were less common etiologies respectively [6, 13]. tb still stays the main reason of hemoptysis worldwide. the differential prevalence varies in different regions. in developing countries statistics show that the most common etiologies are still infections but in the usa and other developed countries the main etiology has changed to bronchiectasis and lung cancer [14]. other uncommon reasons for hemoptysis are bronchial adenoma, systemic hypertension, vasculitis, lipoid pneumonia and radiation pneumonitis or even bronchial artery aneurysm (baa) in only 1% of intermittent hemoptysis patients [13, 15]. in children etiologies are different. the most common causes of hemoptysis in children are foreign body aspiration, infection, bronchiectasis, cf, pulmonary tb and less commonly trauma and cardiovascular abnormalities or even disseminated intravascular coagulation (dic) [16]. hemoptysis happens in 9.1% of cf patients and is usually associated with comorbid diseases. massive hemoptysis happens in 4.1% of cf patients [17]. in this study we concluded that bronchiectasis and tb are the most common etiologies for hemoptysis as h. dabó et al also did. lung is supplied by 2 main arterial systems: bronchial arteries and pulmonary arteries [18]. bronchial arteries receive 1% of cardiac output and supply airways and pulmonary arteries as vasa vasorum. pulmonary arteries are responsible for 99% of lung oxygenation and gas exchange and also supply trachea, bronchi, esophagus and visceral pleura [1, 19]. bronchial artery is responsible for 90% of hemoptysis and the rest 10% is divided between pulmonary and other lung supplying arteries equally [3, 4]. there are a lot of different variations in anatomy of these arteries but the most common variation of bronchial artery pattern was reported by battal et al. using multi-detector computed tomography (mdct) angiography. this pattern is one bronchial artery on each side which is divided into two arteries on right side and one on the left side. normal diameter of bronchial artery is reported to be median 3 millimeter (mm) by the same team but again the diameters are reported to be different in different researches. this points out to the big controversy on bronchial artery anatomy [20]. bronchial arteries typically branch from proximal descending thoracic aorta. they are called as orthotopic when they are originated at t5-t6 vertebral level and ectopic if they are originated elsewhere. 64% of general population show orthotopic pattern in imaging. ectopic bronchial arteries are possible to be originated from aorta directly, subclavian artery, brachiocephalic, thyrocervical and even coronary arteries but the most common site is undersurface of the aortic arch [19]. ectopic bronchial artery pattern is more common in men [21]. patients with massive hemoptysis admitted to emergency room need immediate care since untreated massive hemoptysis can have up to 85% mortality rate [22]. management of these patients mostly needs a team including intensivist, interventionist and thoracic surgeon [11]. the first step of management for these patients is to enhance the patient’s general condition using effective stabilization. the most important step of management is to establish a functional supportive air way and the most available and easy way is intubation using a single lumen endotracheal tube. the next step is to determine the site of bleeding and thus a bronchoscopy is suggested mostly, because it is said to be useful in 2 ways: 1) clearing the airways and 2) establishing an appropriate vision to the bleeding site [6]. it is discussed in different researches that bronchoscopy can also offer useful treatment options in the first steps of management before target therapy or diagnostic work up such as selective intubation, cold saline lavage and applying bleeding control agents. diagnostic work ups are different in massive and non-massive hemoptysis patients and their unit goal is to identify etiology and locate the site of active bleeding. the routine available diagnostic tools are chest x-ray, chest ct scan, bronchoscopy, and laboratory tests [11]. chest radiography is the best diagnostic test for the first step in patients with stable hemodynamics [2]. a descending aortogram is suggested if there is a possibility of bleeding from non bronchial vessels [11]. conservative therapy for hemoptysis mostly consist of controlling coagulopathy state, cardiovascular support and airway protection, the latter is considered as the most important one [14]. conservative therapy for massive hemoptysis is followed by 50-100% of mortality thus after successful stabilization the patient is candidate for definite target therapy which can be achieved within 3 protocols: endovascular intervention (bae), surgical treatment and medical treatment [7, 23]. hemoptysis was not considered to be curable until 1940 and up to approximately 20 years ago surgery was the treatment of choice for massive hemoptysis [1, 16]. since 1973 that bae was first performed by remy et al this endovascular intervention became the treatment of choice in most of acute hemoptysis cases because of both diagnostic and curative benefits [8, 24]. among these options, medical treatment led to 45% mortality, surgical resection led to 7-18% of mortality in selective cases and 40% of mortality in emergency cases [1, 23]. available medical treatment for hemoptysis are not suitable nowadays but tranexamic acid is reported to be useful in controlling patients with bronchiectasis due to cf http://www.ajecr.org/ 329 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org in limited articles. medical treatment using anticoagulants should be in mind only for some pulmonary renal cases such as goodpasture or granulomatosis with polyangitis (gpa). other medical treatments such as anti tb treatment, antifungal or antibiotic agents are not enough and the best results are achieved if they are prescribed along with bronchial artery embolization (bae) or surgical treatments [7]. surgical options for massive hemoptysis is resection which can be done as segmentectomy, lobectomy and pneumonectomy [14]. each of these operations can be done in 3 approaches depending on the time passed since definite hemoptysis diagnosis: emergency, scheduled (is done when bleeding is controlled) and planned (is done in a second admission after discharge) [6]. nowadays surgery remains treatment of choice only in limited cases such as iatrogenic pulmonary artery rupture, chest trauma and aspergilloma except those who cannot survive surgery because of comorbid diseases or emergency conditions [1, 6]. surgery is also useful in patients with lung cancer and idiopathic bronchiectasis after stabilization via bae [14]. immediate success rate of bae method was reported in a range of 80% up to 98.5% [18]. in a study done in 2014, g.r. alexander tried to show that bae can also be used as a temporizing method before surgery rather than main treatment of hemoptysis. but the results demonstrate that although bae is helpful for immediate control of bleeding but recurrence rate in patients with bae as temporizing measure was significantly higher in comparison to patient who underwent surgery without any endovascular intervention (75% compared to 2%) [23]. hemoptysis arising from malignant etiologies are the most failed cases for performing bae and they had the worst long term outcomes [18]. this indicates that maybe in these cases bae is better to be done only for immediate control of bleeding and then the cases can be candidate for surgical resection [14]. some studies indicate that recurrence rate of bae is about 29.8% as an average and some others reported the average recurrence of hemoptysis as 23% in 5 years depending on etiology [3, 18]. our study ended up with a recurrence rate of 17.85% but one of the highest recurrence rates was reported by fruchter et al. which was 57.7% [14]. high recurrence rates of hemoptysis controlled using bae was reported to be related to the underlying lung diseases such as aspergillosis, tb and bronchial carcinoma [25]. in our study the highest recurrence rate was among tb infected patients. a spanish study involving 317 patients revealed that in tb related massive hemoptysis, endovascular treatment had a success rate of 91% and the recurrence rate was 22% and need to second embolization was recorded in only 7% of patients [26]. another study performed about tb associated hemoptysis showed that average age of diagnosis of active tb was 36 years and 25% of patients experience recurrent hemoptysis after the first time of bae. destructed lung tissue, fungal ball and chest ct consolidations were reported as recurrence risk factors in tb infected patients by ryu et al. [27]. there are no certain contraindications to perform bae except 2 relative contraindications including coagulopathy and renal failure or contrast allergy [8]. the most common embolic materials used in the procedures are pva and gelatin sponge and particles larger than 300 microns are mostly used [25]. the most common complications of bae seem to be sub intimal dissection, fever, chest pain, dyspnea, dysphagia headache and etc. [4]. we faced sub intimal dissection and dysphagia in our study. spinal artery ischemia, the most frightening complication of bae that can cause paraplegia is reported to happen in a range of 1.4% up to 6.5% [18, 25]. this complication can be prohibited by using particles larger than 300 microns [25]. we also used large particles in our procedures and thus we didn’t face spinal ischemia. conflict of interest the authors declare no conflicts of interest and no financial support. references 1. larici ar, franchi p, occhipinti m, contegiacomo a, del ciello a, calandriello l, et al. diagnosis and management of hemoptysis. diagn interv radio 20:299309, 2014. 2. earwood js, thompson td. hemoptysis: evaluation and management. am fam physician 2015;91:243-249, 2015. 3. farquharson s. bronchial artery embolization. in: taslakian b, al-kutoubi a, hoballah j (eds). procedural dictations in image-guided intervention: springer, cham. p. 531-535, 2016. 4. shao h, wu j, wu q, sun x, li l, xing z, et al. bronchial artery embolization for hemoptysis: a retrospective observational study of 344 patients. chinese med j 128:58-62 , 2015. 5. ghanaati h, rad as, firouznia k, jalali ah. bronchial artery embolization in life-threatening massive hemoptysis. iran red crescent med j 15(12):e16618, 2013. 6. radchenko c, alraiyes ah, shojaee s. a systematic approach to the management of massive hemoptysis. j thoracic dis 9(suppl 10):s1069-s1086, 2017. 7. renaud s, falcoz p-e, santelmo n, massard g. prise en charge des hémoptysies massives. rev pneumol clin 68:123-130, 2012. 8. o’dell mc, gill ae, hawkins cm. bronchial artery embolization for the treatment of acute hemoptysis. tech vasc interven radiol 20:263-265, 2017. 9. choi j, baik jh, kim ch, song sh, kim sk, kim m, et al. long-term outcomes and prognostic factors in patients with mild hemoptysis. am j emerg med 36:11601165, 2017. 10. ibrahim w. massive haemoptysis: the definition should be revised. eur respir j 32:1131-1132, 2008. 11. rali p, gandhi v, tariq c. massive hemoptysis. crit care nurs q 39:139-147, 2016. 12. gaude g. hemoptysis in children. indian pediatr. 47:245-254, 2010. 13. hirshberg b, biran i, glazer m, kramer mr. hemoptysis: etiology, evaluation, and outcome in a tertiary referral hospital. chest 112:440-444, 1997. 14. fruchter o, schneer s, rusanov v, belenky a, http://www.ajecr.org/ 330 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org kramer mr. bronchial artery embolization for massive hemoptysis: long-term follow-up. asian cardiovascul thorac ann 23:55-60, 2015. 15. ahmad s, jones kr. bronchial artery aneurysm: an unusual cause of intermittent hemoptysis. case reports in the pulmonary circulation: am thoracic soc p. a3940a, 2016. 16. simon dr, aronoff sc, del vecchio mt. etiologies of hemoptysis in children: a systematic review of 171 patients. pediatr pulmonol 52:255-259, 2017. 17. hurt k, simmonds n. cystic fibrosis: management of haemoptysis. ped respirat rev 13:200-205, 2012. 18. dabó h, gomes r, marinho a, madureira m, paquete j, morgado p. bronchial artery embolisation in management of hemoptysis–a retrospective analysis in a tertiary university hospital. rev port pneumol 22:34-38, 2016. 19. walker cm, rosado-de-christenson ml, martinezjimenez s, kunin jr, wible bc. bronchial arteries: anatomy, function, hypertrophy, and anomalies. radiographics 35:32-49, 2015. 20. akgun v, battal b, sari s. bronchial arteries: normal anatomy, variation and radiologic evaluation. surg radiol anat 36:103-104, 2014. 21. yener ö, türkvatan a, yüce g, yener aü. the normal anatomy and variations of the bronchial arteries: evaluation with multidetector computed tomography. can assoc radiol j 66:44-52, 2015. 22. flores rj, sandur s. massive hemoptysis. hospital physician 42:37-39, 2006. 23. alexander gr. a retrospective review comparing the treatment outcomes of emergency lung resection for massive haemoptysis with and without preoperative bronchial artery embolization. eur j cardiothorac sug 45:251-255, 2014. 24. remy j, voisin c, dupuis c, beguery p, tonnel ab, denies jl, et al. treatment of hemoptysis by embolization of the systemic circulation. ann radiol 17:516, 1974. 25. kalva sp. bronchial artery embolization. tech vasc interv radiol 12:130-138, 2009. 26. krishnan b, shaukat a, chakravorty i. fatal haemoptysis in a young man with tuberculous mediastinal lymphadenitis. respiration 77:333-336, 2009. 27. ryu yj, kim s, lee sj, lee jh, chang jh, choi sy, et al. risk factors of recurred hemoptysis after bronchial artery embolization in patients with active pulmonary tuberculosis or post tuberculosis sequelae. d35 rare and interesting cases of lung inflammation, injury, and infection: am thoracic soc p. a5718-a, 2014. 28. okuda k, tanaka j, okamoto j, kishi f, nakagawa j, hino h, et al. two cases of cryptogenic life‐threatening hemoptysis–identification and management of bleeding point. acute med surg 4:114-118, 2017. http://www.ajecr.org/ 331 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2019;6(1):331-336 original article clonidine as early adjunctive therapy for alcohol withdrawal in the emergency department lindsey shannon, christopher leong, ahmed a mahmoud* department of pharmacy, northwestern memorial hospital, east huron street, chicago, illinois, usa abstract. alpha-2-agonists may decrease cumulative benzodiazepines (bzd) requirements in alcohol withdrawal syndrome (aws), leading to a reduction in bzd related adverse events. this study aimed to evaluate the impact of adjunctive clonidine on bzd requirements in patients with aws who presented to the emergency department (ed). a retrospective chart review study from 1/2015 to 12/2016 was performed in patients who were admitted for aws via the ed and would have received at least 24 hours of benzodiazepines. the primary study outcome was the difference of the 12-hour cumulative bzd requirements in lorazepam equivalents (bzd-le) in patients who received clonidine compared to patients who received bzd monotherapy. secondary endpoints included total hospital benzodiazepine requirements, intensive care unit (icu) admission, icu and hospital length of stay, and incidence of hypotension. a total of 11 patients who received clonidine adjunctive therapy and 33 patients to standard management were included in the study. the median 12 hour cumulative bzd-le was 16 mg (iqr 3-19) in the intervention group compared to 7 mg (iqr 4-13) in the control group (p = 0.90). however, the total cumulative bzd-le requirements for the hospital stay was 31 mg (iqr 21-48) in the intervention group compared to 45 mg (iqr 26-71) in the control group (p = 0.28). in conclusion, adjunctive clonidine administration to bzd in aws initiated in the ed was not associated with a decrease in 12 hour bzd requirements. keywords: alcohol withdrawal, clonidine, adjunctive therapy introduction alcohol use disorders remains a significant burden to the healthcare system with an annual estimated 1.2 million hospital admissions related to alcohol abuse [1]. of those 500,000 episodes experience severe withdrawal symptoms that necessitate inpatient treatment. prolonged alcohol abuse leads to the development of physical dependence in which the brain undergoes changes to maintain neurotransmitter homeostasis [2]. as a result patients that abruptly cease alcohol consumption experience autonomic hyperactivity of the central nervous system (cns) [2]. thus, symptoms of alcohol withdrawal syndrome (aws) results from an excessive sympathetic surge in the acute absence of alcohol [1, 3, 4]. depending on the severity of aws, symptoms may be mild such as mild tremors, tachycardia, hypertension or in the case of severe untreated aws exhibiting severe agitation, seizures, hallucinations, and delirium tremens which if left untreated is fatal [1, 5]. benzodiazepines (bzds) are the treatment of choice in aws. although bzds have been used for decades, there is no studies to indicate the ideal bzd nor treatment strategy in the management of aws(1). in an effort to avert aws, clinicians may be aggressive with bzd dosing which may inadvertently cause respiratory depression necessitating mechanical ventilation and an intensive care unit (icu) admission. thus, there is a renewed interest in agents that may be used adjunctively with bzd for the treatment of early aws. centrally acting pre-synaptic α-2 receptor agonists such as clonidine and dexmedetomidine are examples of such adjunctive agents that may be utilized in the management of aws. we sought to investigate the impact of early adjunctive clonidine on bzds requirements in early aws patients who presented to the ed compared to standard management. materials and methods this was a retrospective study conducted at a tertiary care center of patients 18 years of age and older who presented to the emergency department (ed) and were admitted inpatient with the primary diagnosis of aws from january 2015 to december 2016. patients were identified using icd 9 (291.81) and icd 10 (f10.231 & f10.231) billing codes. inclusion criteria were patients admitted for aws, receiving at least 24 hours of bzds (t=0 defined as time from first bzd administration in the ed) and the use of clonidine as adjunctive therapy for the ___________________________________________________________ * corresponding author: ahmed a mahmoud, pharmd, bcccp (amahmoud@nm.org). http://www.ajecr.org/ mailto:amahmoud@nm.org mailto:amahmoud@nm.org 332 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org table 1 lorazepam eqivalents treatment of aws. trauma and or intubated patients, the administration of any other neuromodulating agents (i.e: gabapentin, valproic acid, ketamine, carbamazepine, guanfacine) other than bzds, the administration of clonidine as part of an antihypertensive regimen, patients discharged directly from the ed and patients whom primary diagnosis is other than alcohol withdrawal were excluded from analysis. the primary outcome was defined as the difference in the 12-hour cumulative bzds requirements in patients who have received adjunctive clonidine (ac), compared to patients who received standard bzds monotherapy for the management of their aws (sm). secondary endpoints were defined as the difference in the 24 hour cumulative bzd requirement, total administered bzds requirements during the hospital stay, icu admission, icu and hospital length of stay, rates of respiratory depression and the incidence of hypotension (defined as systolic blood pressure less than 90 mmhg). all benzodiazepines doses were converted to and reported as lorazepam equivalents (bzd-le) (table 1) [6, 7]. all tests were two-tailed with alpha set at p < 0.05. continuous data was analyzed using a t-test. categorical data was analyzed using chi-squared test. all statistical analysis was performed using ibm spss. results a total of 18 patients were screened for inclusion in the ac group. six patients were discharged from the ed and were excluded from further analysis. one patient’s primary diagnosis was not alcohol withdrawal and was excluded. a total of 11 patients were included in the clonidine group for analysis. a total of 42 patients were screened for inclusion in the sm group. nine patients were excluded for receiving adjunctive therapy other than clonidine for the management of their aws. the remainder 33 patients were included into the sm group. baseline characteristics were similar between the two groups (table 2). overall, the majority of patients presenting with aws were men with median systolic blood pressure (sbp) and median heart rate (hr) of 185 mmhg and 132 bpm in the clonidine group compared to the standard management group’s median sbp of 167 and median hr of 120 bpm. serum ethanol levels ranged from undetectable up to 430 mg/dl (median: 29 mg/dl) in the ac group, and from undetectable to 427 mg/dl (median: 101 mg/dl) in the sm group. table 2 baseline characteristics results reported in median (iqr) unless otherwise noted. the median cumulative dose of clonidine administered in the ed was 100 mcg and ranged between 100 mcg and 300 mcg. the median time to clonidine administration was 4 hours, while the median ed stay was 6 hours. clonidine therapy was continued during inpatient therapy in 10 out of 11 patients (91%). as there was no standardized protocol, the resumption of clonidine was left to the discretion of the admitting team in the inpatient service with the majority of patients receiving 100mcg three times daily. the median duration of clonidine therapy was 3 days and ranged from 1 to 4 days. the median 12 hour cumulative bzd-le requirement was 16 mg (iqr 3-19) in the ac compared to 7 mg (iqr 4-13) in the sm group p = 0.90. at 24 hours, the median cumulative bzd-le requirement was 21 mg (iqr 16-30) in the ac group and 18 mg (iqr 11-34) in the sm group p = 0.44. the median total cumulative bzd-le requirement was 31 mg (iqr 21-48) in the ac group compared to 45 mg (iqr 26-71 mg) in the sm group p = 0.28. at 12 hours, the median bzd-le requirements in the ac group were 9 mg higher compared to the sm group, that difference however decreased at the 24 hour mark where the ac group required higher total requirement of 3 mg compared to the sm group. upon patient discharge, the median total cumulative bzd-le dose was 14mg higher in the sm group compared to the ac group (table 3 and fig. 1). the rate of icu admission was not different between the ac group (45%) and the sm group, (42%). neither the median icu days nor the median hospital days was different between the two groups ac group 2 days (iqr 23), 4 days (iqr 3-5), sm group, 2 days (iqr 2-5), 4 days(iqr 4-8) respectively. three patients experienced hypotension in the sm group (9%), and none in the ac group, however that was not statistically significant (table 4). there was no reported respiratory depression necessitating intervention and or intubation. discussion at our institution there is no standardized approach regarding the management of aws. choice, route and frequency of bzd is left up to the discretion of the provider. the treatment approach is then dictated via patient presentation, alcohol blood level and patient vitals. the use of adjunctive therapies are thus optional and varies between medical and pharmacy providers. gaba agonism 1 mg oral lorazepam 0.25 mg oral clonazepam 0.5 mg oral alprazolam 0.5 mg intravenous lorazepam 5 mg intravenous diazepam 10 mg oral chlordiazepoxide intervention group (n=11) control group (n=33) age 50 (38-52) 47 (40-54) sex, male (n, %) 9 (82) 28 (85) weight (kg) 73 (67-78) 77 (69-89) maximum sbp (mmhg) 185 (143-206) 167 (146-194) maximum dbp (mmhg) 101 (92-119) 99 (81-109) maximum hr (bp) 132 (112-138) 120 (107-130) http://www.ajecr.org/ 333 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org table 3 baseline characteristics results reported as milligrams of lorazepam equivalents in median (iqr). remains the only treatment for aws, however, historically providers use the minimum effective dose required for concerns of ade. thus, we sought to investigate if the use of early clonidine administration may translate to reduced bzd requirements. our results indicate that the use of clonidine as adjunctive therapy in early aws did not reduce 12 hour bzd requirements compared to patients receiving standard management. however, at 24 hours and at time of discharge, patients receiving clonidine adjunctive therapy required less bzd compared to patients that did not receive clonidine. it has been well established that the neurotransmitter effects of chronic ethanol use leads to a decrease in γaminobutyric acid subtype-a (gabaa) receptor functioning. subsequently, an increase in effort to adapt to the increased inhibitory gabaa stimulation, glutamate, nmda receptors are upregulated [8]. the downregulation of gabaa receptors is compounded by the change of the change of the gaba receptor subunit that is available for activation, making them less responsive to synaptic neurotransmitters. norepinephrine and dopaminergic levels have been shown to be elevated with chronic ethanol intake and the dysregulation of the neurotransmitters explains the sympathetic surge witnessed in aws [9]. upon abrupt cessation of ethanol intake, the unopposed increased n-methyl-d-asparte (nmda) receptor activity coupled with the downregulated, less sensitive gabaa receptors places the patient at a high risk for aws [8]. excessive dopamine, and norepinephrine levels unopposed by alcohol cessation have been demonstrated in aws [10]. pre-synaptic neuron releases catecholamines such as norepinephrine and dopamine resulting in the over activation of the sympathetic nervous system, leading to an increased anxiety, tremors and sympathetic tone exhibited by hypertension and tachycardia [10]. individuals that chronically intake ethanol are at a higher risk for aws, as ethanol is highly specific to the gabaa receptors, inducing a higher tolerance [6, 7]. thus these patients generally may require higher doses of bzd for aws symptom control [7]. although bzd are the treatment of choice for symptom management of aws, there has not been any evidence to suggest superiority of one bzd over the other [1, 11-13]. rapid escalation of doses may be required in the refractory patient, however adverse effects such as respiratory depression and sedation necessitating intubation may occur, though rare. adjunctive therapy may be of interest in the setting of severe alcohol withdrawal to mitigate the use of high dose table 4 secondary endpoints results reported as median (iqr) unless otherwise noted. bzds. thus, there has been a renewed interest in the use of adjunctive therapy in aws patients, in an effort to reduce the total amount of bzd. the addition of such agents may blunt the sympathetic response to aws by decreasing the norepinephrine and epinephrine levels during aws, leading to an overall bzds sparing effect [1, 14-16]. agents such as dexmedetomidine, clonidine, carbamazepine and valproic acid have all been studied in this setting [17-19]. aside from one study that compared gabapentin to lorazepam for the treatment of mild to moderate aws, the treatment of choice of aws remains to be bzds [20, 21]. clonidine, approved in 1974 is the oldest central alpha 2 agonist, via creating a negative feedback loop and binding to the pre-synaptic neuron decreases the release of catecholamines. this unique mechanism of action is how clonidine may be of benefit in the body’s pro-excitatory state in the setting of aws [22, 23]. thus, there is a proposed benefit to add clonidine in patients presenting with hypertension, tachycardia anxiousness, agitation, diaphoresis, and requiring aggressive doses of bzds [1, 7]. the first reported study for the use of adjunctive clonidine in aws patients, admitted in a non-icu setting was published in 1975 by bjorkqvist. the author compared the use of clonidine vs. placebo in patients receiving standard care for aws and evaluated a “nurses evaluation score” which graded the patient’s general condition, sleep disturbance, movements and behavior for each patient. additionally, patient vitals, and tremor severity was recorded. the clonidine dose was 0.15mg, and was given as tid with a rapid taper over 4 days. the use of clonidine was associated with a lower nurses evaluation score by day 2 (p<0.01), less tremulous and less incidence of hypotension compared to the placebo group [13]. there was no difference in the incidence of adverse drug reactions. the author highlighted that clonidine should not be the sole therapy for aws, rather “a useful aid” in aws. walinder et al. investigated clonidine compared to standard care (bzd) in 26 patients, in a randomized open label study in 1981 [24]. this non-icu study demonstrated equal efficacy of clonidine compared to bzd with no difference in side effects. limitations to this study included using a different scoring system and patients receiving clonidine had a higher alcohol use prior to study initiation [24]. a subsequent study in 1985 by manhem et al. sought out to compare clonidine (0.15mg-0.3mg every 6 hours) to intervention group (n=11) control group (n=33) p-value 12 hrs cumulative bzd (mg) 16 (3-19) 7 (4-13) 0.9 24 hrs cumulative bzd (mg) 21 (16-30) 18 (11-34) 0.44 total cumulative bzd (mg) 31 (21-48) 45 (26-71) 0.28 intervention group (n=11) control group (n=33) icu admission (n, %) 5 (45) 14 (42) icu length of stay (days) 2 (2-3) 2 (2-5) hospital length of stay (days ) 4 (3-5) 4 (4-8) hypotension (n, %) 0 (0) 3 (9) http://www.ajecr.org/ 334 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org figure 1 cumulative benzodiazepine trend. chlormethiazole (a non-bzd hypnotic that has been used for the management of aws) in 20, non-icu aws patients [25]. the authors investigated the effect of therapies on blood pressure, an alcohol withdrawal assessment scale. seventeen patients who completed the study, the use of standard treatment and clonidine was associated with a significant decrease in lowering systolic blood pressure, heart rate and the alcohol withdrawal assessment scale compared to standard treatment. the authors drew plasma norepinephrine and epinephrine levels and compared it between clonidine and nonclonidine recipients. patients’ norepinephrine and epinephrine levels who received clonidine were lower compared to non-clonidine patients (p<0.01), and proved the basis for clonidine’s potential for use in aws. two subsequent studies by baumgartner et al and robinson et al were conducted, both concluded that the use of clonidine as adjunctive to standard treatment was associated with lower alcohol withdrawal syndromes, systolic blood pressure and heart rate [26, 27]. none of these studies initiated the use of clonidine in the emergency department. with the introduction of dexmedetomidine, a highly selective intravenous alpha-2 agonist, the use of adjunctive modalities for the treatment of aws was reignited. a randomized prospective double blind, placebo controlled study by mueller et al compared patients treated for aws with lorazepam with or without dexmedetomidine [28]. the primary efficacy outcome was the change in total lorazepam requirements 24 hours prior to and 7 days preceding the initiation of the intervention while the primary safety outcome was rates of hemodynamic instability (bradycardia and or hypotension). patients initiated on dexmedetomidine required less lorazepam in the first 24 hours (p = 0.037) compared to placebo, but at 7 days, they difference was not statistically different. bradycardia unsurprisingly was much more common in the dexmedetomidine group compared to placebo. vanderweide et al. conducted a retrospective, cohort study evaluating the impact of adjunctive dexmedetomidine on bzd us in the critically ill patients presenting with aws. patients either received dexmedetomidine plus standard alcohol withdrawal protocol versus the institutions standard alcohol withdrawal protocol. the authors found that adjunctive dexmedetomidine demonstrated a bzd sparing effect as compared to the standard alcohol withdrawal protocol, however failed to demonstrate an impact on clinical outcomes such as incidence or duration of mechanical ventilation and length of icu or hospital stay [22]. although beneficial as adjunctive therapy in aws, alpha 1 agonists should not be used as monotherapy. this was demonstrated in a study by robinson et al, where clonidine monotherapy was associated with higher rates of hypotension and lack of efficacy [26]. adinoff et al. compared clonidine vs diazepam vs alprazolam vs placebo for alcohol withdrawal, and found that clonidine monotherapy was essentially as effective as placebo [29]. we sought to evaluate the impact of adjunctive clonidine on bzd requirements in patients with aws who presented to the ed, and if suppressing the catecholamine surge in the ed may confer clinical and disposition benefits (i.e., icu vs non-icu admission, excessive lethargy). currently at our institution, there is no standard of care regarding the management of aws. choice, route and frequency of bzd is left up to the discretion of the provider, in which presentation, alcohol blood level and patient vitals plays a role in the approach and management of these patients. the use of adjunctive therapies are optional and varies between medical and pharmacy providers. this is different than most studies in which patients were initiated on adjunctive therapy upon admission or due increasing bzd requirements [25]. we hypothesized that the use of early clonidine would have decreased the total amount of bzd use early in the management of aws. our results demonstrated that the ac group had higher bzd requirements early on aws management compared to the sm group at 12 hours, although by the end of the hospital stay the sm group ultimately required more bzds. the higher bzd requirements may be due to these patient experiencing more severe aws early in the hospital stay increasing the 12 hour cumulative bzd doses. there may also be some component of a “therapy delay effect” defined as time delta from ed admission with aws to clonidine administration which was 4 hours in our study. therapy delay effect may extend further depending on the onset of action and up titration depending to response and tolerability. this hypothesis would account for the trend observed in that the intervention group ultimately requiring less cumulative bzd use than the control group despite the higher 12 hour requirements. although statistically non-significant difference in the bzd requirements, there was an overall trend of increasing bzd in the sm group vs. ac group by the end of the hospitalization (table 3). additionally, the rate of clonidine continuation was higher than expected highlighting the potential role of clonidine therapy as adjunctive in aws. our study had several limitations. first, due to the small sample size, the study was not powered to detect a statistical difference. second, our institution does not have a standardized approach regarding the use of bzd and adjunctive therapy in aws, thus clonidine may have been preferentially used in patients with more severe aws. thirdly, due to limitations with the electronic medical record at the time, the documentation of ciwas were http://www.ajecr.org/ 335 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org lacking and rarely documented, making a direct aws comparison challenging. there is a possibility that patients with more severe aws led to the selection for the use of clonidine in these patients. thus selection bias for the administration of clonidine to severe aws compared to mild or moderate aws is a possibility. lastly, the discretion to initiate clonidine therapy was determined by the bedside physician as there was no institutional protocol. this study did not attempt to differentiate the efficacy of once clonidine dose from another, and although there studies had clonidine doses starting with 150mcg up to 300mcg, 100mcg is a reasonable starting dose which may be repeated to target response based on the practitioner’s assessment [16]. conclusion adjunctive clonidine administration to bzd in aws in the ed was not associated with a decrease in 12 hour bzd requirements, icu admission, or icu or hospital days. clonidine was safe and well-tolerated, larger prospective studies are needed to further evaluate the use of adjunctive clonidine in the ed for aws management. conflict of interest the authors declare no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. long d, long b, koyfman a. the emergency medicine management of severe alcohol withdrawal. am j emerg med 35:1005-1011, 2017. 2. jesse s, brathen g, ferrara m, et al. alcohol withdrawal syndrome: mechanisms, manifestations, and management. acta neurol scand 135:4-16, 2017. 3. kosten tr, o’connor pg. management of drug and alcohol withdrawal. n engl j med 348:1786-1795, 2003. 4.schuckit ma. alcohol-use disorders. the lancet 373:492-501, 2009. 5. american psychiatric association. diagnostic and statistical manual of mental disorders [internet]. arlington, virginia, usa, p. 991, 2013. 6. barr j, zomorodi k, bertaccini ej, shafer sl, geller e. a double-blind, randomized comparison of i.v. 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dexmedetomidine for alcohol withdrawal syndrome. ann pharmacother 49: 1336-1342, 2015. 19. muzyk aj, fowler j a, norwood dk, chilipko a. role of α2-agonists in the treatment of acute alcohol withdrawal. ann pharmacother 45:649-657, 2011. 20. a. c, o.p. r, s. cc, s. cc. a comparative study of efficacy & tolerability of lorazepam and gabapentin in the treatment of alcohol withdrawal syndrome. int j pharm sci rev res 3:80-84, 2010. 21. kaim sc, klett cj rb. treatment of the acute alcohol withdrawal state: a comparison of four drugs. am j psychiatry 125:1640-1646, 1969. 22. vanderweide la, foster cj, maclaren r, kiser th, fish dn, mueller sw. evaluation of early dexmedetomidine addition to the standard of care for severe alcohol withdrawal in the icu. j intensive care med 31:198-204, 2016. 23. frazee en, personett ha, leung jg, nelson s, dierkhising ra, bauer pr. influence of dexmedetomidine therapy on the management of severe alcohol withdrawal syndrome in critically ill patients. j crit care 29:298-302, 2014. 24. walinder j, balldin j, bokstrom k, karlsson i, lundstrom b, svensson th. clonidine suppression of the alcohol withdrawal syndrome. drug alcohol depend 8:345-348, 1981. 25. manhem p, nilsson lh, moberg a ‐l, wadstein j, hbkfelt b. alcohol withdrawal: effects of clonidine treatment on sympathetic activity, the renin-aldosterone system, and clinical symptoms. alcohol clin exp res 9:238-2343, 1985. 26. robinson bj, robinson gm, maling tj jr. is http://www.ajecr.org/ 336 am j exp clin res, vol. 6, no. 1, 2019 http://www.ajecr.org clonidine useful in the treatment of alcohol withdrawal? alcohol clin exp res 13:95-98, 1989. 27. baumgartner gr, rowen rc. clonidine vs chlordiazepoxide in the management of acute alcohol withdrawal syndrome. arch intern med 147:1223\-1226, 1987. 28. mueller sw, preslaski cr, kiser th, fish dn, lavelle jc, malkoski sp, et al. a randomized, double blind, placebo-controlled dose range study of dexmedetomidine as adjunctive therapy for alcohol withdrawal. crit care med 42:1131-1139, 2014. 29. adinoff b. double-blind study of alprazolam, diazepam, clonidine, and placebo in the alcohol withdrawal syndrome: preliminary findings. alcohol clin exp res 18:873-878, 1994. http://www.ajecr.org/ 127 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(4):127-132 original article prevalence of benign tumors among patients with multiple sclerosis aryan rafiee zadeh 1 , mehrdad farrokhi 1 , masoud etemadifar 1 , ali amani beni 1 , nastaran dastravan 2, 3 1 department of neurology, school of medicine, isfahan university of medical sciences and multiple sclerosis and neuroimmunology research center, isfahan, iran 2 department of biology, faculty of sciences, university of isfahan, isfahan, iran 3 nour danesh institute of higher education, meymeh, isfahan, iran abstact. multiple sclerosis (ms), an inflammatory autoimmune disease, affects almost 1% of world’s population in which myelin sheaths of neurons are targeted by immune cells. association of different factors and diseases with ms provides new insights into possible pathogenesis and treatment for this disease. in this regard, we investigated the association of benign tumors with ms disease by studying total isfehan multiple sclerosis (tims) records for ms patients registered in isfahan multiple sclerosis society (imss) who had developed any kind of benign tumors whether before ms diagnosis or after it. this study was performed in isfahan province, third large province of iran, with 4,815,863 populations located 1590 meters above sea level between latitudes 30 and 34 degrees north of the equator, and longitude 49-55 east. among 4950 registered patients, 28 patients were discovered to have three types of benign tumors; pituitary adenoma in 22, meningioma in 5 and lipoma in 1 patient. the incidence rate of developing pituitary adenoma and meningioma were higher than in general population (or 95%ci: 1.110; range: 0.731-1.685 and 1.035; range: 0.431-2.487 respectively) but these findings were not statistically significant (p= 0.624 for pituitary adenoma and p= 0.939 for meningioma). but the incidence rate for lipoma was lower among ms patients (or 95%ci: 0.020; range: 0.003-0.143) which was statistically significant (p <0.001). keywords: multiple sclerosis, benign tumors, pituitary adenoma, meningioma, lipoma introduction multiple sclerosis (ms) is a chronic inflammatory autoimmune disease of central nervous system (cns) and a leading cause of neurologic disability in young adults. although, the exact etiology of ms still remains unknown but based on studies crucial role of immune system and immune cells in targeting myelin sheaths of neurons have been proven. autoactivated t-cells and exacerbated cellular immune responses are considered as potential causes of ms. also imbalanced th1/th2 cell population and increased th1 inflammatory cytokines play a pivotal role in ms pathogenesis [1]. the prevalence of ms varies widely in different geographical regions. for instance, higher latitude is associated with higher prevalence of ms based on what kurtzke reported [2]. iran is located between latitude 32°00 ′ n longitude 53°00 ′ in middle east with increasing both incidence and prevalence of ms especially in females. benign tumors are mass of tissue without the ability to invade neighboring tissues or metastasize. most of benign tumors are surrounded by a capsule and can be recognized by different mechanisms including detecting tumor associated antigens, magnetic resonance image (mri), clinical symptoms and other methods. pituitary adenoma, meningioma and lipoma are all considered as benign tumors with different incidence rates. pituitary adenoma affects almost 0.4% of general population, meningioma has incidence rate of 97.5 per 100,000 and lipoma affects 1% of general population. in this study we endeavored to compare the incidence rate of growing benign tumors in public with ms patients in order to determine whether incidence rate of benign tumors in those who suffer from ms is higher than general population or not. materials and methods our study was performed in isfahan which is the third large province of iran (province (107 003 km 2 ). isfahan is located in the center of iran with 1590m above sea level between latitudes 30 and 34 degrees north of the equator, longitude 49–55 east. the climate of isfahan is dry and temperate with a mean of daily temperature which varied widely from 5.3 in january to 27.2 in august. the population of isfahan was estimated 4,815,863 according to the iranian central bureau of statistics (icbs) reports. the socioeconomic proportions, current demographic characteristics and lifestyles are all the same like other ___________________________________________________________ *corresponding author: mehrdad farrokhi, md (mehrdadfarokhi72@yahoo.com) http://www.ajecr.org/ mailto:mehrdadfarokhi72@yahoo.com 128 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org table 1 clinical and paraclinical features of the cases sex female 27 96.4 male 1 3.6 first sign weakness 7 25 sensory 4 14.3 visual 14 50 cerebellar 3 10.7 edss <2 25 89.3 >2 3 10.7 ms type rr 26 92.9 pp 1 3.6 sp 1 3.6 medication cinovex 14 50 imuran 5 17.9 betaferon 7 25 avonex 2 7.1 cancer family present 27 96.4 absent 1 3.6 tumor type meningioma 5 17.9 lipoma 1 3.6 pituitary adenoma 22 78.6 parts of the country. the major health services in this province consist of private physicians and hospitals, district health centers, government, university hospitals, and clinics. in this retrograde study, we reviewed the case records of those patients diagnosed with definite ms based on mcdonald criteria [3] who were also residents of isfahan. all these patients had been previously registered in isfahan multiple sclerosis society (imss), the only referral center for isfahanian ms patients. the diagnosis of patients is performed by the neurologists of imss based on the 2010 version of international panel in ms diagnosis which is commonly known as mcdonald. mcdonald criteria are made up of three distinct broad groups: “definite ms,” “possible ms,” and “not ms.” definite ms diagnosed patients were included in our study. also disability of these patients was assessed by expanded disability status scale (edss) [4]. due to the supports for insurance, laboratory investigations, treatment, and rehabilitation, all diagnosed ms patient from all health sectors in isfahan province has been registered by imss since 2005. also it should be noted that the information of some small number of patients who prefer private care services might possibly be missed. the case records of patients, which were used as data base in our study, include glorious information taken from each patient by neurologists of imss such as: background variables, clinical records including disease pattern (relapsing-remitting ms (rrms), primary progressive ms (ppms), secondary-progressive ms (spms), and progressive-relapsing ms), symptoms, signs and relapse history (date, duration and type), therapeutic protocols, edss, and any significant complication in patients’ status. we first checked the total isfehan multiple sclerosis (tims) records looking for ms patients with benign tumors diagnosed at any stage of their life span by oncologists. we investigated these records for those patients who had developed any kind of benign tumors whether before ms diagnosis (by searching in clinical history records) or after it (by searching in imss follow-up records). all detected patients were called back to imss center for final clinical/paraclinical examinations and signing the written informed consent. the study was also approved by the ethic committee of the university. we then compared by χ2-statistics the rate of developing benign tumor in general population with the frequency of benign tumors in ms population in order to determine whether the association of ms and benign tumors in these diagnosed patients happened merely incidental or not. the size of this population was estimated by summation of pituitary adenoma, meningioma and lipoma incidence ranges which are 0.4% for pituitary adenoma, 97.5 in 100,000 for meningioma and 1% for lipoma respectively serving as the upper and lower values worldwide respectively multiplied by isfahan population resulting the incidence rate of developing benign tumors among general population. data were analyzed by spss, version19. results have been reported as a mean (±sd) and number (percent). all tests were two-tailed, and a p-value of <0.05 was considered as significance threshold. results of the 4950 registered ms patients in imss, three types of benign tumors (meningioma, lipoma and pituitary adenoma) had been developed in 28 ms patients (0.565%) with the mean age of 33.61±6.93 years. their ms condition was considered rrms in 92.9% of them with the edss ≤2 with frequency of 89.3% (n=25) and half of patients (50%) were on therapeutic period with cinovex. 27 (96.4%) of these patients were female adults and only one patient (3.6%) was male. the first symptom of ms observed in these patients was visual, weakness, sensory and cerebellar (table 1). from these 28 ms patients, five patients (17.9%) had developed meningioma, one (3.6%) diagnosed with lipoma and 22 of these ms patients (78.6%) had been developing pituitary adenoma. it should be noted that 27 of these patients (96.4%) had familial backgrounds. the results from comparing rates of developing benign tumor among patients with ms and non-ms yielded the incidence rate of developing meningioma to be 1.035 but this result was not statistically significant due to the p=0.939. another result of our study showed that the incidence rate for lipoma was 0.020 among ms patients which was statistically meaningful (p<0.001). this result suggests that lipoma occurs more likely in ms patients than in general population. for pituitary adenoma, the results indicated an incidence rate of 1.110 which was not http://www.ajecr.org/ 129 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org table 2 comparison of prevalence rate of benign tumors between ms patients and general population tumor or p-value present absent (95%ci) total no. 4950 4810913 meningioma present 5 4695 absent 4945 4806218 1.035 (0.4310.939 lipoma present 1 48158 absent 4959 4762755 0.02 (0.003-0.143) <0.001 pituitary adenoma present 22 19263 absent 4928 4791650 1.110 (0.7310.624 ms table 3 surveying factors affecting benign tumors tumor type factor beta or (95%ci) p-value meningioma age 0.408 1.51 (0.951-24.012 0.033 ms onset age 0.379 1.46 (0.975-22.188) 0.036 edss 2.08 8.00 (0.823-77.82) 0.043 cancer family 24.193 30.01 (0.901-1.011) 0.20 pituitary adenoma age -0.222 0.801 (0.66-0.968) 0.021 considerable because of the p-value of 0.624 (table 2). a survey on the risk factors effective in developing the three reported benign tumors using “logistic regression” with “background” method indicated that the following factors can be effective in developing meningioma: age, ms onset age, edss and familial background serving as the most important of all. the same survey performed for pituitary adenoma demonstrated that the only effective factor is age with an adverse relation. due to only one reported lipoma, no significant risk factor could be considered for developing this benign tumor. age deliberations made this fact distinct that the age of ms patients with meningioma is at the higher end of our data and on the other side pituitary adenoma stands which means younger ms patients or those ms patients with lesser onset age are more susceptible for developing meningioma and the ms patients with higher years of age are more likely to develop pituitary adenoma (table 3). another finding of our research was that the ms disease process was low in most of ms patients (89.3%) with benign tumors based on edss scale (≤2) meaning they had a slow disease process. discussion there have been some sporadically reports claiming the coincidence of a benign tumor and ms in the literature [58] which suggest a higher incidence of brain tumors in patients who suffer from ms compared to general population. although the reason for this suggested association between the two factors is not yet definitely clear, there are some hypothesis claiming the possible association between ms and developing a benign tumor might be due to the plaques formation in the brains of patients with ms or maybe developing a benign tumor may be caused by immunosuppressive treatments taken by ms patients [9, 19]. in most reported patients, occurrence of ms is prior to developing a benign tumor with a long time gap between them but in few case reports including markou’s [11], it has been indicated that ms occurs shortly after treating a benign tumor. our research team tried to find out the relationship between the two factors. in this regard we studied the tims records of ms patients registered in imss in order to find any case of ms disease with any kind of benign tumors and compare it to the data about incidence rate of developing benign tumor in nonms patients. since we couldn’t find any survey in which the incidence rate of developing benign tumor in isfahan general population had been determined, we compared our findings with the world’s incidence rate for benign tumors (i.e. pituitary adenoma: 0.4%, meningioma: 97.5 in 100,000 , lipoma: 1%). as mentioned above, three types of benign tumors (i.e. meningioma, pituitary adenoma and lipoma) were found in 0.565% of ms patients. data analysis demonstrated a higher incidence rate of meningioma and pituitary adenoma among ms patients but these findings were not statistically meaningful. on the other hand, incidence rate of lipoma was found to be significantly lower among ms patients. furthermore, a survey on disease process and severity based on final edss score indicated a low disease process in majority of ms patients who also suffer from developing benign tumor. taken together, these findings help us to conclude that there may not be a direct correlation between these two diseases, although they may affect each other in some ways. it’s been well documented in many research studies that ms is an autoimmune disease of central nervous system in which immune cells attack to myelin sheaths of nerves [12]. both t-cell and b-cell activation with antibody formation have been shown to play a role in ms pathogenesis [13]. on the other hand, developing tumors is highly associated with a defect in immune system or weakness in any aspect of immune responsibility against tumors. so there can be a relationship in developing tumors and ms disease. pituitary adenoma is a benign tumor of pituitary glandin the brain that is mostly associated with higher amount of prolactin production. prolactin production is regulated by some regulatory hormones produced in hypothalamus including: thyrotrophin releasing hormone (trh), somatostatin (ghih) and prolactin inhibitory hormone (pif). prolactin is well known for its effects on breast feeding and milk production (14) but it has some other roles in immune system and immune cell maturation by its receptors spread throughout immune cells such as tcells and b-cells [15, 16]. prolactin is recently known to play an important role in some autoimmune diseases such as systemic lupus erythematous (sle), rheumatoid arthritis (ra), sjogren’s syndrome (ss), hashimoto’s thyroiditis (ht), celiac disease (cd), graves’ disease (gd), lymphocytic hypophysitis (lh), addison’s disease (ad), diabetes mellitus (dm) type i, and ms [17-19]. as some recent studies indicate, prolactin level in serum and csf is http://www.ajecr.org/ 130 am j exp clin res, vol. 2, no. 4, 2015 http://www.ajecr.org increased in of ms patients [19-21]. furthermore, they claim that higher levels of prolactin in ms patients are associated with higher edss score. on the other hand, some researchers including markianos report no correlation between prolactin level and the disease [22]. although studies on the correlation between pituitary adenoma and ms hasn’t yet drawn much attention among researchers and the definite correlation between ms and pituitary adenoma is still lacking, in this article we found higher incidence of pituitary adenoma in ms patients compared to with general population. the effects of prolactin in ms might be due to, at least in part, progressing inflammation and inflammatory processes. the association of higher level of prolactin and autoimmune diseases is mediated via different influences of prolactin on different aspects of immune system. increased level of prolactin has been demonstrated to interfere with b-cell tolerance, clonal deletion and decreasing the threshold for activation of anergic b-cell as saha et al. [23] indicated, hyperprolactinemia interferes with bcr-mediated deletion, receptor editing and anergy as three main mechanisms of b-cell tolerance which can induce production of autoreactive b-cells. another mechanism that prolactin helps to improve autoimmune diseases is by enhancing the release of thymocytes from thymus and activating th1 lymphocytes and producing inflammatory cytoines such as inf-g [24, 25]. other inflammatory cytokines, in particular, il-1, tnfa, il-6 and il-13 are able to induce prolactin from pituitary which in part helps more inflammatory responses of immune system. as shown by matera, increased antigen presenting activity by dendritic cells and expression of cd40 cells are also induced by prolactin that in turn improves autoimmune activity [26]. taken together, these findings demonstrate a considerable influence of prolactin on improving inflammation and autoimmune diseases such as ms. we indicated in our study that the incidence of pituitary adenoma as a benign tumor is higher among ms patients than in general population but this increased risk was not statistically significant. few confirmed risk factors have been associated with meningiomas which account for 38% and 20% of all cranial tumors in women and men respectively [27]. many researchers believe that autoantibodies play an important role in pathogenesis of meningiomas [28]. although the association of meningioma with ms is not yet completely vivid, batay and al-mefty [29] believe that occurrence of meningioma is correlated with ms and the exacerbation of meningioma was observed during interferon treatments as it was observed by others too[30]. they also believe that therapies with type-1 interferon could be the possible cause of meningioma growth due to autoimmune aggression with lymphocytic infiltration [29]. association of cns tumors growth including meningioma and use of glatiramer acetate was also suggested by kleinpaul and colleagues [31] in a case report study. on the other hand costa et al. [32] suggest that meningioma symptoms are usually remitted after corticosteroid therapy in ms patients. in this article our research team indicated a higher incidence rate of meningioma in ms patients but these results were not statistically considerable. in another study performed by schneider and colleagues [33], it's been shown that meningioma is associated with some other diseases such as diabetes mellitus and hypertention but not with rheumatoid arthritis. the higher incidence of meningiomas in women (2:1) can be caused by hormonal factors including estrogen and progesterone however consequently some suggest that administering oral contraceptives and/or hormone replacement therapy can cause meningiomas [34, 35]. data from case-control and cohort studies indicate controversial results about the impact of oral contraceptive use and risk of meningioma. while some demonstrated increased risk of meningioma associated with the use of long acting hormonal contraceptives [36], others show no definite correlation [34]. similar studies with different results have been performed to consider the association between the use of contraceptives and ms disease [37-39]. some of these studies resulted in positive effects of oral contraceptive use on ms onset and severity while others found no considerable relation. optic nerve sheath meningiomas are formed in the anterior visual pathway resulting in visual deficit, color blindness and finally complete loss of vision [40, 41] with some similarity in symptoms with ms. another finding of our study was significant lower incidence of lipoma among ms patients. lipomas are benign tumor of mature adipocyte tissue mostly found in gastrointestinal tract especially colon. one important characteristic of lipomas is that these types of neoplasm are often asymptomatic and are found incidentally at radiological investigation, colonoscopy, surgery or autopsy [42, 43]. furthermore, lipomas are painless and do not draw much attention in public which results in ignorance. therefore, not all of patients with lipoma visit doctors or go to therapeutic centers meaning lower reported cases. the correlation between lipoma and ms disease is not completely clear yet and much more basically researches are demanded in order to consider the molecular relation between ms and lipoma. in conclusion, of the three types of benign tumors found in ms patients in our study, two (pituitary adenoma and meningioma) were considered to have higher incidence rate among those suffering from ms than in general population but these findings were not statistically significant. on the other hand, lipoma had a lower incidence rate in ms patients which was statistically significant. association of those benign tumors with ms disease which was also reported in other studies could be explained via reviewing the mechanisms and outcomes of these tumors but further studies are required for definite explanations. conflict of interest the authors declare no 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multiple sclerosis. mult scler 19:15-23, 2013. 22. markianos m, koutsis g, evangelopoulos me, mandellos d, sfagos c. serum and cerebrospinal fluid prolactin levels in male and female patients with clinicallyisolated syndrome or relapsing-remitting multiple sclerosis. j neuroendocrinol 22:503-508, 2010. 23. saha s, gonzalez j, rosenfeld g, keiser h, peeva e. prolactin alters the mechanisms of b cell tolerance induction. arthr rheumat 60:1743-1752, 2009. 24. de mello-coelho v, villa-verde dm, dardenne m, savino w. pituitary hormones modulate cell-cell interactions between thymocytes and thymic epithelial cells. j neuroimmunol 76(1-2):39-49, 1997. 25. matera l, contarini m, bellone g, forno b, biglino a. up-modulation of interferon-gamma mediates the enhancement of spontanous cytotoxicity in prolactinactivated natural killer cells. immunol 98:386-392, 1999. 26. matera l, mori m, galetto a. effect of prolactin on the antigen presenting function of monocyte-derived dendritic cells. lupus 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vasconcelos jc, ferret-sena v, pedrosa r. oral contraceptive use and clinical outcomes in patients with multiple sclerosis. j neurol sci 317:47-51, 2012. 38. villard-mackintosh l, vessey mp. oral contraceptives and reproductive factors in multiple sclerosis incidence. contraception 47:161-168, 1993. 39. holmqvist p, hammar m, landtblom am, brynhildsen j. age at onset of multiple sclerosis is correlated to use of combined oral contraceptives and childbirth before diagnosis. fertil steril 94:2835 2837, 2010. 40. berman d, miller nr. new concepts in the management of optic nerve sheath meningiomas. ann acad med singapore 35:168-174, 2006. 41. bosch mm, wichmann ww, boltshauser e, landau k. optic nerve sheath meningiomas in patients with neurofibromatosis type 2. arch ophthalmol 124:379385, 2006. 42. fernandez mj, davis rp, nora pf. gastrointestinal lipomas. arch surg 118:1081-1083, 1983. 43. jeong hk, cho sb, seo tj, lee kr, lee ws, kim hs, et al. autoamputation of a giant colonic lipoma. gut liver 5:380-382, 2011. http://www.ajecr.org/ american journal of 2 am j exp clin res, vol. 1, no. 1, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(1):2-5 original article cancer incidence of larger thyroid nodules and the diagnostic value of palpation and ultrasound guided fine needle aspiration biopsy umut rıza gündüz,* arif aslaner, rojbin karakoyun demirci, hasan çalış, osman zekai öner, nurullah bülbüller department of general surgery, antalya training and research hospital, antalya, turkey abstract. in this prospective study, we aimed to determine the cancer incidence of thyroid nodules larger than 3cm and compare the efficacy of palpation and ultrasound guided fine needle aspiration biopsy (fnab). the study was conducted at general surgery departments of istanbul okmeydanı and antalya training and research hospitals of turkey from march 2008 to december 2011. in our clinics, between the years 2008 and 2011, both palpation and ultrasound guided fnab were performed in all 55 patients having thyroid nodules larger than 3 cm. the results were determined as inadequate, benign, suspicious and malignant. histopathological findings were compared with the results of both palpation and ultrasound guided fnab and sensitivity, specificity and accuracy rates were calculated. the specificity (98%) and accuracy rate (92.72%) of ultrasound guided fnab corresponded to those from the literature, except the rate of sensitivity (40%). this is explained by fnab performed only on the dominant nodule. ultrasound guided fnab is an efficient and reliable diagnostic method when performed by experienced clinicians. hematoma and pain are such rare complications. ultrasound plays an important role in taking biopsy from the true solid thyroid nodules. we conclude that ultrasound guided fnab is the gold standard method in the diagnosis of thyroid nodules. keywords: thyroid, nodule, fine needle aspiration biopsy, ultrasonography introduction thyroid nodules are one of the common clinical problems. in the light of epidemiological studies conducted abroad, with palpation the prevalence was determined as 5%, and with high-resolution ultrasound, it was reported to be from 19% to 67% [1]. several autopsy series have identified the thyroid gland nodules in 37% to 57% of cases and detected small nodules by ultrasound in thyroids that were palpated as normal, which the importance of prevalence of nodular goiter [2]. despite the high prevalence of thyroid nodules malignancy found only 5% of histopathological findings [2]. despite the low ratio early diagnosis of thyroid cancer is life threatening. to assess the thyroid nodule, there are several options including thyroid function tests, thyroid scintigraphy and thyroid ultrasonography. cytological examination with fnab is highly recommended for all palpable solitary or dominant nodules independent of their size. it is more helpful and cost effective in the diagnosis and discrimination of lesions if we could establish whether they are benign or malignant. fine needle aspiration biopsy (fnab) in conjunction with other diagnostic methods which facilitates clear information about the pathology of the thyroid nodules has been frequently used in recent years [3]. in addition, fnab has a high sensitivity, specificity, and accuracy rate as related to malignant thyroid nodules and it can also prevent unnecessary thyroidectomy [4]. in this study we aimed to determine the incidence of thyroid cancer in patients which have thyroid nodule larger than 3 cm and make comparison between manual palpation and ultrasound guided fnab. materials and methods patients diagnosed with thyroid nodules larger than 3 cm were included in this prospective study conducted at the general surgery departments of istanbul okmeydanı and antalya research and training hospitals of turkey between the dates from march 2008 to december 2011 according to the ethical standards as in an appropriate version of the declaration of helsinki and the subsequent amendments. we studied on 55 patients who gave informed consents prior to inclusion in the study. of these patients, 40 (72.7%) were females and 15 (27.3%) were males. the mean age was 47 and ranged from 23 to 72 years old. ultrasound and palpation guided fnab were performed by four different surgeons without local anesthesia, by using a 21 gauge needle attached to a 10 mlsyringe after cleaning the skin with antiseptics. ___________________________________________________________ * corresponding author: umut riza gündüz, md (utg1@yahoo.com). mailto:utg1@yahoo.com 3 am j exp clin res, vol. 1, no. 1, 2014 http://www.ajecr.org smear preparations were evaluated after fixation with 95% ethyl alcohol and staining with pappanicolau procedure. cytological diagnoses were classified into four main groups: low (enough to make a diagnosis), benign (regressive changes, nodular hyperplasia, colloidal goiter, etc.), suspicious (follicular neoplasm, etc.) and malignant (papillary carcinoma, follicular carcinoma, etc.). in all patients who underwent thyroidectomy, the histopathological findings were classified as benign or malignant. the cytological and histopathological diagnoses were compared and sensitivity, specificity, positive predictive value and accuracy rates of ultrasound and palpation guided fnab were calculated. statistical analysis statistical analysis of the findings of was evaluated by using the spss (statistical package for social sciences) program for windows 15.0. results there were 55 fnab cytology samples from 40 females (72.7%) and 15 males (27.3%). the mean age was 47.11 ± 11.78 years (range 23-72 years). the distribution of characteristics of the cases is shown in table 1. solitary nodules were found in 11 patients (20%). the other 44 patients had multinodular goitre (80%). according to the histopathological results of the specimens, the incidence of cancer was calculated to be 9.09%; 3 of these 5 patients were men (60%) and 2 were female (40%). the results of fnab were classified as inadequate, benign, suspicious and malignant. the results of fifty five palpation guided fnab reports showed malignancy for only 1 (1.81%), benign for 28 (50.9%) and insufficient material and blood cells for 26 (47.27%) biopsy samples. the results of fifty five ultrasound guided fnab reports showed malignancy for 2 (3.63%), benign for 49 (89.09%), and suspicious for 1 (1.81%) whereas the remaining 3 (5.45%) reports stated inadequacy of biopsy samples. in the statistical classification of fnab, insufficient material reported as benign, and suspicious results reported as malignant. postoperative histopathological findings, compared with the results of fnab; a) five histopathologically malignant findings were reported as benign and one malignant histopathology result was reported as benign in 55 palpation guided fnab results. b) three histopathologically malignant findings were reported as benign and one malignant histopathology result was reported as benign in 55 ultrasound guided fnab results. the sensitivity, specificity and the accuracy rate of the ultrasound guided fnab were 40%, 98% and 92.72%, respectively. of the 5 samples which were confirmed as malignant on histopathological findings, initially 2 were thought to be benign and 3 were thought to have inadequate material. for this reason, sensitivity of palpation guided fnab was not calculated but specificity and accuracy rate were calculated as 98% and 89.09%, respectively (table 2). discussion thyroid nodules are commonly seen and hard to diagnose clinically. the majority of the thyroid glands are normal by palpation, but detection of small nodules by ultrasound shows the importance of the prevalence of nodular goiter. however although thyroid nodules are very common, thyroid malignancies are rare and constitutes 1% of all malignant neoplasms [5]. with widespread use of ultrasound in the diagnosis of thyroid nodules, the number of asymptomatic patients is increasing [6-11]. a main problem is the distinction of detected thyroid nodules as benign or malignant and prevention of unnecessary surgical procedures. to diagnose thyroid nodules, thyroid function tests, thyroid scintigraphy and thyroid ultrasonography are used routinely. but the benign or malignant distinction cannot be done accurately with these methods. fnab, when evaluated with other diagnostic methods, provide more accurate information about the pathology of thyroid nodules [12-14]. preoperative diagnosis of differentiated thyroid cancer is a difficult task and a definitive diagnosis is made by histopathological examination. because of its safety and cost effectiveness, cytologic diagnosis with fnab has been the method of choice [1-4, 15]. in contrast, studies in the literature seem to doubt the reliability of fnab. in a previous study on 441 patients with single nodule guided fna and 15-year follow-up period, cancer has been identified in 6.4% of non-growing nodules and 26.3% of growing thyroid nodules [16]. however, the mayo clinic study followed 680 of 8000 patients who had undergone surgery and observed that fnab gave false negative results in 15% of cancer patients [17]. in another study, the false negative fnab results in nodules larger than 3cm seen as cystic, solid, semi-solid and in overall were 17%, 25%, 30% and 11%, respectively [18]. the aim of our study was to determine the incidence of cancer of thyroid nodules larger than 3cm and to compare table i patents’ characteristics characteristics value age (years) mean range 47.14 ± 11.78 23 72 gender female (%) male (%) 40 (72.7) 15 (27.3) table 2 sensitivity, specificity and accuracy rate of fine needle aspiration biopsy characteristics palpation guided ultrasound guided sensitivity 0 40 specificity 98 98 accuracy rate 89.09 92.72 4 am j exp clin res, vol. 1, no. 1, 2014 http://www.ajecr.org the outcomes of palpation and ultrasound guided fnab. we detected malignancy in 5 of the 55 patients (9.09%). this rate is similar to the rates reported in the literature [19-22]. inadequate material reported in the literature varies between 10% and 28.2%, [12, 23, 24]. besides our rates were 47.27% in palpation guided and 5.45% in ultrasound guided fnab. for palpation guided fnab, it is noteworthy that the ratio was in an unexpected range values. we attribute this situation due to the experience and skills performed by different clinicians without the use of any guidance of screening methods such as ultrasound. a randomized controlled trial of office based surgeonperformed ultrasound-guided fnab of palpable lesions yielded a statistically significant higher diagnostic rate compared to standard palpation technique. this finding supports the utility of surgeon-performed ultrasound as a core competency in clinical practice [25]. in our study, sensitivity of palpation guided fnab was not calculated because of the 5 samples which were confirmed as malignant on histopathological finding; initially 2 were thought to be benign and 3 were thought to have inadequate material. in the palpation guided fnab, the accuracy rate was 89.09% and the specificity was 98%. these results were compatible with the results reported in the literature for fnab i.e. the accuracy rates of 53-98% and specificity of 72-100% [26-28]. the precision rate for the true-positive results cannot be calculated because of the previously stated reasons. in ultrasound guided fnab, the sensitivity was 40%, specificity is 98% and the accuracy rate was 92.72%, respectively. these results are consistent with rates reported in the literature except for the sensitivity [26-28]. ultrasound guided fnab has an important role in the diagnosis of thyroid malignancies. with the distinction between malignant and benign lesions by fnab, unnecessary surgical procedures or surgical approaches can be prevented and decisions made according to the result of fnab. accordingly, patients undergo surgery when the cytology is reported as suspicious or malignant, and undergo followed-up for certain periods of time if cytology is reported as benign. as shown in the present study, fnab is still the most reliable method for the diagnosis of thyroid malignancy. in our study, the findings from palpation guided fnab are similar to those from the literature except for the sensitivity. a high rate of insufficient material in palpationguided fnab result can be explained by inexperienced clinicians, inappropriate patient positioning, fault in mass immobilization, insufficient negative pressure and the ineffective movements along the long axis of the needle. we conclude that performing fnab under the guidance of ultrasound is highly important in the diagnosis of thyroid gland malignancies for nodules larger than 3cm. conflict of interest the authors declare no conflicts of interest. references 1. american thyroid association (ata) guidelines taskforce on thyroid nodules and differentiated thyroid cancer, cooper ds, doherty gm, haugen br, kloos rt, lee sl, mandel sj, mazzaferri el, mciver b, pacini f, schlumberger m, sherman si, steward dl, tuttle rm. revised american thyroid association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. 1167-1214, 2009. 2. pinchot sn, al-wagih h, schaefer s, sippel r, chen h. accuracy of fine-needle aspiration biopsy for thyroid nodules in predicting neoplasm or carcinoma 4 cm or larger. arch surg 144:649-655, 2009. 3. gharib h, papini e, paschke r, duick ds, valcavi r, hegedus l, vitti p. american association of clinical endocrinilogists, associazione medici endocrinilogi, and european thyroid association medical guidelines for for clinical practice for the diagnosis and management of thyroid nodules: executive summary of recommendations. endocr pract 16:468-475, 2010. 4. kuru b, gulcelik ne, gulcelik ma, dincer h. the false-negative rate of fine-needle aspiration cytology for diagnosing thyroid carcinoma in thyroid nodules. langenbecks arch surg 395:127-132, 2010. 5. frates mc, benson cb, charboneau jw, cibas es, clark oh, coleman bg, cronan jj, doubilet pm, evans db, goellner jr, hay id, hertzberg bs, intenzo cm, jeffrey rb, langer je, larsen pr, mandel sj, middleton wd, reading cc, sherman si, tessler fn. society of radiologists in ultrasound: management of thyroid nodules detected at u.s.society of radiologists in ultrasound consensus conference statement. radiology 237:794-800, 2005. 6. ezzat s, sarti da, cain dr, braunstein gd. incidentelomas thyroid. prevalence by palpation and ultrasonography. arch intern med 154:1838-1840, 1994. 7. brander a, viikinkovski p, nickels j, kivisaari l: thyroid gland: u.s. screening in a random adult population. radiology 181:683-687, 1991. 8. berker d, aydin y, ustun i, gul k, tutuncu y, isik s, delibasi t, guler s. the value of fine-needle aspiration biopsy in subcentimeter thyroid nodules. 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[a clinical pathological study of thyroid nodules detected by physical examinations]. zhonghua nei ke za zhi 47:189-192,2008. 12. gharib h, goellner jr. fine-needle aspiration biopsy of the thyroid: ann intern med 118:282-289, 1993. 13. accurso a, rocco n, palumbo a, feleppa c. use http://www.ncbi.nlm.nih.gov/pubmed?term=%22american%20thyroid%20association%20(ata)%20guidelines%20taskforce%20on%20thyroid%20nodules%20and%20differentiated%20thyroid%20cancer%22%5bcorporate%20author%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22american%20thyroid%20association%20(ata)%20guidelines%20taskforce%20on%20thyroid%20nodules%20and%20differentiated%20thyroid%20cancer%22%5bcorporate%20author%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22american%20thyroid%20association%20(ata)%20guidelines%20taskforce%20on%20thyroid%20nodules%20and%20differentiated%20thyroid%20cancer%22%5bcorporate%20author%5d http://www.ncbi.nlm.nih.gov/pubmed?term=cooper%20ds%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=doherty%20gm%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=haugen%20br%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=kloos%20rt%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=lee%20sl%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=mandel%20sj%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=mazzaferri%20el%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=mciver%20b%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=pacini%20f%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=schlumberger%20m%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=sherman%20si%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=steward%20dl%5bauthor%5d&cauthor=true&cauthor_uid=19860577 http://www.ncbi.nlm.nih.gov/pubmed?term=tuttle%20rm%5bauthor%5d&cauthor=true&cauthor_uid=19860577 5 am j exp clin res, vol. 1, no. 1, 2014 http://www.ajecr.org c. usefulness of ultrasound-guided fine-needle aspiration cytology in the diagnosis of non-palpable small thyroid nodules: our growing experience. j endocrinol invest 32:156-159, 2009. 14. accurso a, rocco n, palumbo a, leone f. usefulness of ultrasound-guided fine-needle aspiration cytology in the diagnosis of non-palpable small thyroid nodules. tumor 91:355-357, 2005. 15. ashcraft mw, van herle aj. management of thyroid nodules. ii: scanning techniques, thyroid suppressive therapy, and fine needle aspiration. head neck surg 3:297-322, 1981. 16. grant cs, hay id, gough ir, mccarthy pm, goellner jr. long-term follow-up of patients with benign thyroid fine-needle aspiration cytologic diagnoses. surgery 106:980-985, 1989. 17. kuma k, matsuzuka f, kobayashi a, hirai k, morita s, miyauchi a, katayama s, sugawara m. outcome of long standing solitary thyroid nodules. world j surg 16:583-587, 1992. 18. meko jb, norton ja. large cystic / solid thyroid nodules: a potential false-negative fine-needle aspiration. surgery 118:996-1003, 1995. 19. papini e, guglielmi r, bianchini a, crescenzi a, taccogna s, nardi f, panunzi c, rinaldi r, toscano v, pacella cm. risk of malignancy in nonpalpable thyroid nodules: predictive value of ultrasound and color-doppler features. j clin endocrinol metab 87:1941-1946, 2002. 20. cochand-priollet b, guillausseau pj, chagnon s, hoang c, guillausseau-scholer c, chanson p, dahan h, warnet a, tran ba huy pt, valleur p. the diagnostic value of fine-needle aspiration biopsy under ultrasono graphy in nonfunctional thyroid nodules: a prospective study comparing cytologic and histologic findings. am j med 97:152-157, 1994. 21. hagag p, strauss s, weiss m. role of ultrasoundguided fine-needle aspiration biopsy in the evaluation of nonpalpable thyroid nodules. thyroid 8:989-995, 1998. 22. leenhardt l, hejblum s, franc b, fediaevsky ld, delbot t, le guillouzic d, ménégaux f, guillausseau c, hoang c, turpin g, aurengo a. indications and limits of ultrasound guided cytology in the management of nonpalpable thyroid nodules. j clin endocrinol metab 84: 24-28, 1999. 23. demicco c. assessment and prospects of thyroid cytology. ann endocrinol 54:258-263, 1993. 24. mandreker srs, nadkarni ns, pinto rgw, menesez s. the role of fine needle aspiration cytology as the initial modality in the investigation of thyroid lesions. acta cytol 39:898-904, 1995. 25. robitschek j, straub m, wirtz m, klem c, sniezek j. diagnostic efficacy of surgeon-performed ultrasoundguided fine needle aspiration: a randomized controlled trial. otolaryngol head neck surg 142:306-309, 2010. 26. caraway np, sneige n, hay na. diagnostic pitfalls in thyroid fine-needle aspiration: a review of 394 cases. cytopathol diag 9:345-50, 1993. 27. harach hr, sb zumsan. cytologic findings in the follicular variant of papillary carcinoma of the thyroid. acta cytol 36:142-146, 1992. 28. sidawy mk, del vecchio dm, knoll sm. fineneedle aspiration of thyroid nodules: correlation between cytology and histology and evaluation of discrepant cases. cancer 81:253-259, 1997. 157 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(2):157-160 original article effect of life skills education on nurses’ happiness farahnaz farnia1, fateme rahighee yazdi1*, maryam salehzadeh abarghouei2 1department of nursing, school of nursing and midwifery, shahid sadoughi university of medical sciences, yazd, iran 2departments of psychology and educational sciences, university of yazd, yazd, iran abstract. happiness is known as a common goal for all the human being and an effective factor in reducing and coping with stress. nursing is one of the stressful jobs in healthcare facing various occupational stressors. life skills education is an intervention used to promote mental health and confront psychosocial injuries. the present study was conducted to examine the effect of life skills education on nurses’ happiness at shahid sadoughi hospital in yazd, 2015. this is a semi-experimental study with preand post-test and a control group. a total of 32 volunteer nurses were selected and assigned into control (n=16) and case (n=16) groups. the case group participated in an 8-hour course of life skills education (including effective communication, self-awareness, anger management, and coping with stress). data collection was carried out using fordyce happiness scale and demographic questions completed before and one week after the course by both groups and analyzed using chi square and t-test. according to the findings of this study the mean (±sd) of nurses’ happiness in the control and case groups were 131.25 ± 11.96 and 130.44 ± 12.75 at the baseline and 130.06 ± 28.10 and 146.25 ± 13.70 one week later, respectively (p = 0.038). this difference was not significant in control group (p = 0.163). the results of this study indicated that life skills education could significantly increase nurses’ happiness and thus it can be used as a beneficial intervention. keywords: happiness, nurse, education, life skills introduction happiness is the most basic human issue [1] and an undeniable pillar in every organization [2] leading to socioeconomic development, better task performance, increased production, and decreased costs of abandonment of duty [3]. in addition to self-esteem and appropriate social relations, happy people have greater organizational commitment [4]. happiness results in higher environmental constancy and lower tension while affecting the staff spirit and making the organizational environment more appealing. when the staff happiness is originated in their organization, they become more devoted to their career and perform their tasks in the best level, thus feeling well for being positive for their organization and searching for more progress and development [5]. happiness decreases feeling the stress and increases staff ability for work [6]. research shows that happy people have less occupational burnout and absenteeism and unlikely to leave their jobs [7]. other benefits mentioned in the studies for happiness include positive self-concept, mental and physical health, emotional balance, increased hope for the future, favorable attitude towards one's self and others, promotion in social relations, more tendency to help others, better decision making, and more creativity [7] nursing is one of the stressful jobs in healthcare where various occupational stressors endanger mental health of the [8]. evidence shows that depression and anxiety are common problems among nurses [9].the national survey of the work and health of nurses reported that nurses had higher rate of depression (1 in 10) as compared with their counterparts [10]. only one seventh of the nurses are happy at work environment [11]. peterson suggested that hospital nurses became depressed twice as much as ordinary people [5]. on the other hand, nurses' stress has been increasing during the last decade [12]. mirzaei tanshizi et al. [13] believed that depression significantly reduced well-being and caused low happiness and life satisfaction, while happiness is one of the factors in decreasing and coping with stress. life skills are abilities helping to behave appropriately and wisely in different situations and adaptively communicate with one's self and others. these skills assist in solving the problems with no aggression and feeling happy in life while being successful. learning these abilities yields mental health promotion, human relations ___________________________________________________________ * corresponding author: fateme rahighee yazdi, msc (rahighee@gmail.com). http://www.ajecr.org/ mailto:rahighee@gmail.com 158 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org enrichment, and increased healthy behaviors. this program is introduced by who for promoting mental health and confronting psychosocial injuries which consists of 10 skills [14]. these skills enable individuals to convert their knowledge, value, and attitudes to practical abilities while they are effective in creating and developing abilities such as decision making, motivating, accepting responsibility, effective relationships, self-esteem, problem solving, selfsufficiency, and mental health [15]. this program has shown to be helpful in decreasing anxiety and anger as well as preventing depression, and increasing self-esteem and flexibility against changes [16]. life skills education increases ability in controlling problems and the person voluntarily calms down [17]. in the present study, based on the nurses' needs, four skills were presented including selfawareness, effective communication, coping with anger, and coping with stress. nurses are exposed to tensions, stress, and job emotions leading to anger and aggression. learning coping with emotions, coping with stress, and effective communication can improve inappropriate behaviors. considering the importance of this issue, the present study was conducted to examine the effect of life skills education on nurses’ happiness at shahid sadoughi hospital in yazd. materials and methods this is a semi-experimental study with preand posttest and a control group. study population included nurses working at shahid sadoughi hospital. in the pilot study standard deviation of happiness score was calculated as 0.5. considering n = 2(z 1− α 2 +z1−β) 2 δ2 d2 , α = 0.05, β = 0.2, and minimum difference of 0.5 between the intervention and control groups, 16 nurses from both control and experiment groups were selected. case group participated in an 8-hour course of life skills education (including effective communication, self-awareness, anger management, and coping with stress). data collection was carried out using fordyce happiness scale and demographic questions completed before and one week after the course by both groups and analyzed by chi square and t-test. inclusion criteria were defined as informed consent of the nurses for participating in the study, attendance in all the sessions of life skills course in the case group, having a bachelor's or master's degree in nursing with minimum 6month work experience, not attending life skills education course before, having no mental disease and medicines consumption. those with major stressful problems in life such as recent death of relatives or close friends or divorce were excluded from the study. data collection was carried out using fordyce emotions questionnaire including two parts: 1) demographic variables such as age, sex, work experience, income, and secondary job; 2) 47 questions of fordyce happiness scale table 1 frequency distribution of demographic characteristics in case and control groups * mt: million tomans. table 2 comparison of nurses’ happiness in case and control groups at the baseline and one week after life skills education on a four-point likert scale (agree, partly agree, partly disagree, and disagree) developed by eysenck [18] and translated and revised at isfahan university. argyle [19] reported the internal consistency of this questionnaire as 0.73-0.93 with mean of 0.86. validity of the happiness test in persian was determined using oxford happiness questionnaire. the correlation between these two scales was 0.78 and significant [20] with reliability of 0.92 [21]. with permission from the institutional ethics committee, volunteer nurses who met the inclusion criteria were selected and after obtaining informed consent voluntarily participated in the control or case groups. case group attended two 4-hour sessions of life skills education. participants of both groups completed happiness questionnaire before and one week after the classes. happiness in each step was determined and compared. statistical analysis data were analyzed using descriptive statistics (mean, sd, percentage) and differential statistics (chi-square, ttest). a probability (p) value at the level of less than 0.05 was considered statistically significant. results most of the participants were married women with bs cont rol case no. (%) no. (%) male 5 (31.2) 3 (18.8) female 11 (68.8) 13 (81.2) single 2 (12.5) 2 (12.5) married 14 (87.5) 14 (87.5) bs degree 15 (93.8) 15 (93.8) msc degree 1 (6.2) 1 (6.2) morning 1 (6.2) 3 (18.8) rot at ing 15 (93.8) 13 (81.2) 1 t o 2 mt 10 (62.5) 9 (43.8) 2 t o 3 mt 3 (18.8) 5 (56.3) >3 mｔ 3 (18.8) 2 (0.0) yes 1 (6.2) 1 (6.2) no 15 (93.8) 15 (93.8) 1.00 group pdemographic variables income* 0.307 secondary job 1.00 educat ion 1.00 work shift 0.2 gender 0.44 marit al st at us group no. baseline 1 wk post -educat ion mean (sd) mean (sd) cont rol 16 131.25±11.96 130.44±12.75 -1.42 0.163 case 16 130.06±28.10 146.25±13.70 -2.17 0.038 st at ist ics t p happiness http://www.ajecr.org/ 159 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org degree working at rotating shift. the mean ± sd for age was 34.25 ± 6.68 in control and 36.65 ± 5.68 in case group. the results of chi-square test showed no considerable difference in demographic characteristics in the two groups (table 1). average happiness score in control group was 131 ± 11.96 at the baseline and 130.44 ± 12.75 one week after the course, while in case group the average happiness score raised from 130.06 ± 28.10 to 146.25 ±13.70 which was statistically significant (table 2). discussion the present study was designed to examine the effectiveness of life skills education on nurses' happiness working at shahid sadoughi hospital. the results indicated that life skills education could significantly increase happiness in case group which confirmed the results of similar studies [22-27]. mahdavi haji et al. evaluated the effectiveness of life skills education on happiness, quality of life, and emotional regulation in students showing meaningful difference in all items in the case group [22]. javadi et al. reported that life skills education could considerably improve nurses' quality of life in the case group [23]. ghorbanshiroodi et al. studied life skills education on decreasing occupational stress and staff anxiety where it could decrease staff anxiety [24]. ghavipanjeh et al. examined the effect of problem solving skill on students' depression. case group participated in a 6-session course in groups of 6 to 8 members in three weeks [25]. the average depression score decreased significantly in the case group. in the present study four skills of effective communication, self-awareness, anger management, and coping with stress were presented. self-awareness education helps the individuals recognize their strengths and weaknesses and find better recognition of themselves, trying to remove their weaknesses and improve the strengths. techniques of coping with stress use more efficient ways to confront problems with less stress. according to anger management, if temper is not controlled it can negatively affect mental and physical health. and finally effective communication is about how to inform others of our needs and sensations in a way that while we meet our desire the other person is satisfied as well. these skills help to have an appropriate and wise behavior in different situations and adaptively communicate with one's self and others. also they assist in solving problems with no aggression and feeling happy in life while being successful. on the other hand, group learning can have a positive effect in decreasing mental pressure. congregating and feeling that others have the same problems while using their experience in coping with stress are effective in reducing stress and increasing happiness. also muscle relaxation was practiced in these classes which decreases muscle tension following a stress. regarding the effectiveness of these skills on nurses' happiness, it is recommended to put these skills in cultural and educational programs for the nurses. acknowledgement the authors would like to express their gratitude to the nurses of shahid sadoughi hospital especially mr. ahmad kamali zarbandi for his kind help in data collection. conflict of interest the authors declare no conflicts of interest. references 1. sharifi k, sooky z, tagharrobi z, akbari h. happiness and its related factors among the students of kashan university of medical sciences in 2006-7. feyz, j kashan univ med sci 14:62-69, 2010. 2. nasaran broujeni i, asadi h. investigation of the relationship between happiness and commitment of the staff of the ministry of sport and iranian youth. sport manage 6:545-557, 2013. 3. fisher cd. happiness at work. int j manage rev 12:384-412, 2010. 4. dehaghi mr. happiness as an effective factor in organizational commitment of managers. afr j bus manage 6:9460-9468, 2012. 5. peterson c. the future of optimism. am psychol 55:44-55, 2000. 6. carr a. positive psychology: the science of happiness and human strengths. 2nd ed, routledge london, new york 2011. 7. cohen s, herbert tb. health psychology: psychology factors and physical disease from the perspective of human psychoneuroimmunology. annu rev psychol 47: 113-142, 1996. 8. badryzadh a, frhady a, tarahi mj, saki m, biranovand gr. evaluation of mental health nurses in public hospitals of horramabad. j lorestan univ med sci 15:62-69, 2013. 9. nakakis k, ouzouni ch. factors influencing stress and job satisfaction of nurses working in psychiatric units: a research review. health sci j 2:183-195, 2008. 10. marilyn c, michael s, dorothy a. depression in nurses. can j nurs res 42:66-82, 2010. 11. duffin c. only one in seven nurses happy in their job, work survey reveals. nurs stand 28:7, 2014. 12. moustaka a, constantinidis tc. sources and effects of work-related stress in nursing. health sci j 4:210-219, 2010. 13. mirzaei tanshizi p, et al. comparison of the effectiveness of subjective well-being program and fordyce cognitive-behavioral program in reducing depression among high school students in isfahan. j shaheed sadoughi univ med sci 17:291-302, 2009. 14. who/cpa. school health education to prevent aids and std. school health education to prevent aids and std resource package for curriculum planners world health organization global program on aids geneva. 1994. 15. ashoori m, jalilabkenar s, hasanzadeh s, pourmohamadreza tajrishi m. effectiveness of life skill instruction on the mental health of hearing loss students. rehabilitation 13:48-57, 2013. 16. wodarski js, feit md. adolecent preventive health. a social and life group skills. j fam ther 24:191208, 2000. http://www.ajecr.org/ 160 am j exp clin res, vol. 3, no. 2, 2016 http://www.ajecr.org 17. albertyn rm, kapp ca, groenewald cj. patterns of empowerment in individuals through the course of a life-skills programme in south africa. stud educ adult 33:180-200, 2001. 18. eysenck m. happiness: fact and myths. uk: erlbaum, 1990. 19. argyle m. the psychology of happiness. london: rutledge, 2001. 20. bakhtiar nasrabadi ha, pahlevan sadegh a. impact of education and training programs to enhance the vitality fordyce. j edu psychol 15:21-40, 2012. 21. liaghatdar mj, abedi mr, jafari e. standardization of isfahan-fordyce happiness inventory in university students. j psychol 13:183-96, 2008. 22. mahdavi haji t, mohammadkhani s, hahtami m. the effectiveness of life skills training on happiness, quality of life and emotion regulation. procedia-soc behav sci 30:407-411, 2011. 23. javadi m, sepahvand mj, mahmudi h, sori a. the effect of life skills training on quality of life in nurses of khorramabad hospitals. sci j hamadan nurs midwifery facul 21:32-40, 2013. 24. ghorbanshiroudi s, maddahi me, mahmoudi e, ghorbannejad f, keikhayfarzaneh mm. the relationship between job stress and anxiety level of the employees of tehran municipality and determining the effectiveness of life skills training in reducing them. eur j sci res 65:444452, 2011. 25. ghavipanjeh s, ebrahimi h, barzanjeh atri s, pakpour v, tarverdizadeh p. effect of problem-solving skills on depression scores in nursing and midwifery students. tehran univ med sci j nurs midwifery 20:3846, 2014. 26. hartati anis s, gusaptono h. the role of life skills training on self-efficacy, self esteem, life interest, and role of behavior for unemployed youth. global j manage bus res 10:132-139, 2010. 27. yadav p, iqbal n. impact of life skill training on self-esteem, adjustment and empathy among adolescents. j indian acad appl psychol 35:61-70, 2009. http://www.ajecr.org/ 206 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(2):206-209 original article neurodevelopmental outcome in perinatal asphyxia ashaq h thoker 1 *, mushtaq sheikh 1 , parvez a thoker 2 , mukhtar thoker 2 1department of pediatrics, sher-i-kashmir institute of medical sciences, soura, kashmir,india 2 abstract. perinatal asphyxia is an important cause of neonatal morbidity and mortality. it might lead to neurologic handicaps in children. to find out the incidence of hypoxic ischemic encephalopathy (hie) in hospital delivered patients, we studied the correlation between apgar score hie stage and neurodevelopmental outcome at one year of age. in this prospective study 93 asphyxiated babies admitted to the neonatal intensive care unit (nicu) during the period of oct 2012 to nov 2013 were followed for a period of 1 year. the m ale to female ratio in hie was 1.16:1. the incidence of hie in hospital delivered patients (at trust hospital) was 2.2%. death rate in the nicu was 8.4%. hie was accounting for 22.4% of total nicu deaths. head circumference at 1 year was significantly low in hie patien ts (especially in hie iii). seizures were present more frequently in patients with hie ii and hie iii than in hie i patients. neurodevelopmental delay was present in 31.2% of patients at the end of 1 year in hie patients. more number of hie iii patien ts had developmental delay as compared to hie i and hie ii. low apgar score at 5 minutes and low admission ph were predictors of poor neurodevelopmental outcome. apgar score at 1 minute was poorly related to neurodevelopmental outcome. it was concluded that the birth asphyxia still remains a major cause of morbidity and mortality during neonatal period. keywords: perinatal asphyxia, hypoxic ischemic encephalopathy, neurodevelopment, outcome introduction neonatal hypoxic ischemic encephalopathy (hie) is the term used most frequently to designate the clinical and neuropathologic findings thought to occur in the full term infant following either intrapartum or neonatal asphyxia. perinatal hie occurs in one to three per 1000 live full-term births [1]. perinatal asphyxia is defined as failure to initiate and sustain breathing at birth as per who definition [2]. the national neonatal perinatal database (nnpd) defined it as moderate birth asphyxia or an apgar score of 4-6 at 1 minute of age; or severe birth asphyxia or an apgar score of 0-3 at 1 minute of age [3]. it continues an important cause of neonatal morbidity and mortality. manifestation of hie seen in 1.5% live births and is responsible for 20% of neonatal deaths as per nnpd [3]. seizures occur in 50% of patients of hie ii and 100% of patients with hie iii [4]. forty-ei ght percent of patients had neurodevelopmental delay in hie ii [5]. delayed neurodevelopmental outcome was present in 100% of patients with hie iii [6]. asphyxia during the perinatal period led to the development of ulegyria in children who, as neonates, survi ved a perinatal insult. the cycle of hypoxia leading to lact ate dependent cellular damage is likely causal in brain damage. as the cells swell with secondary loss of membrane transfer integrity, ischemi a simultaneously, cardiac muscle is being affected, resulting in decreased cardiac output and further hypoperfusion [7]. the outcomes of hie are devastating and permanent, making it a major burden for the patient, the family, and society. the aim of this study was to find out the incidence of hie in hospital delivered patients and correlation between apgar score or hie stage and neurodevelopmental outcome at one year of age. materials and method all term infants admitted in neonatal intensive care unit (nicu) of sher-i-kashmir institute of medical sciences (skims) hospital with the diagnosis of hie i, ii and iii during oct 1, 2012 to oct 31, 2013 (total 13 months) were included in the study. exclusion criteria included infants whose examination was not possible during follow up for at least 2 different examinations were excluded from the study. every patient was assigned a number, apgar score, hie stage, number of seizures if present as well as all metabolic parameters were recorded as per the proforma. values of head circumference, height and weight were plotted in nchs charts104 anthropometry. they were called back between 1 and 6 months once, and then 2 monthly until one year. on follow up, through ___________________________________________________________ * corresponding author: dr. ashaq h thoker (ashaqhussain22@gmail.com). http://www.ajecr.org/ mailto:ashaqhussain22@gmail.com 207 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org examination was done and development was assessed by tri vendrum developmental screening chart. those who didn’t turn up were repeatedly contacted. all children were managed in nicu at skims hospital, and they were called on follow up at high risk clinic for neurological examination. all those who developed even a minor deficit were advised for stimulation therapy, physiotherapy as and when required. eeg, mri, ct scan, us g and bera was advised whenever indicated ophthalmic examination was performed on discharge and on first follow up and repeated as and when required. all patients were i mmunized as per national schedules and advised optional vaccination. statistical analysis statistical analysis was done using spss v20 (chicago, il) expressing group variables as mean ± standard deviation. student’s t-test was applied to independent samples of continuous variables, and chisquare or fisher’s exact test was used for categorical values. statistical significance was set at p < 0.05. results total number of deli veries at trust hospital were 3000 cases. sixty-six of the total patients developed hie-i, ii, iii after deli very in trust hospital. number of patients with hie-i, ii, iii referred to other hospitals were 85 cases. total number of patients admitted with hie-i, ii, iii were 151 cases. total nicu admissions were 1267 cases. total deaths were 107 cases. death due to hie i, ii, iii were 24 cases. total patients discharged with hie-i, ii, iii were 127 cases (151 admissions minus 24 deaths). cases enrolled for follow up were 102 cases with 46 being hie i, 42 being hie ii and 14 being hie iii. ninety-three cases were successfully followed for 1 year and included in the study. males were 50 and females being 43 cases with 45 being hie i, 40 had hie ii and 8 had hie iii. nine patients were lost to follow up out of which 3 belonging to group hie iii died at home as told by relati ves on phone interview. one of the probable reason for lower number of female babies could be the biased attitude of the parents towards girl children. incidence of hie at trust hospital was 2.2% (66/3000×100). death rate in the nic u at skims hospital, soura was 8.4% (107/1267×100). contribution by hie to total nic u deaths was 22.4% (24/107×100). overall, mortality in neonates admitted at skims nic u was 8.4% with major causes including sepsis, prematurity and hie > hie was responsible for 22.4% deaths. when followed over a period of time (1 year), it was found that head growth was significantly affect ed hie i patients as seen by statistically p value (p<0.0001). head growth was affected more in patients with hie iii as it is evident from mean ofc values. among the 93 neonates included in the study, 28 patients convulsed at any stage from admission to the follow up. most convulsions occurred during first 3 days of life. convulsions occurred in all patients, 8 (100%) belonging to group hie iii. convulsions were more in group ii patients, 17 (42.5%) than group i patients, 3 (6.7%). the patients of group i who convulsed were not included in group ii because they convulsed after discharging them from ward while on follow up. there was a statistically significant relation between hie stage and occurrence of convulsion (p < 0.0001). twent y-nine patients had neurodevelopmental delay in the form of delayed attainment of milestones or abnormal neurological examination at the end of one year. all 8 patients (100%) of sarnat stage iii hie had neurodevelopmental delay at the end of 1 year while 21 (52.5%) patients of stage ii and no patient of stage i were affect ed during the same period. there was statistically significant relation between sarnat stage of hie and neurodevelopmental outcome at 1 year (p<0.0001). blood sugar and serum calcium were checked at admission, at 24 hours and again at 48 hours. any patient with symtomatic hypoglycemi a <45 mg/dl was treated with 2 ml/kg of dextrose 10% in water. all those neonates with blood sugar <40mg/dl were enrolled as having hypogl ycemia. a total of 10 patients at admission were hypoglycemic while as only 2 patients at 24 hours and 1 patient at 48 hours was hypogl ycemic. there was statistically no significant relation between sarnat hie stage and blood sugar at admission, at 24 hours and at 48 hours of life; p=0.854; p=0.905; p=0.512. there was statistically a significant relation bet ween ofc at 1 year and neurodevelopmental outcome (p=0.0001). patients with neurodevelopmental delay were more likely to have lesser ofc (mean ofc=40.52cm) as compared to unaffected patients (mean ofc=45.86cm). apgar score at 1 minute had statistically no significant relation with neurodevelopmental outcome (p=0.092), while apgar score at 5 minutes had statistically significant relation with neurodevelopmental outcome (p=0.0001). blood ph at admission had statistically significant relation with neurodevelopmental outcome (p=0.0001). patients with lower admission blood ph had poor neurodevelopmental outcome as compared to patients with higher admission blood ph. discussion perinatal hypoxic-ischemic encephalopathy (hie) is an important cause of brain injury in the newborn and can result in long-term devastating consequences. perinatal hypoxia is a vital cause of long-term neurologic complications varying from mild behavioral deficits to severe seizure, mental retardation, and/or cerebral palsy in the newborn. hie is an i mportant cause of morbidity and mortality in the neonatal period and of cerebral palsy as a late neurologic sequela in the postnatal period. although intervention is limited and mostly supporti ve at this ti me, it is still important to promptly and accuratel y identify neonates who have sustained a hypoxic-ischemic brain injury to facilitate optimal management as per nnpd [3] data manifestation of hie were seen in 1.5% and as per cloherty frequency of perinatal asphyxia is approxi matel y 1% to 1.5% of live births [8]. thornberg et al. reported incidence of birth asphyxia in swedish population was 0.54% [9]. in the present study, http://www.ajecr.org/ 208 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org incidence of hie in hospital delivered patients at trust hospital was 2.2% (66/3000×100) which is comparable to nnpd [3]. higher incidence in present study compared to national data may be because facilities for continuous antenatal fetal monitoring are not available in most hospitals of the valley. hie was responsible for 20% of neonatal deaths as per nnpd whereas the study by lee et al. in southern nepal observed that birth asphyxia accounted for 30% of neonatal mortality [3, 10]. as per lawn et al., birth asphyxia was responsible for 23% neonatal deaths [11]. as per bang at et al., birth asphyxia was responsible for 25% neonatal mortality rate in rural gadchiroli, india [12]. in the present study, asphyxia was responsible for 22.4% (24/107×100) of neonatal deaths which is comparable to studies of nnpd and lawn et al. [3, 11]. mortality in the present study was lesser than the mortality found by lee et al. and bang et al. [10, 12]. more mortality in bang et al. study may be because the study was conducted in rural area [12]. early recognition and management of complications in hie patients in our nicu was responsible for lesser mortality than other studies. in the present study, it was found that there is a statistically significant relation between hie and ofc at 1 year of age. there was more decrease in ofc at 1 year of age with increase in stage of hie. similar results were found in the study by mercuri et al. where 53% of neonates with hie had suboptimal head growth, with a statistically significant relation between hie and suboptimal head growth [13]. cordes et al. also found that initial slow head growth in patients with hie resulted in development of microcephal y [14]. pisani et al. found seizures in 50% of patients of hie ii and 100% patients with hie ii and in the begum et al. study seizures were present in 35% patients with birth asphyxia [4, 15]. in present study, seizures were found in 30.1% patients with hie. seizures were present in 100 % cases with hie iii and 42.5% patients with hie ii. seizures were also present in 6.7% cases with hie i, but all these patients developed seizures during follow up and not during hospital stay. seizures in our study were less than the other studies and may be due to regular monitoring and better management of patients. carli et al. observed that 52% of neonates who suffered birth asphyxia hie ii were normal at the end of 1 year and 48% patients had neurodevelopmental delay ranging from mild to severe delay [5]. as per robertson et al., the study in which patients with hie were followed up to 3.5 years, all patients with hie i were normal while delayed neurodevelopmental outcome was present in 21.3% of patients with hie ii and 100% patients with hie iii [6]. the results in the present study were comparable with the study of carli et al. with neurodevelopmental delay present in 52.5% patients with hie ii [5]. but in the present study outcome in patients with hie ii was delayed in 52.5% as compared to patients with hie ii in study by robertson et al. (21.3%). this can be explained because in our study there was shorter follow up ti me [6]. in the present study, no significant relation was found between hie and hypoglycemia. this is in contrast to the study made by bassu et al. who found that mean blood glucose was significantly lower in asphyxiated patients than in non-asphyxiated neonates [16]. different results in present study may be attributed to strict blood sugar monitoring during admission. on the other hand, nadeem et al., while studying relation between hypoglycemi a and neurodevelopmental outcome in hie patients, found that 83.8% of patients were normogl ycemic while in our study 89.2% were normogl ycemic [17]. dong et al. studied the relation between birth asphyxia and hypocalcemia [18]. no significant relation was found between the two. in the present study also no significant relation was found between hie and hypocalcemia. in the present study, there was a statistically significant relation between ofc at 1 year and neurodevelopmental outcome (p<0.0001). patients with neurodevelopmental delay were more likely to have lesser ofc (mean ofc = 40.52cm) as compared to unaffected patients (mean ofc = 45.86cm). ofc at admission had poor relation with neurodevelopmental outcome at one year of age. similar results were found by mercuri et al., who found that suboptimal head growth at 1 year was significantl y associated with pattern of brain lesion and neurodevelopmental delay in patients with hie [13]. also in the study by coronado et al., it was found that mini mum head circumference and head circumference drop were relevant markers of neurological i mpairment [19]. in misra et al. study, there was delayed neurodevelopmental outcome in patients with low apgar scores at 5 and 10 minutes [20]. also there was not a significant relation between apgar score at 1 minute and neurodevelopmental outcome. ehrenstein et al. found that a fi ve-minute apgar score < 7 has a consistent association with prevalence of neurologic disability and with low cogniti ve function [21]. in the present study, results were comparable to studies by misra et al. and ehrenstein et al. [20, 21]. in our study, no significant relation was found between apgar score at 1 minute and outcome whereas a significant relation was found between apgar score at 5 minutes and neurodevelopmental outcome. relation between apgar score at 10 minutes and outcome was not studied. in the present study, we found a statistically significant relation between blood ph at admission and neurodevelopmental outcome in hie patients at 1 year of age. mean blood ph at admission in affected patients was lower than the unaffected patients. graham et al. also found a significant relation between neurologi cal morbidity and blood ph at birth in patients with hie. gemma et al. also found that low arterial cord ph showed strong, consistent, and temporal associations with clinically i mportant neonatal outcomes [22, 23]. conclusion birth asphyxia still remains a major cause of morbidit y and mortality during neonatal period in india. overall mortality was 22.4%, which clearl y indicates the need for early detection of maternal risk factors, better obstetric http://www.ajecr.org/ 209 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org management and the prompt resuscitation measures. conflict of interest the authors declare no conflicts of interest. references 1. lai mc, yang sn. perinatal hypoxic-ischemic encephalopathy. j biomed biotechnol 2011; 2011:609813. 2. world health organization, perinatal mortality: a listing of available information. frh/msm.96.7 geneva: who, 1996. 3. report of the national neonatal perinatal database (national neonatology forum, india) 2000. 4. pisani f, orsini m, braibanti s, copioli c, sisti l, turco ec. development of epilepsy in newborns with moderate hypoxic-ischemic encephalopathy and neonatal seizures. brain dev 31:64-68, 2009. 5. carli g, reiger i, evans n. one-year neurodevelopmental outcome after moderate newborn hypoxic ischaemic encephalopathy. j paediatr child health 40:217-20, 2004. 6. robertson c, finer n. term infants with hypoxic ischemic encephalopathy: outcome at 3.5 years. dev med child neurol 27:473-84, 1985. 7. myers re. experimental models of perinatal brain damage: relevance to human pathology. in gluck l (ed.): intrauterine asphyxia and the developing fetal brain. chicago, year book medical publishers, inc., 1977. 8. cloherty jp, eichenwald ec, stark ar. manual of neonatal care, 6th edition: lippincott williams & wilkins pp 518-528, 2008. 9. thornberg e., thiringer k., odeback a. and milsom i. birth asphyxia: incidence, clinical course and outcome in a swedish population. acta paediatrica, 84: 927-932, 1995. 10. lee ac, mullany lc, tielsch jm, katz j, khatr y sk, leclerq sc, adhikari rk, shrestha sr, darmstadt gl. risk factors for neonatal mortality due to birth asphyxia in southern nepal: a prospective, community based cohort study. pediatrics 121:e1381-90, 2008. 11. lawn je, cousens s, zupan j. lancet neonatal survi val steering team. 4 million neonatal deaths: when? where? why? lancet 365:891-900, 2005. 12. bang at, reddy hm, bang ra, deshmukh md. why do neonates die in rural gadchiroli, india? (part ii): estimating population attributable risks and contribution of multiple morbidities for identifying a strategy to prevent deaths. j perinatol suppl 1:s35-43, 2005. 13. mercuri e, ricci d, cowan fm, lessing d, frisone mf, haataja l, counsell sj, dubowitz lm, rutherford ma. head growth in infants with hypoxic-ischemi c encephalopathy: correlation with neonatal magnetic resonance i maging. pediatrics 106:235-43, 2000. 14. cordes i, roland eh, lupton ba, hill a. early prediction of the development of microcephaly after hypoxic-ischemic encephalopathy in the full-term newborn. pediatrics 93:703-707, 1994. 15. begum ha, rahman a, anowar s, mortuza a, nahar n. long term outcome of birth asphyxiated infants. mymensingh med j 15:61-65, 2006. 16. basu p, som s, choudhuri n, das h. contribution of the blood glucose level in perinatal asphyxia. eur j pediatr 168:833-838, 2009. 17. nadeem m, murray dm, boylan gb, dempsey em, ryan ca. earl y blood glucose profile and neuro developmental outcome at two years in neonatal hypoxicischaemic encephalopathy. bmc pediatr 11: 10, 2011. 18. dong n, wu pyk, siassi b, viray l. perinatal asphyxia and postnatal changes in serum total and ionized calcium. pedtr res 18:356a-356a, 1984. 19. coronado r, giraldo j, macaya a, roig m. head circumference growth function as a marker of neurological impairment in a cohort of microcephalic infants and children. neuropediatrics 43:271-274, 2012. 20. misra pk, srivastava n, malik gk, kapoor rk, srivastava kl, rastogi s. outcome in relation to apgar score in term neonates. indian pediatr 31:12151218, 1994. 21. ehrenstein v, pedersen l, grijota m, nielsen gl, rothman kj, sørensen ht. association of apgar score at fi ve minutes with long-term neurologic disability and cogniti ve function in a prevalence study of danish conscripts. bmc pregnancy and childbirth 9:14, 2009. 22. graham em, ruis ka, hart man al, northington fj, fox he. a systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy. am j obstet gynecol 199:58795, 2008. 23. gemma l malin, rachel k morris, khalid s khan. strength of association bet ween umbilical cord ph and perinatal and long term outcomes: systematic review and meta-analysis. brit med j 340:c1471, 2010. http://www.ajecr.org/ 194 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(1):194-196 case report neonatal infectious hemophagocytic lymphohistiocytosis boutrid nada 1 , rahmoune hakim 1 *, amrane mounira 2 , boutaghane noureddine 3 , bousfiha ahmed aziz 4 , bioud belkacem 1 1department of pediatrics, setif university hospital, setif-1 university, algeria 2department of biochemistry, setif university hospital, setif-1 university, algeria 2neonatoal unit, n. hammoud universityhospital, algiers-1 university, algeria 4pediatric clinical immunology unit, casablanca children hospital, hassan ii university, morocco abstract. hemophagocytic lymphohistiocytosis (hlh) is a disorder of the mononuclear phagocytic system and a severe, life-threatening syndrome due to an excessive immune activation. this hyperinflammatory condition is often triggered by a variety of agents or events, mostly genetic or infectious; and its scarcity in the neonatal period often delay diagnosis and adequate management. we report a peculiar neonatal case with multivisceral involvement, managed promptly and aggressively. discussing the most important relevant points in both diagnosis and therapy of this peculiar disorder is highlighted in purpose of increasing awareness and enhancing early determinant recognition. keywords: hemophagocytic lymphohistiocytosis, neonatal period, life-threatening, diagnosis, therapy introduction hemophagocytic lymphohistiocytosis (hlh) is a severe, life-threatening syndrome due to an excessive immune activation and inflammation, most frequently affecting infants less than 18 months of age. it can be triggered by a variety of events that disrupt immune homeostasis [1]. the rarity of this syndrome, its scarcity in the neonatal period, combined to the lack of specificity of the clinical and laboratory findings often delay diagnosis and adequate management [2]. case presentation a male, term newborn initially admitted for perinatal asphyxia, presented at day 3 an acute, severe combination of multivisceral deficiency (hepatic, renal and cardiac) associated to a myriad of clinical and biological signs including anemia, thrombocytopenia and febrile splenomegaly. the balance sheets show a marked inflammation (serum ferritin > 1500 ng/ml); hyponatremia (117 meq/l) and hypertriglyceridemia (> 3 g/l). a probable infectious neonatal hlh was then highly considered. a holistic approach, with antibiotics (ceftriaxone: 100 mg/kg/day, and amikacin: 15 mg/kg/day, twice), antiviral (acyclovir*: 1 g/m²), and steroids (methyl prednisolone: 04mg/kg/day, every 6h) is ordered. a close monitoring and supportive care with transfusion and glucose 10% perfusion were also conducted. the newborn recovered within 5 days. total duration of treatment was of 15 days of antibiotics (ceftriaxone) and antiviral. steroids were tapered from the 7th day. one month after discharge, the infant did well and was free of symptoms. discussion hemophagocytic lymphohistiocytosis (hlh) is a disorder of the mononuclear phagocytic system [3]. it represents a severe hyperinflammatory condition with the cardinal symptoms: prolonged fever, cytopenias, hepatosplenmegaly, and hemophagocytosis by activated, morphologically benign macrophages. biochemical markers include elevation of ferritin and triglycerides, and low fibrinogen [4] (table 1). this syndrome, first described in 1939 by scott and robb-smith, is classically divided into primary (genetic) hemophagocytic syndrome and secondary (reactive) hemophagocytic syndrome [5] (table 2). according to the last international classification of primary immune deficiency, hlh ought to be considered first among the “diseases of immune dysregulation” [4]. in fact, impaired function of natural killer (nk) cells and cytotoxic t-cells (ctl) is characteristic for both genetic and acquired forms of hlh [5]. however, both primary ___________________________________________________________ * corresponding author: dr. rahmoune hakim (trahmounehakim@gmail.com). http://www.ajecr.org/ mailto:rahmounehakim@gmail.com 195 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org table 1 diagnostic criteria for hemophagocytic lymphohistiocytosis (ref. 4) table 2 hemophagocytic syndrome classification (ref. 5) and secondary syndromes can be precipitated by an infection, and the latter can lead to severe hlh [6], even in the newborn. initially, and more specifically in the very young infants (i.e. < 3 months), hlh may masquerade as any infection. awareness of the clinical symptoms and of the diagnostic criteria of hlh is important to start life-saving therapy with immunosuppressive/immunomodulatory agents in time [3, 5, 6]. systemic infections and/or sepsis share many features with hlh, such as fever, cytopenias, and hepatic involvement, disseminated intravascular coagulation and cytokine abnormalities. however, ferritin levels tend to be static in patients with infections, but are prone to dramatic increases in case of hlh [1]. fever is, however, less common in newborns and therefore is not essential for the diagnosis of hlh [7]. the clinical dilemma for the physician then is to distinguish between this systemic hyperinflammatory condition and other hyperferritinemia causes like sepsis or perinatal hemochromatosis, so that sharp and timely therapy could be initiated [8]. a review of ferritin levels in pediatric patients found a cut off of 10 000μg/l to be 90% sensitive and 96% specific for the diagnosis of hlh [9]. a rapid and reliable pathway would also evaluate serum triglycerides and blood cells: mahlaoui et al. [10] reported high triglyceride levels in 89% and decreased blood counts in 95%of the hlh population. among infectious triggering agents in this period of age, mostly are viral (herpes viruses) and bacterial. in japan, up to 36% of neonatal hlh were found in association with herpes simplex virus infection [2]. a polymerase chain reaction, pcr-search for infectious agents (including herpes simplex virus (hsv), epstein barr virus (ebv), cytomegalovirus (cmv) has been recommended by some authors [11]. moderate/mild cases of infectious hlh tend to be self-limiting, requiring chiefly a prompt supportive (i.e. antibiotic/antiviral) management. immunomodulation (i.e. steroids, immunosuppressive agents or polyvalent immunoglobulins) are the cornerstone of lifesaving therapies for severe cases / late recognition. better responses have been reported with high dose steroids (2 to 4 mg/kg/day of prednisolone) [12]. lack of specific therapy or even late management may be fatal. whatever the treatment is based on, it has to be continued until all clinical (fever and visceromegaly) biochemical and hematological abnormalities subside. outcome depends largely on the infectious trigger and the delay of diagnosis [1, 5, 6]. in conclusion, hlh is a rare, life-threatening condition characterized by clinical (fever, visceromegaly, multiorganic deficiency) and biological features (cytopenias, low fibrinogen, high plasma triglycerids, and hyperferritinaemia), associated to pictures of hemophagocytosis. infection, most frequently by viruses, is an important driver of acquired hlh; genetic causes of hlh being the most renowned source of neonatal hlh [1, 13]. the potential infectious agents must, therefore, be actively searched and promptly treated. organ failures must be supported and immunomodulation (i.e. corticosteroids) therapy is also crucial [13]. conflict of interest the authors declare no conflicts of interest. references 1. mcclain k. clinical features and diagnosis of hemophagocytic lymphohistiocytosis. up to date: topic. 87499, 2014. 2. suzuki n, morimoto a, ohga s, kudo k, ishida y, ishii e, hlh/lch committee of the japanese society of pediatric hematology. characteristics of hemophagocytic 1. familial disease or known genetic defect 2. clinical and laboratory criteria (5/8 criteria) 1. fever 2. splenomegaly 3. cytopenia , at least 2 cell lines a. hemoglobin < 9.0 g/dl (below 4 weeks < 120 g/l) b. platelets < 100 · 000/ml c. neutrophils < 1000/ml 4. hypertriglyceridemia and/or hypofibrinogenemia a. fasting triglycerides ≥ 3 mmol/l b. fibrinogen < 1.5 g/l 5. ferritinemia > 500 mg/l 6. scd25 ≥ 2400 u/ml 7. decreased or absent nk-cell activity 8. hemophagocytosis in bone marrow, csf or lymph nodes supportive evidence : cerebral symptoms with moderate pleocytosis and/or elevated protein elevated transaminases, bilirubin, ldh. 1. primary or genetic hemophagocytic syndrome familial hemophagocytic lymphohistiocytosis immune deficiency syndromes chediak-higashi syndrome griscelli syndrome x-linked lymphoproliferative syndrome wiskott-aldrich syndrome severe combined immunodeficiency lysinuric protein intolerance hermansky-pudlak syndrome 2. secondary or reactive hemophagocytic syndrome infection associated hemophagocytic syndrome viral herpes viruses (herpes simplex virus, varicella zoster virus, cyto megalovirus, epstein-barr virus, human herpesvirus 6 and 8) hiv other viruses (adenovirus, hepatitis viruses, parvovirus, influenza) other infections bacteria including mycobacteria and spirochetes parasites fungi malignancy-associated hemophagocytic lymphohistiocytosis (mainly lymphoma) macrophage activation syndrome (in autoimmune diseases) http://www.ajecr.org/ 196 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org lymphohistiocytosis in neonates: a nationwide survey in japan. j pediatr 155:235-238, 2009. 3. bousfiha a et al. the 2015 iuis phenotypic classification for primary immunodeficiencies. j clin immunol 35:727-738, 2015. 4. tanoshima r, takahashi h, hokosaki t, yamaguchi k, goto s, kai s. hemophagocytic lymphohistiocytosis in very young infants. pediat blood cancer 52:137-139, 2009. 5. janka ge. hemophagocytic syndromes. blood rev 21:245-253, 2007. 6. rouphael ng et al. infections associated with haemophagocytic syndrome. lancet infect dis 7:814-22, 2007. 7. freeman hr, ramanan av. review of haemophagocytic lymphohistiocytosis. arch dis child 96:688-693, 2011. 8. kapoor s. distinguishing hemophagocytic lymphohistiocytosis from hemochromatosis in patients with hyperferritinemia. pediatr blood cancer. 50:1287 1288, 2008. 9. allen ce, yu x, kozinetz ca, mcclain kl. highly elevated ferritin levels and the diagnosis of hemophagocytic lymphohistiocytosis. pediatr blood cancer 50:1227-1235, 2008. 10. mahlaoui n, ouachée-chardin m, desaint basile g et al. immunotherapy of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins: a single-center retrospective report of 38 patients. pediatrics 120:e622-628, 2007. 11. ansuini v, rigante d, esposito s. debate around infection-dependent hemophagocytic syndrome in paediatrics. bmc infect dis 13:15, 2013. 12. pramanik s, pal p, das pk, chakrabarty s, bhattacharya a, banerjee s. reactive haemophagocytic lymphohistiocytosis. indian j pediatr. 76: 643-645, 2009. 13. gonzalez f, vincent f, cohen y. syndrome d’activation macrophagique d’origine infectieuse: étiologies et prise en charge. (article in french). réanimation 18:284-290, 2009. http://www.ajecr.org/ 165 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(3):165-169 original article polysomnographic findings in patients with sleep apnea syndrome in different body positions during sleep shahla afsharpaiman1, ensiyeh vahedi2, mohammad-hasan alaghemand2, amin saburi2, 3* 1health research center, baqiyatallah university of medical sciences, tehran, iran 2chemical injuries research center, baqiyatallah university of medical sciences, tehran, iran 3birjand atherosclerosis and coronary artery research center, birjand university of medical sciences, birjand, iran abstract. there are some evidences that have approved the role of body position on various diseases and their causes. respiratory diseases particularly disorders associated with respiratory rate and rhythm can be affected by the body position. dizziness could be categorized as one of the most common medical complaints of patients referred to the neurology clinics. in this study, we aim to study polysomnographic findings in patients with sleep apnea syndrome in different positions during sleep. a cross-sectional study was conducted on 155 patients with sleep breathing disorders and epworth sleepiness scale above 10 referred to the sleep clinic of baqiyatallah hospital during 2009–2011. after confirming the diagnosis of obstructive sleep apnea, polysomnography containing sleep breathing apnea-hypopnea indices (ahis), different body positions, respiratory movements, oximetry pulse cases, emg, eog, and eeg were done by alice device and the method was split night test. the mean age and bmi of patients were 50.62 ± 11.65 years old and 32.48 ± 7.19 respectively. there was a significant difference between ahi in supine position (25.78 ±21.01) versus lateral position; right (16.28 ± 22.40; p<0.001) or left (18.05 ± 21.04; p=0.007) but there was no significant difference between ahi in right versus left position (p = 0.782). according to the results of this study, it seems that apnea-hypopnea index in supine position could be worse than the left or right side sleeping position. however, this index value in each left and right lateral positions was not more than the other. in addition, desaturation value in supine position was significantly different from two others, but there were no preference in left or right positions. keywords: polysomnography, sleep apnea syndrome, body positions, sleep introduction sleep apnea syndrome is a type of clinical sleep disorder that is developing from recurrent apneas during sleep. sleep apnea is categorized into three different types, namely, obstructive, central, and compound with total prevalence of 24% [1–4]. polysomnography is recognized as a standard method of diagnosing sleep apnea. polysomnographic definition of respiratory disorders during sleep is based on the abnormal respiratory events in every hour of the sleep that is called apnea-hypopnea index (ahi). usually up to five apnea-hypopnea events per hour is considered as being normal, but in children even one apnea event per hour is abnormal [3–5]. different treatments including continuous positive airway pressure (cpap), surgical procedures, using oral device, and changing the lifestyle are recommended for these patients. some of the changing lifestyle that is approved for treatment of these patients include avoiding alcohol and muscle relaxants, losing excess body weight, stopping smoking habit, and elevating upper body 30 degree during the sleep [6]. unlike supine position, lateral position is recommended for these patients [7, 8]. there is evidence that shows the effect of different body positions on sleep apnea solutions (sas). it is shown in many patients that the obstructive sleep apnea (osa) will exacerbate in supine position. according to the past studies, ahi is doubled in supine position than the lateral one [9, 10], and the intensity of apnea (apnea duration, minimal non saturate oxygen, arousal frequency, and duration) also increasing in the supine position [49]. pressure to re-establish the flow of air is more in supine position [6]. mechanism of exacerbation of sleep respiratory disorders in the supine position is not clear. some studies considered the effectiveness of gravity on the shape and size of upper respiratory tract, but no consistent evidence reported the relationship between other body positions during sleep and sas [12–14]. in addition, there are recommendations for sleeping positions in islamic sources that can be considered as advises to improve sleep of these patients. although there are studies aimed at investigating the probable relationship between sleeping positions and polysomnographic indices, supplementary ___________________________________________________________ * corresponding author: amin saburi, md (aminsaburi@yahoo.com). http://www.ajecr.org/ mailto:aminsaburi@yahoo.com 166 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org table 1 apnea-hypopnea (ahi) and desaturation in terms of sleep positions* * ci: confidence interval, sd: standard deviation. work need to be carried out on this (15). in this study, we wanted to study polysomnographic findings in patients with sleep apnea syndrome in different positions during sleep. materials and methods a cross-sectional study was done on the patients with sleep breathing disorders referred to sleep clinic of baqiyatallah hospital and has been under the polysomnography during 2009–2011. after clinical examinations of patients complaining of sleep disorders and approval of indication of psg through epworth sleepiness scale (ess) tests (usually with a score above 10) by physician, they were nominated for doing polysomnography. after visiting the sleep lab, the polysomnography started from 6 p.m. to 6 a.m. and their different sleep parameters including sleep breathing ahi, different body positions, respiratory movements, oximetry pulse cases, electromyogram (emg), electrooculogram (eog), and electroencephalogram (eeg) during sleep were recorded. tests were done by alice device and the method was split night test. the final number of patients was 155. the informed consents were obtained from all patients and the study was approved by the institutional review board of baqiyatallah university of medical sciences, tehran, iran. statistical analysis data collected were entered and analyzed using the spss software version 16 (spss inc., chicago, il). tables and charts were extracted. descriptive statistics were calculated for quantitative data and the qualitative variables were expressed as counts and percentage. the relationship between qualitative variables was examined through chi squared statistical test, whereas the quantitative variables used t-test and analysis of variance (anova). also, some nonparametric tests such as wilcoxon test, mann–whitney u test, and friedman test were used. data are presented as mean ± sd. results the mean age of the patients was 50.62 ± 11.65 years, body mass index (bmi) was 32.48 ± 7.19, neck circumference was 41.41 ± 3.64 cm, the waist circumference was 111.51 ± 17.07 cm, total sleep time (tst) was 465.6 ±72.93 h, sleep efficacy 1 was 86.64 ± 10.65, sleep efficacy 2 was 89.43 ± 9.86, sleep efficacy 3 was 29.64 ± 12.02, average sleep duration at supine was 297.47 ± 139.20 min, sleep duration at left side was 127.98 ± 86.56 min, sleep duration at right side was 120.38 ± 89.44 min, ahi in supine was 25.78 ±21.01, ahi in left was 18.05 ± 21.04, ahi in right was 16.28 ± 22.40, desaturation in supine was 127 ± 146.45, desaturation in left position was 42.5 ± 66.09, desaturation in right position was 34.70 ± 62.1. mean desaturation and the sd were 92.58 and 3.15, respectively. the minimal amount of desaturation was 76.79 with sd of 15.40. among all of the patients, 109 (70.3%) were men and 46 (29.7%) were women. the mean age among women and men were 56.63 ± 12.79 and 48.0 ± 10.18 years, respectively (table 1). there was a significant difference between the age (p < 0.001, r = −4.4.08, confidence interval (cl): 95%: 12.36– 4.71), bmi (p < 0.001, r = −3.756), neck circumference (p = 0.003, r = 2.98), waist circumference (p = 0.076, r = −1.78), and tst (p = 0.811, r = 0.239). the results in the examination of ahi in terms of sleep positions (supine, right, and left) are reported in the following section. there was a significant difference in amount of ahi in different positions with p < 0.001. ahi in supine position was significantly different from left lateral one (p = 0.007). also, the amount of ahi in supine was clearly different from right lateral position (p = 0.001) as well as difference of desaturation in left, right, and supine positions (table 2). but there were no significant differences in ahi between right and left lateral positions (p = 0.782). there was a significant difference between desaturation in right, left, and supine positions but not between right and left lateral positions alone (p < 0.001). in this case, there was a statistically significant association between desaturation lo wer b o und up p er b o und sup ine 154 2 5.78 4 4 2 1.0 18 6 2 2 2 .4 3 8 3 2 9 .13 0 5 left 13 8 18 .0 50 7 2 1.4 0 0 9 1 14 .4 4 8 3 2 1.6 53 1 r ig ht 13 0 16 .2 8 8 5 2 2 .4 0 715 12 .4 0 0 2 2 0 .176 7 to t al 4 2 2 2 0 .3 3 0 1 2 1.9 3 3 2 2 18 .2 3 14 2 2 .4 2 8 8 sup ine 154 1.2 8 e+0 2 14 6 .4 50 3 10 4 .2 76 4 150 .9 0 54 left 13 8 4 2 .6 0 14 6 6 .0 9 6 54 3 1.4 754 53 .72 75 r ig ht 13 0 3 4 .70 77 6 2 .18 18 3 2 3 .9 174 4 5.4 9 8 to t al 4 2 2 71.18 4 8 110 .6 50 2 6 0 .59 73 8 1.772 4 variab le 9 5% c i to t al ahi des at urat io n po s it io n no m ean sd http://www.ajecr.org/ 167 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org table 2 probability (p) values of apnea index and desaturation in terms of sleep positions and ahi values in supine positions (p < 0.001, r = 0.602). there was no significant association between ahi values in supine position and sleep efficacy (p = 0.291). also, there was no significant association between desaturation values in supine position and sleep efficacy 3 (p < 0.422). there was a significant association between ahi values and desaturation in left lateral position (p < 0.001, r = 0.781), but not between them and sleep efficacy 3 (p = 0.646). however, there is no significant association between desaturation in left position and sleep efficacy (p = 0.407). also, there was a significant association between ahi and desaturation values in right lateral position (p < 0.001, r = 0.784), and these values were not associated with sleep efficacy 3 (p = 0.729). also, there was no significant association between desaturation in right lateral position and sleep efficacy (p = 0.681). there was no significant association between bmi and sleep efficacy 3 values (p = 0.868). although there was no significant association between bmi and right ahi (p < 0.001, r = 0.373) and also desaturation in right lateral, (p < 0.001, r = 0.510) there was a significant association between bmi values and ahi in left lateral position (p < 0.001, r = 0.367), ahis (p = 0.002, r = 0.245), desaturation in left lateral position (p < 0.001, r = 0.507), and desaturation in supine position (r = 0.403 and p < 0.001). also, there was a significant association between age and the amount of desaturation in right position (p = 0.008, r = 0.233), but not between age and amounts of ahis (p = 0.231), ahil (p = 0.067), ahir (p = 0.186), desaturation in supine (p = 0.260), and left (p = 0.187) positions. the linear regression showed that the only predictors of ahi values were neck circumference (p = 0.004, r = 2.923) and desaturation in supine position (p < 0.001, r = 8.216). about this index in the left position, age (p = 0.041, r = 2.066) and desaturation (p < 0.001, r = 8.145) in left and about the index in the right position, bmi (r = 2.562, p = 0.012), neck circumference (p = 0.006, r = 2.779), waist circumference (p = 0.005, r = 2.843), and desaturation (p < 0.00, r = 4.513) in right were predictors. discussion according to the results of this study, it seems that ahi in supine situation could be worse than the left or right side sleeping position. however, this index values in each left and right lateral positions was not more than the other. in addition, desaturation value in supine position was significantly different from two others, but there were no preference in left or right positions. bmi was significantly associated with ahi value in the right situation and the right desaturation. neck and waist circumference and desaturation in left and right were associated with ahi, and also, there was an association between age and right desaturation. in multivariate analysis, it seems that ahi in supine position is associated with neck circumference and desaturation in this position and also as the left ahi with age and left desaturation and right ahi with bmi, neck and waist circumference, and desaturation. in a paper published in 2012 by bahammam [16] reviewing stories, verses, and islamic traditions, he tried to make medical recommendations about situation and direction of sleep. in this paper, the right side has been particularly emphasized in early sleep, but there is no emphasis on the possible effects of it. in addition, it has been found that sleeping in supine, right, and left lateral decubitus positions could be effective on the health of heart that is nearly compatible with low ahi value that could have effects such as arrhythmia on heart health. however, past studies showed that sleeping in right lateral decubitus can effect on vague nerve, and so, it inhibits the arrhythmia and improves cardiac function in patients with chf that is not comparable with current study, because this study does not examine the effects on autonomic nervous system. but generally, it can be reasonable according to the lowest amount of right desaturation among different sleeping positions; however, the effects of desaturation on making arrhythmia and probably disorders of nervous system of the heart [16]. in a study on 131 patients with osa by ozeke in 2011 [15], right-sided sleeping position (rssp) compared to left-sided sleeping position (lssp) and supine position provided greater reduction in frequency of apnea-hypopnea events in patients with moderate or severe osa, and this is compatible with our study. in that study, the average ahi in supine, lssp, and rssp were 60.4 ± 36.2, 30.2 ± 32.6, and 23.6 ± 30.1 h, respectively. but there was no difference between lssp and rssp in patients with mild apnea (p = 0.130), and again these results and values in different positions are consistent with our study [15]. in another study on 16 patients with mild to moderate osa by lee in 2009 in korea [17], the influence of lateral position with cervical support by head tilting and scapula support in reduction of ahi have been reported that has not been discussed in this study, but this support can be examined in different sleeping positions in future studies. a study by fan maanen in 2011 [18] on 30 patients examined the function of an intelligent neck brace on a variable p osit ion direct ion p value left 0.007 right 0.001 supine 0.007 right 0.782 supine 0.001 left 0.782 left 0.00 right 0.00 supine 0.00 right 0.803 ssupine 0.00 left 0.803 supine left right desat urat ion supine left rright t ot al ahi http://www.ajecr.org/ 168 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org patient with osa during the sleep. this device was shaking in supine position, so the patient would change his/her status. in that study, the patient’s ahi have been reduced from 27.7 ± 2.4 to 12.8 ± 2.2 h without any reduction in sleep duration and the ahi became less than 5 in 7 patients [18]. therefore, he concluded that the supine position increases the apnea that can be justified by relaxation of muscles in the upper respiratory tracts and it is compatible with our results. different mechanisms have been suggested for ahi reduction in right lateral decubitus in patients with sleep apnea including the influence on reduction of unwanted flow by patient foramen oval (pfo) that is very common in these patients. however, sleeping in the right side can decrease the liver weight on the input of blood to the heart, right to left shunt, and pulmonary blood pressure by increasing the valsalva maneuver and vagus system stimulation, so the desaturation will be decreased. in addition, sleeping in right lateral decubitus can be associated with reduction in pressure of central airways that would accompany with smaller amounts of osa [19]. also, the right lung volume is greater than the left that can provide more oxygenation. sleeping in right lateral decubitus increase blood flow to right lung more than the left one and therefore the amounts of oxygenation increase. the contents related to cardiac findings in patients with osa and the effect of position on it have been mentioned just for discussion and explanation of possible causes. supplementary studies focusing on results of cardiac tests are required to definitely prove the subject and the relationship between above items. in conclusion, according to the results of our study, it is better for patients with sleep apnea to sleep in the right and left lateral decubitus, although there is no difference between right and left positions. in addition, due to the association of higher rates of apnea, hypopnea and desaturation with overweight and the resulted high appleshaped waist circumference and high neck circumference, so it seems logical to advise these patients to lose weight. it is suggested to examine sleep disorders by comparing ahi, desaturation, sleep effectiveness, and so on between sleep disorders and other physical disorders. acknowledgement we would like to thank patients who participated in this study. we also thank personnel of research department of baquiyatallah hospital, tehran and sleep department in this center. conflict of interest the authors declare no conflicts of interest and no financial support. references 1. young t, peppard pe, gottlieb dj. epidemiology of obstructive sleep apnea: a population health perspective. am j respir crit care med 165:1217-1239, 2002. 2. young t, palta m, dempsey j, skatrud j, weber s, badr s. the occurrence of sleep-disordered breathing among middle-aged adults. n engl j med 328:1230-1235, 1993. 3. bearpark h, elliott l, grunstein r, cullen s, schneider h, althaus w, sullivan c. snoring and sleep apnea: a population study in australian men. am j respir crit care med 151:1459-1465, 1995. 4. punjabi nm. the epidemiology of adult obstructtive sleep apnea. proc am thorac soc 5:136-143, 2008. 5. rombaux p, bertrand b, boudewyns a, deron p. standard ent clinical evaluation of the sleep-disordered breathing patient: a consensus report. acta otorhinolaryngol bleg 56:127-137, 2002. 6. neill am, angus sm, sajkov d, mcevoy rd. effects of sleep posture on upper airway stability in patients with obstructive sleep apnea. am j respir crit care med 155:199-204, 1997. 7. loord h, hultcrantz e. positionera method for preventing sleep apnea. acta otolaryngol 127: 861-868, 2007. 8. szollosi i, roebuck t, thompson b, naughton mt. lateral sleeping position reduces severity of central sleep apnea/cheyne-stokes respiration". sleep 29:10451051, 2006. 9. cartwright rd. effect of sleep position on sleep apnea severity. sleep 7:110-114, 1984. 10. oksenberg a. positional vs nonpositional obstructive sleep apnea patients: anthropomorphic, nocturnal polysomnographic, and multiple sleep latency test data. chest 112:629-639, 1997. 11. oksenberg a. association of body position with severity of apneic events in patients with severe nonpositional obstructive sleep apnea. chest 118:1018-1024, 2000. 12. isono s. lateral position decreases collapsibility of the passive pharynx in patients with obstructive sleep apnea. anesthesiol 97:780-785, 2002. 13. fouke jm. effect of position and lung volume on upper airway geometry. j appl physiol 63:375-380, 1987. 14. kairaitis k. tracheal traction effects on upper airway patency in rabbits: the role of tissue pressure. sleep 30:179-186, 2007. 15. ozeke o, erturk o, gungor m, hızel sb, aydın d, celenk mk, dıncer h, ilıcın g, ozgen f, ozer c. influence of the rightversus left-sided sleeping position on the apnea-hypopnea index in patients with sleep apnea. sleep breath 16:617-620, 2011. 16. bahammam as. sleep from an islamic perspective. ann thorac med 6:187-192, 2011. 17. lee jb, park yh, hong jh, lee sh, jung kh, kim jh, yi h, shin c. determining optimal sleep position in patients with positional sleep-disordered breathing using response surface analysis. j sleep res 18:26-35, 2009. 18. van maanen jp, richard w, van kesteren er, ravesloot mj, laman dm, hilgevoord aa, de vries n. evaluation of a new simple treatment for positional sleep apnea patients. j sleep res 21:322-329, 2011. 19. fujita k, wada y, aono t et al. sleep apnea syndrome in patients with cardiac disease. rinsho byori 56:767-771, 2008. http://www.ajecr.org/ 210 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(2):210-213 original article usefulness of chest radiographs in the management of acute asthma in adult asthmatics in a developing country piyusha milani atapattu1*, chemindra biyanwila2, manamalabaduge upul jerard fernando3 1department of physiology, faculty of medicine, university of colombo, colombo 8, sri lanka 2outpatient department, provincial general hospital, badulla, sri lanka 3lady ridgeway hospital for children, dr. denister de silva mawatha, colombo 08, sri lanka abstract. chest radiographs are widely available, even in most hospitals in developing countries, and can detect a variety of lung path ologies. when adults are admitted to hospital with acute asthma, chest radiographs are often obtained. studies on the usefulness of chest radiographs in acute asthma have shown varying results. this study aimed at evaluating the usefulness of chest radiographs in the management of acute asthma in adult asthmatics in a developing country. plain chest radiographs performed within 24 hours of admission in 115 consecutive adult asthmatic patients (asthma defined by asthma education and prevention programme nih-1997) admitted to national hospital of sri lanka were reviewed prospectively for one year. the relationship between radiographic findings and clinical findings and leucocyte count were evaluated. significance was calculated using fisher exact test. among 115 participants there were 56 males and 59 females, with mean age of 47.77 years. chest radiographs were abnormal in 40.9%, the abnormalities including hyperinflation (8.7%), consolidation (15.7%), segmental/greater atelectasis (5.2%), pneumomediastinum (0.9%), features of obstructive lung disease (6.1%), solitary lung no dule (0.9%), and pulmonary edema (3.5%). there was no significant association of abnormal radiographs with leucocytosis (p=0.322), elevated body temperature (p=3.109), and high pulse rate (p=0.157). the incidence of abnormalities on admission chest radiographs in patients with acute asthma was high, though immediate management was influenced in only less than half of the patients with abnormal radiographs. chest radiographic abnormalities had no significant association with elevated blood leukocyte count, body temperature, and pulse ra te. using clinical criteria to identify those requiring chest radiographs within the first 24 hours in adults admitted with acute asthma may improve the cost effectiveness of chest radiographs without compromising patient care. keywords: acute asthma, chest radiograph, developing country introduction patients with acute severe asthma require hospital admission for immediate management, as inadequate treatment may lead to respiratory failure, coma and death [1] with up to 8% mortality in admissions to intensive care [2]. exacerbations of asthma may be triggered by underlying pulmonary pathology [3], with one study reporting 37% with respiratory tract infection [4]. rate of relapse varies from 7-15% and depends on the aggressiveness of the treatment given [2]. chest radiographs are widely available, even in most hospitals in developing countries, and can detect a variety of lung pathologies, thus when an adult is admitted to hospital for acute asthma, chest radiographs are often obtained. chest radiographs may reveal expected findings in asthma, including hyperinflation and prominent perihilar markings, which do not require specific management [5], or more sinister pathologies such as consolidation, pneumothorax, pulmonary edema, pulmonary tuberculosis and lung collapse which necessitate additional specific management in addition to the standard treatment for asthma [5, 9]. studies on the usefulness of chest radiographs in acute asthma have shown varying results, with some authors recommending admission chest radiographs be obtained for all patients hospitalized with acute asthma [6, 10], whereas other studies suggesting that routine admission chest radiographs are unnecessary [11]. several studies of chest radiographs in adults have reported that the results of chest radiographs influenced treatment in only 1 to 5 percent of patients [11, 12]. obtaining unnecessary chest radiographs is costly and exposes the patients to radiation. in addition, facilities such as additional labor and transportation of patient or x-ray machine are required to obtain a radiograph. it is therefore useful to know how important chest radiography is in the management of acute asthma. we reviewed the usefulness ___________________________________________________________ * corresponding author: dr. piyusha milani atapattu (piyushaatapattu@yahoo.com). http://www.ajecr.org/ 211 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org of admission chest radiographs on the management of 115 adult patients with acute asthma, in a tertiary care hospital in sri lanka. materials and methods the study was conducted in two medical wards admitting males and females at the national hospital of sri lanka over a period of one year. one hundred and fifteen consecutive adult patients over the age of 12 years, admitted to the wards with acute asthma (asthma defined by asthma education and prevention programme nih-2007)[13] were recruited for the study. all patients were managed as for acute asthma, using the standard protocol. plain chest radiographs (posteroanterior) were performed within 24 hours of admission in all patients. if abnormalities were detected in the chest radiographs, patients were treated accordingly. all chest radiographs were reported by the same consultant radiologist. all patients had oral temperature and pulse rate measured, and white blood count evaluated as a standard procedure on admission. approval for study was obtained by the ethics review committee of the national hospital sri lanka. informed consent was obtained from the patients or immediate family member, to participate in the study. women who were pregnant and patients not wishing to undergo a chest radiograph were excluded from study. the relationship between radiographic findings, clinical parameters and leucocyte count were evaluated using spss version 20 (spss chicago, il). data were analyzed using mean, standard deviation, and percentages for descriptive statistics and fisher’s exact test for comparison of groups. p < 0.05 was considered statistically significant. results one hundred and fifteen consecutive patients (56 males, 59 females), with acute asthma were recruited. the mean age was 47.77 (sd ±  1.78) years (range 13-86 years). most patients [n=78 (67.8%)] had chest radiographs that were compatible with uncomplicated asthma, which included normal chest radiographs in 68 (59.1%) and chest radiographs with hyperinflation in 10 (8.7%). thirty-seven radiographs (32.2%) were interpreted as positive; in which the radiographic findings were not necessarily seen routinely in uncomplicated asthma, and included consolidation, atelectasis, pneumomediastinum, features of obstructive lung disease, solitary lung nodule and pulmonary edema (table 1). there was no significant relationship between abnormal radiographs with consolidation and leucocytosis (p=0.322), elevated temperature (p=3.109), or high pulse rate (p=0.157) (table 2). all patients with radiographic evidence of consolidation were commenced on antibiotics addition to bronchodilators and steroids. the patients with pulmonary edema were treated accordingly. the pneumomediastinum resolved with conservative management. the patient with the solitary nodule was directed for further investigation in the surgical unit. there were no mortalities in the study group. table 1 chest radiograph findings in 115 patients discussion: abnormal chest radiographs were detected 40.9% cases, which higher than that reported in other studies: 14% (dalton, 1991), 23% (ismail et al, 1994), 25% (aronson et al, 1989) and 34% (white et al, 1991) [5, 6 ,8, 11]. however, only 32.3% were interpreted as positive; in which the radiographic findings were not necessarily seen routinely in uncomplicated asthma. the higher rate of abnormal chest radiographs in this study could be attributed to patient behavior in urban sri lanka, as many patients with asthma opt to seek treatment from widely available private practitioners, and present to government hospitals only when the disease is severe or unresponsive to treatment. furthermore only patients who are unresponsive to treatment offered at the emergency treatment units in government hospitals are admitted to the wards, which implies that severe disease or complicated asthma was more likely to be included in this study population. this is supported in a study by aaronson et al, 1989, in which chest radiograph abnormalities were reported only among patients with complicated asthma, with no abnormal chest radiographs detected in patients with uncomplicated asthma [11]. the much higher rates of abnormal chest radiographs reported in older studies, eg. 72% by rebuck (1970) may be attributed to inadequate outpatient care and poor social circumstances at those times, which could have contributed to the higher mortality rates caused by asthma prior to late 1980s [14, 15] . the abnormalities detected in the chest radiographs in this study are comparable other published data [6, 14, 16] : number of patients with pneumonia (15.7%) was similar to published data reporting 16-20% of patients [14]. the incidence of pneumomediastinum and solitary lung nodule chest radiograph findings no. percent normal 68 59.1 hyperinflation 10 8.7 consolidation 18 15.7 segmental/greater atelectasis 6 5.2 pneumomediastinum 1 0.9 features of obstructive lung disease 7 6.1 solitary lung nodule 1 0.9 pulmonary edema 4 3.5 total 115 100 http://www.ajecr.org/ 212 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org table 2 comparison between chest radiograph findings with white blood cell count, oral temperature and pulse rate in adults with acute asthma is comparable with other series [14, 16]. in these patients, chest radiographs were essential in the diagnosis. one reason for the variation in radiographic findings could be the selection criteria in different studies. white et al, 1991 included patients who failed to respond to a 12 hour course of bronchodilator therapy in the emergency ward [6], and aronson et al, 1989 reported his findings in patients with complicated asthma [11]. another could be the classification of normal and abnormal chest radiographs, where hyperinflation and increased perihilar markings may or may not be considered as abnormal. it was interesting to note that there was no relationship between the presence of consolidation in the chest radiograph with the leucocyte count, oral temperature or pulse rate. lack of correlation of radiographic focal opacities with white cell count or body temperature has been reported by white et al, 1991 [6]. it has been reported that only a minority of patients admitted with acute asthma with recent respiratory tract infection had fever or leucocytosis [4]. it is also possible that treated respiratory tract infection or pneumonia may have resulted in the presence of consolidation the radiographs with normal white cell count and temperature, as the resolution of a consolidation on chest radiograph lags behind clinical resolution [17]. this could have resulted in over-treatment of such patients with antibiotics, when they had only radiographic evidence of consolidation. multiple abnormalities may be present in the lungs of patients with asthma, mostly related to bronchial destruction, i.e. reversible abnormalities such as mucoid impactions, acinar pattern, and lobar collapse and irreversible abnormalities including bronchiectasis, bronchial wallthickening, sequellar line shadows, and emphysema [18]. such expected abnormalities were found in 37% of chest radiographs and 71.9% of ct scans in patients with chronic asthma [18]. thus it is likely that routine chest radiographs may pick up radiographic findings that may not require any treatment. chest radiographs within the first 24 hours of admission are useful for acute asthma management. however, in the current study which identified a high rate of abnormalities by routine admission chest radiography in patients with acute asthma, only a minority appears to have lead to the diagnosis of unsuspected but clinically important disease. gentile et al, 2003 have shown that devising protocols for identifying patients requiring chest radiography in patients admitted with asthma to emergency department, significantly reduced the request for chest radiographs without compromising patient care [19]. in conclusion, it may be appropriate to perform chest radiographs in selected groups of patients admitted to hospital with acute asthma, so that a higher yield of underlying respiratory pathologies requiring treatment is obtained, while reducing the cost of patient management which is burdening the developing countries. acknowledgement we thank the staff of wards 45 and 48a of national hospital of sri lanka. conflict of interest the authors declare that they have no competing interests and no financial support. references 1. papiris sa, manali ed, kolilekas l, triantafillidou c, tsangaris i. drugs 69:2363, 2009. 2. mcfadden er jr. acute severe asthma. am j respir crit care med 168:740-59, 2003. 3. rodrigo gj, rodrigo c, hall jb. acute asthma in adults: a review. chest 125:1081-102, 2004. 3. teichtahl h, buckmaster n, pertnikovs e. the incidence of respiratory tract infection in adults requiring hospitalization for asthma. chest 112:591–596, 1997. 5. dalton am. a review of radiological abnormalities in 135 patients presenting with acute asthma. arch emerg med 8:36-40, 1991. 6. white cs1, cole rp, lubetsky hw, austin jh. acute asthma. admission chest radiography in hospitalized adult patients. chest. 1991 jul;100(1):14-6. 7. reed s, diggle s, cushley mj, sleet ra, tattersfield ae. assessment and management of asthma in an accident an accident and emergency department. thorax 40:897902, 1985. ≤12 000 >12 000 normal * elevated <100 >100 without consolidation 71 26 92 5 89 8 with consolidation 12 6 15 3 16 2 total 83 32 107 8 105 10 x-ray findings white blood cell count x 10 9 /l significance fisher's exact test temperature (f 0 ) pulse rate/minute p=0.322 p=3.109 p=0.157 http://www.ajecr.org/ http://www.ncbi.nlm.nih.gov/pubmed/?term=rodrigo%20c%5bauthor%5d&cauthor=true&cauthor_uid=15006973 http://www.ncbi.nlm.nih.gov/pubmed/?term=hall%20jb%5bauthor%5d&cauthor=true&cauthor_uid=15006973 http://www.ncbi.nlm.nih.gov/pubmed/15006973?dopt=abstract 213 am j exp clin res, vol. 4, no. 2, 2017 http://www.ajecr.org 8. ismail y , loo cs , zahary mk. the value of routine chest radiographs in acute asthma admissions. singapore medical j 35:171-172, 1994. 9. pickup cm, nee pa, randall pe. radiographic features in 1016 adults admitted to hospital with acute asthma. j accid emerg med 11:234-237, 1994. 10. petheram is, kerr ih, collins jv. value of chest radiographs in sever acute asthma. clin radiol 32:281-82, 1981. 11. aronson s, gennis p, kelly d, landis r, gallagher j. the value of routine admission chest radiographs in adult asthmatics. ann emerg med 18:1206-08, 1989. 12. blair dn, coppage l, shaw c, medical imaging in asthma. j thorac imag 1:23-25, 1986. 13. us department of health and human services. national asthma education and prevention program. expert panel report 3: guidelines for the diagnosis and management of asthma. summary report 2007. nih publication no.: 08-5846 [online]. url: http://www.nhlbi.nih.gov/guidelines/asthma/asthsu mm.pdf 14. rebuck as. radiology aspects of severe asthma. aust j radiology 14:264-8, 1970. 15. wijesinghe m, weatherall m, perrin k, crane j, beasley r. international trends in asthma mortality rates in the 5to 34-year age group: a call for closer surveillance. chest 135:1045-9, 2009. 16. dattwyler rj, goldman ma, bloch kj. pneumomediastinum as a complication of asthma in teenage and young adult patients. j allergy clin immunology 63:412-6, 1979. 17. bruns ahw, oosterheert jj, prokop m, lammers jwl, hak e, hoepelman aim. patterns of resolution of chest radiograph abnormalities in adults hospitalized with severe community-acquired pneumonia. clin infect dis 45: 983-991, 2007. 18. paganin f, trussard v, seneterre e, chanez p, giron j, godard p, sénac jp, michel fb, bousquet j. chest radiography and high resolution computed tomography of the lungs in asthma. am rev respir dis 146:1084-1087, 1992. 19. gentile nt, ufberg j, barnum m, mchugh m, karras d. guidelines reduce x-ray and blood gas utilization in acute asthma. am j emerg med 21:451–453, 2003. http://www.ajecr.org/ http://www.nhlbi.nih.gov/guidelines/asthma/asthsumm.pdf http://www.nhlbi.nih.gov/guidelines/asthma/asthsumm.pdf http://www.sciencedirect.com/science/article/pii/s0735675703001657 http://www.sciencedirect.com/science/article/pii/s0735675703001657 http://www.sciencedirect.com/science/article/pii/s0735675703001657 http://www.sciencedirect.com/science/article/pii/s0735675703001657 http://www.sciencedirect.com/science/journal/07356757/21/6 25 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2014;1(2):25-30 original article age-related changes in membrane fluidity and fluorescence intensity by tachykinin neuropeptide nkb and aβ (25-35) with 17β estradiol in female rat brain rashmi jha 1,2 abbas ali mahdi 2 shivani pandey 2 najma z baquer 1 and sudha m cowsik 1 * 1 2 school of life sciences, jawaharlal nehru university, new delhi, india department of biochemistry, king george’s medical university, lucknow, uttar pradesh, india abstract. changes in the fluidity of membrane lipids are known to occur during aging and by lipid peroxidation. it is well documented that the fluidity state of the lipid phase in a membrane is important for the activity of intrinsic membrane proteins. oxidants and fluidity of membrane lipids play a significant role in aging and age related neurodegenerative diseases. the aim of the present study was to determine the effect of tachykinin neuropeptide, neurokinin b (nkb) and amyloid beta fragment aβ (25-35) on 17β estradiol (e2) treated aging female rat synaptosomes of different age groups. aging brain functions were measured by membrane fluidity and fluorescent intensity with neuropeptides. an in-vitro incubation of aβ (25-35) in e2 treated brain synaptosomes showed toxic effects on all the parameters. these effects of aging and aβ (25–35) on membrane fluidity were restored by nkb and combined nkb and aβ (25–35) with e2. furthermore, we measured the tryptophan (trp) fluorescence to monitor changes in proteins and to make inferences regarding structure and dynamics. trp is a sensitive marker of protein oxidation and its fluorescence significantly increased in e2 treated synaptosomes of aging rats. furthermore, to evaluate the effect of oxidative stress on the membrane and protein conformation, fluorescent probe 1-anilino8-naphthalenesulfonate (ans) were used. an increase in ans fluorescence in e2 treated synaptosomes of aging rats indicated that e2 is associated with significant conformational changes and surface hydrophobicity of membranes and proteins. keywords: aging, neurokinin b, amyloid beta (23-35), estradiol introduction aging is defined as a universal, progressive and deleterious process occurring in cells and tissues, affecting most of the living organisms. during aging, most organs and systems undergo a gradual loss of physiological function usually associated to the imbalance of redox status and alterations in cellular signaling pathways [1]. the free radical or “oxidative stress” theory holds that oxidative reactions are the factors underlying these changes [1]. highly reactive oxygen species (ros) cause a wide spectrum of cell damage, including lipid peroxidation, inactivation of enzymes, alteration of intracellular oxidation–reduction state, and dna damage in the aging brain [2]. the ovarian steroid hormone estradiol (e2) is one of the most important hormones and it can protect neurons against aβ toxicity, oxidative stress and excitotoxicity [35]. most of the studies showed that e2 is neuroprotective in neurodegenerative disorders such as stroke, alzheimer disease (ad) and parkinson disease (pd) [6]. the effect of e2 is primarily mediated by erα and erβ which are members of the nuclear receptor superfamily of ligandactivated transcription factors [7]. e2 modulates multiple functions of the brain, via activation of erα and erβ including development, cognition and memory [8] highlighting its protective effects against neuronal damage [9]. mammalian tachykinins comprise a family of regulatory peptides including substance p (sp), neurokinin a (nka) and neurokinin b (nkb) [10, 11]. they are known to reduce oxidative stress in the brain [12-14]; to reverse the neurotoxic effects of aβ in neurons and play a role in neurodegenerative diseases [15, 16]. nkb has biological importance such as regulatory role in pre-eclampsia [17], neuroprotective agent [16, 18 and 19] and as a potential antioxidant molecule [12, 13]. several studies have proposed that aβ binds to the cell surface via direct membrane interactions, thereby initiating both neurotoxicity and plaque formation. recently, it was demonstrated that aβ deposition is initiated in a plasma membrane-bound form, resulting in diffuse plaque formation [20]. many small peptides are able to exist in dynamic equilibria between unfolded and folded structures, depend ___________________________________________________________ * corresponding author: sudha mahajan cowsik (scowsik@yahoo.com). mailto:scowsik@yahoo.com 26 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org ing on the solvent polarity and their interaction with the membrane phase [21, 22]. this is known for a variety of neurotransmitter, peptides and hormones for which the importance of the ordered structures has been recognized in relation to binding to g proteincoupled membrane receptors [23]. furthermore, when small peptides permeate the membrane to fulfill physiological requirement, a certain folding may be required for a favorable interaction with the lipid moiety [24]. decreases in membrane fluidity could hamper the functioning of cell surface receptors and ion channel protein; such decreases have been associated with cellular toxicity [25]. the fluidity parameters of synaptosomal membranes are linked to neuronal signal transduction pathways, channels and enzymes [26]. changes in the fluidity of membrane lipids are known to occur during aging and by lipid peroxidation. it was evidenced that the fluidity state of the lipid phase in a membrane is important for the activity of intrinsic membrane proteins. fluorescence is the result of the three-stage process that occurs in a particular molecule called fluorophores. a fluorescent probe is a fluorophore designed to localize within a region of a biological specimen or to respond to a stimulus. there are three amino acids with intrinsic fluorescence properties, phenylamine (phe), tryptophan (trp), tyrosine (tyr), but only tyr and trp are used experimentally because their quantum yield (emitted photons/excited photons) is high enough to give a good fluorescence signal. in a hydrophobic environment, trp has high quantum yield and fluorescence intensity. in contrast, in a hydrophilic environment its quantum yield decreases leading to low fluorescence intensity. for trp residue, there is a strong stoke shift dependent on the solvent, meaning that the maximum emission wavelength of trp depending on the trp environment. 1-anilino-8naphthalenesulfonate (ans), an amphipathic dye, with hydrophobic naphthalene and phenyl groups and a charged sulfonate group, is frequently used for the investigation of equilibrium, and kinetic protein folding intermediates [27, 28]. e2 treatment had beneficial effects on antioxidant enzymes in aging rat tissues as reported earlier by jha et al 2013 and baquer et al 2009 [2, 29]. in the present study, we examine the neuroprotective effect of nkb and e2 against aβ (25-35) toxicity on the membrane fluidity and fluorescence intensity of the brain synaptosomes of aging female rats. materials and methods animals the present study was conducted on female albino rats of the wistar strain in different age groups (3, 12 and 24 months). animals were maintained in the animal house facility of jawaharlal nehru university (jnu), new delhi, india at a constant temperature of 25˚ c, humidity 55% and 12h dark and light cycle. the animals were fed standard chow rat feed (hindustan leaver ltd., india) and given tap water until the time of sacrifice. the institutional animal ethics committee (iaec) of jnu approved all the animal experiments; all institutional guidelines for care of animals were followed. hormone administration subcutaneous injections of e2 (0.1 μg/g body weight) were given daily for one month, to the aged rats (12 and 24 months old; n=8 for each group). e2 was dissolved in propylene glycol in appropriate concentrations [30]. control animals received an equal volume of vehicle. there was no treatment on the day of the sacrifice. animals of all the groups were sacrificed and brains were isolated for further study. preparation of synaptosomes the animals from control and e2 treated groups were sacrificed by cervical dislocation. the whole brain was excised and washed in ice-cold saline (0.9 % nacl). tissue homogenates were prepared as described by mayanil et al 1982 [31]. tissues were soaked, dried on blotting paper and weighed, minced and homogenized in nine volumes of homogenizing buffer containing 0.25 m sucrose, 0.02 m triethanolamine (ph 7.4) and 0.12mm dithiothreitol. the pellet obtained after centrifugation at 12,000 (rpm) containing synaptosomes and mitochondria were taken for the present study. the whole procedure was carried out at 4˚c. treatment of synaptosomes with nkb and aβ (25-35) each sample containing ~100 µg protein of isolated rat brain synaptosome was incubated with nkb, aβ (25–35) and nkb+ aβ (25–35) in microfuge tubes at 37 ˚ c for 60 min in a shaking water bath with 0.1, 1 and 5 µm concentration of each of the peptides. all incubations were performed in four combinations; control (without any peptide), aβ (25–35), nkb and nkb+aβ (25–35) in three age groups of control and e2 treated rats at three peptide concentrations. measurement of fluorescence anisotropy the synaptosomes prepared from the rat brains of different age groups were diluted in 50mm tris-hcl, to a protein concentration of ~100µg. 1ml of synaptosomal membrane was mixed with 1, 6-diphenyl-1, 3, 5-hexatriene (dph), a fluorescent probe and the mixture was incubated at 37°c for 30 min and fluorescence intensity was recorded using an excitation wavelength of 365nm and emission wavelength of 428nm [32]. polarization (p) measurements were carried out on polarization spectrofluorometer as described by mantha et al. 2006 [13]. fluorescence measurement the fluorescence intensity was measured in the synaptosomes prepared from rat brains of different age groups. fluorescence measurements were performed in a solution containing 100µg proteins per ml, 10mmol.1 hepes, 100 mmol/kcl (ph 7.0) at room temperature using shimadzu rf 540 spectrofluorimeter. the fluorescence emission spectra (from 300 to 450 nm, 5nm slit width) of trp were measured by excitation at 280nm 27 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org (2nm slit width) [33, 34]. ans fluorescence was measured following 15min incubation of the ans probe with synaptosomes. the excitation and emission wavelength for ans measurement were 365 and 480nm, respectively (5nm slit width) [33]. protein estimation protein was estimated in the synaptosomes by the method of bradford, 1976 [35] using bovine serum albumin (bsa) as standard. statistical analysis data have been presented as mean ± standard error of mean (sem). the data were analyzed using one way anova to test for differences between different treatments at different age groups. differences between the means of the individual groups were assessed by dunnett's multiple comparisons test. a value of p <0.05 was considered to be statistically significant. chemicals all substrates, standards, nkb and aβ (25–35) peptide fragment were purchased from sigma chemicals company, usa. all other chemicals were of analytical grade and purchased from srl and qualigens, india. results membrane fluidity or fluorescence anisotropy fluorescence anisotropy is inversely proportional to the membrane fluidity. the changes in the anisotropy (r) monitored by using dph probe for membrane fluidity were measured in rat brain synaptosomes, in different age groups with and without e2 treatment, at different concentration aβ (25–35), nkb and nkb+ aβ (25–35). the results are shown in figure 1 (a), (b), and (c). effect of e2 and varying concentration of aβ (25-35) on membrane fluidity the membrane fluidity was found to be decreased in the synaptosomes of control rats (e2 untreated), when incubated with different concentrations of aβ (25-35). however, this decrease in the activity was less in e2 treated rat brain synaptosomes. in the synaptosomes of e2 treated 12 and 24 month rats, the membrane fluidity decreased with incubation of aβ (25-35) at 5µm concentration (p<0.01 and p<0.001). results are shown in figure 1 (a). effect of e2 and varying concentration of nkb on membrane fluidity the membrane fluidity was observed to increase in all control age groups with nkb incubation, whereas this increase in membrane fluidity was more significant in e2 treated rats, as compared to age matched control group. the membrane fluidity in the synaptosomes of e2 treated 12 month rats showed an increase when treated with the concentration of 5µm of nkb (p<0.01). there was a significant increase in membrane fluidity in the synaptosomes of e2 treated 24 months rats with the incubation of 5µm concentration of nkb (p<0.001). results are shown in figure 1(b). effect of e2 and varying concentration of combined nkb and aβ (25-35) on membrane fluidity the membrane fluidity in control synaptosomes (e2 untreated) was observed to increase when treated with a combination of nkb and aβ (25-35), but this increase was more significant in synaptosomes of e2 treated 12 and 24 months aging rats. there was a significant increase of membrane fluidity in the synaptosomes of 12 months e2 treated rats, with the combined dose of 5µm concentration of nkb and aβ (25-35) (p<0.001). the combined dose of nkb and aβ (25 35) at 5μm concentration in 24-month e2 treated rats showed a significantly raised level in membrane fluidity activity as compared to matched control (p < 0.001). results are shown in figure 1 (c). a b c figure 1: percentage changes in anisotropy in the synaptosomes of 3, 12 and 24 months control (cont) and estradiol (e2) treated aging female rats in presence of (a) aβ (25-35) (b) nkb and (c) nkb+aβ (25-35). peptide concentrations are 0.1, 1.0 and 5.0 µm. statistical significance: a p<0.001, b p<0.01, c p<0.05 comparing age matched control (untreated) versus peptide treated; d p<0.001, e p<0.01, f p<0.05 comparing e2 treated versus peptide treated. 0 20 40 60 80 100 120 140 3 cont 12 cont 12 e2 24 cont 24 e2 % a n is o tr o p y c h a n g e ages of rats cont 0.1μm aβ 1μm aβ 5μm aβ a a a c c b e e b df 0 20 40 60 80 100 120 3 cont 12 cont 12 e2 24 cont 24 e2 % a n is o tr o p y c h a n g e ages of rats cont 0.1µm nkb 1µm nkb 5µm nkb b a c b b a e e b a f f d e 0 20 40 60 80 100 120 3 cont 12 cont 12 e2 24 cont 24 e2 % a n is o tr o p y c h a n g e ages of rats cont 0.1aβ+nkb 1µm aβ+nkb 5µm aβ+nkb c b a b b b e c b f e e d c d 28 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org fluorescence measurement the trp fluorescence was used as a sensitive marker of protein oxidation. trp fluorescence increased significantly in e2 treated synaptosomes of aging rats. a fluorescent probe ans was used to evaluate the effect of oxidative stress on the membrane and protein conformation. a change in ans fluorescence in e2 treated synaptosomes of aging rats indicated that e2 treatment is associated with significant conformational cha nges and surface hydrophobicity of membranes and proteins. results are shown in figure 2 (a) and (b). fluorescence intensity of trp in control and e2 treated rat synaptosomes the trp fluorescence of control and e2 treated rats is observed to be significantly changed. the level of tryptophan fluorescence intensity increased 76% (p<0.001), 87% (p<0.001), 81% (p<0.01) and 90% (p<0.01) in 12 month control, 12 month e2 treated, 24 months control and e2 treated rats respectively when compared with 3 month young rats. results are shown in figure 2 (a). fluorescence intensity of ans in control and e2 treated rat synaptosomes the ans fluorescence in synaptosomes of aging rats was observed to be significantly changed when compared with synaptosomes from young rats. the fluorescent intensity of ans probe increased 65% (p<0.01), 85% (p<0.01), 72% (p<0.05) and 88% (p<0.01) in 12 month control, 12 months e2 treated, 24 months control and e2 treated rats respectively when compared with 3 month young rats. results are shown in figure 2 (b). a b figure 2: percentage changes in the fluorescence intensity in synaptosomes of 3, 12 and 24 months control (cont) and estradiol (e2) treated aging female rats in presence of (a) tryptophan intensity (b) ans intensity. stastical significane: a p<0.001, b p<0.01, c p<0.05 comparising age matched control verses e2 treatment, $ p<0.001, # p<0.01, * p<0.05 verses 3 months. discussion changes in the fluidity of membrane lipids are known to occur during aging and lipid peroxidation. it has been observed that the fluidity state of the lipid phase in a membrane is important for the activity of intrinsic membrane proteins. the fluorescence (polarization) anisotropy of membrane-bound dph, which is inversely correlated with membrane fluidity, was measured in isolated rat brain synaptosomes at different ages. results showed a significant decrease in membrane fluidity as a function of age as seen earlier by muller et al, 2001 [36]. the membrane fluidity was measured at different concentrations of nkb, a (25-35) and combined peptide nkb and a (25-35) in different age groups of control (without e2 treated) and e2 treated rats. in the present study, when we examined the effects of a (25-35) on rat brain synaptosomes of various age group of control (without e2 treated) rats, there was a decrease in membrane fluidity. this result seems to be significant since it has been shown that brain aging enhances amyloid neurotoxicity in a concentration dependent manner for different age groups studied [37]. aβ (25-35) showed less reduction in fluidity in the synaptosomes of e2 treated rats as compared to age matched control rats. this result suggested that neurotoxic effect of aβ is probably related to the amplifying effect of aβ on membrane fluidity or the induction of oxidative stress [13]. the changes in fluidity measured in the presence of nkb in control and e2 treated rat brains of different age groups. incubation of nkb in the synaptosomes of e2 treated showed a significant increase in fluidity as compared to age match control rats. the combined treatment of nkb and a (25-35) in the synaptosomes of control and e2 treated rats brain showed an increase in fluidity and this increase are more significant in e2 treated rats as compared to age matched control. these results show that interaction nkb with e2 may represent an important contributing factor in aging and its membrane-mediated functions in the brain tissue. electrostatic interactions (including hydration) are vital to the structure and function of proteins [38-40]. fluorescence from the amino acid trp has long been known to be sensitive to the polarity of its local environment [41-43]. the intensity, quantum yield, and wavelength of maximum fluorescence emission of trp are very solvent dependent. the fluorescence spectrum shifts to shorter wavelength and the intensity of the fluorescence increases as the polarity of the solvent surrounding the trp residue decreases. trp fluorescence wavelength is widely used to monitor changes in proteins and inferences regarding local structure and dynamics. in this study, we investigated the potential effects of aging on oxidative modifications of proteins in the synaptosomes of control and e2 treated rats of different age group. we measured trp fluorescence as a sensitive marker of protein oxidation. the progressive increase in trp content was observed in the synaptosomes of different age group of rats. an increased fluorescence intensity of trp was observed in e2 treated brain synaptosomes of aging rats. the results of trp fluorescence measurements indicated that aging is associated with accumulation of 0 10 20 30 40 50 60 70 80 90 100 3 cont 12 cont 12 e2 24 cont 24 e2 t ry p to p h a n in te n si ty ages of rats $ a # b 0 10 20 30 40 50 60 70 80 90 100 3 cont 12 cont 12 e2 24 cont 24 e2 a n s in te n s it y ages of rats # b * b 29 am j exp clin res, vol. 1, no. 2, 2014 http://www.ajecr.org fluorescent products within the brain and support the view that protein modification mediated trp. ans is an amphipathic dye, with hydrophobic naphthalene and phenyl groups, and a charged sulfonate group. it is frequently used for the investigation of equilibrium, and kinetic protein folding intermediates [27, 28]. when ans is bound to a protein in a nonpolar environment, there is a large increase in the fluorescence quantum yield [44]. ans has been used extensively as a probe for protein folding intermediates, especially molten globules, because their partially structured nature provides access for ans to bind exposed hydrophobic regions whereas ans has a very weak affinity for fully unfolded or folded proteins. ans has also been widely used as a probe for kinetic intermediates in protein folding [27]. to evaluate the effect of oxidative stress on the membrane and protein conformation we used ans, an anionic probe, for membrane surfaces and protein cavities. an increase in ans fluorescence in the synaptosomes of aging rats indicates that aging is associated with significant conformational changes, results in the increased surface hydrophobicity of membranes and proteins. the result showed significant changes in ans fluorescence in the synaptosomes isolated from e2 treated rats. these results support the view that aging is associated with changes in brain tissue. in summary, this study demonstrates agedependent increases in protein oxidation in brain synaptosomes of e2 treated rats. this observation suggests protein oxidation may contribute to deterioration of protein, cellular and organ function. acknowledgements financial grant from university grant commission, new delhi, india in the form of project is gratefully acknowledged. conflict of interest the authors declare that they have no competing interests. references 1. harman d. role of free radicals in aging and disease. ann n y acad sci 673:126-41, 1992. 2. baquer nz, taha a, kumar p, mclean p, cowsik sm, kale rk, singh r, sharma d. a metabolic and functional overview of brain aging linked to neurological disorders. biogerontology 10: 377-413, 2009. 3. brann dw, krishnan d, chandramohan 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brand l. quantative estimation of protein binding site polarity. fluorescence of narylaminonaphthalenesulfonates. biochemistry 7: 3381-90, 1968. 214 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(3):214-218 original article emergency contraception: are reproductive-aged women well informed? ana catarina massa*, tania meneses, ana isabel machado, maria jose alves maternidade dr alfredo da costa, centro hospitalar de lisboa central, lisbon, portugal abstract. despite available effective contraception methods, unintended pregnancy remains a significant health issue. emergency contraception (ec) is used to prevent pregnancy after an unprotected or inadequately protected sexual intercourse. the aim of this study was to evaluate knowledge and personal experience with ec among reproductive -aged women. a prospective survey among a sample of portuguese women at reproductive age was performed. the survey was anonymous, voluntary and included questions regarding sociodemographic characteristics, obstetrical history, known emergency contraception methods, timing to use ec, personal experience and efficacy of available methods. after completing the survey, participants were offered written information about ec. a total of 92 women participated on the survey but 3 were excluded due to incorrect filling. mean age was 30.52 years and most were single, multiparous and with no previous abortions. awareness rate of ec was 95.5% but only 56.4% knew that it prevented pregnancy. just 16.5% stated that both hormonal oral method and iud were the available methods and 67% believed that it was effective within 1 day after unprotected sexual intercourse. ec was previously used by 29.4% and to 65.9% participants the hormonal oral method was considered the most effective. over 90% said that ec does not protect against sexually transmitted infections. despite the high rate of awareness of ec our population had some limitations concerning the purpose and different forms of ec as well as the proper time limit to use it. educating reproductive-aged women and health care providers about ec is important. keywords: emergency contraception, survey, pregnancy, awareness introduction despite available effective contraceptive methods the high number of unintended pregnancies worldwide is an important cause of women requesting an abortion or having an unwanted child. reasons for unplanned pregnancy include failure to use contraception or its incorrect use (e.g. condom breakage or slippage, missed or late doses of hormonal contraceptives), unplanned consensual intercourse and rape [1-4]. emergency contraception (ec) refers to any intervention used to prevent pregnancy after an unprotected or inadequately protect episode of sexual intercourse [1-8]. although ec does not protect against sexually transmitted infections (sti) it offers reassurance for millions of women who rely on condoms for contraception in case of condom breakage or slippage [6, 7]. ec was first investigated in the 1960s and received approval by the u.s. food and drug administration (fda) in 1998 [1]. the first described method of ec was published in 1974 by yuzpe and colleagues which contained combined estrogen-progestin oral contraceptive pill [9]. since then different regimens were developed and are nowadays available: progestin-only contraceptives, anti-progestins (either mifepristone or ulipristal acetate) and the copper-releasing intrauterine device (iud). combined estrogen-progestin regimen this method consists of two doses of 100 μg ethinyl estradiol and 0.5 mg levonorgestrel (lng) taken 12 hours apart. the first dose of the yuzpe regimen should be taken within 72 hours of unprotected sexual intercourse (upsi). further data demonstrated efficacy of norgestrel and norethindrone as optional progestins in this regimen [2, 4, 5, 7, 9]. several clinical studies have shown that combined estrogen-progestin pills can inhibit or delay ovulation. it is associated with a 75% reduction of the risk of pregnancy although this efficacy decreases with time [5-7]. few side effects have been associated: nausea (50%) and vomiting (20%) are the most common ones [2-5, 7]. progestin-only regimen levonorgestrel has been the only progestin studied for ___________________________________________________________ * corresponding author: ana catarina massa, md (anacmassa@gmail.com). http://www.ajecr.org/ mailto:anacmassa@gmail.com 215 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org table 1 sociodemographic and obstetrical characteristics charact erist ics no. (%) 95% ci mean age (yrs) 30.52 ±7.95 28.8-32.2 marit al st at us single 43 (48.3) 38.8-58.9 married 29 (32.6) 22-40.9 not married but in a relat ionship 10 (11.2) 5.6-17.3 divorced 6 (6.7) 1.8-12.8 p arit y nuliparous 22 (24.7) 14.6-35.3 p rimiparous 25 (28.1) 19.7-39.3 mult iparous 42 (47.2) 34.8-57.3 p revious abort ion(s) no 64 (71.9) 64-82.5 yes 25 (28.1) 17.5-36 ci, confidence interval. usage in ec. two treatment schedules have been approved: two 0.75 mg pills taken 12 hours apart and a single-dose of 1.5 mg lng. the single dose regimen or the first dose should be taken as soon as possible up to 72 hours after upsi. efficacy is similar among both treatment schedules without significantly increasing adverse effects [2-7]. the mechanism of action is similar to the one described previously for the combined estrogen-progestin regimen. the closer to ovulation treatment is given the less likely the probability of success [8]. this method is associated with higher pregnancy reduction (about 85%) and with fewer adverse effects when compared to the yuzpe regimen. like the latter most data supports that its efficacy decreases with time. some reports demonstrated a 50% and 70% lower incidence of nausea and vomiting, respectively [4, 5, 7]. anti-progestin regimen two anti-progestins have been effective for ec: mifepristone and ulipristal acetate (upa). in portugal mifepristone is used for medical abortion and, although highly effective, it is not licensed for ec. upa, a selective progesterone receptor modulator, was approved by the fda in 2010 for ec. it is used as a 30 mg single-dose and is indicated up to 120 hours (5 days) after upsi [1, 3, 5, 8, 10]. to date it is the only oral form of ec approved for the 72-120 hour period after upsi. upa’s primary mechanism of action is thought to be inhibition or delay of ovulation. the reported pregnancy reduction rate is greater than 85% and maintains its efficacy throughout 120 hours making it the most effective method of oral ec [5]. the most common adverse effects are headaches, nausea and abdominal pain [8, 10]. since upa is in pregnancy category x [3], prior to its administration women should be informed of the risks if pregnancy occurs. table 2 knowledge of emergency contraception quest ion no. (%) 95% ci have you ever heard of ec? no 4 (4.5) 1.1-8.4 yes 85 (95.5) 91.6-98.9 which met hods of ec do you know? hormonal oral cont racept ive pill 70 (82.3) 71.3-88.2 iud 1 (1.2) 0-3.5 bot h 14 (16.5) 9.4-24.5 does ec prevent pregnancy? no 35 (41.2) 29.6-52.3 yes 48 (56.4) 45.9-69.3 unanswered 2 (2.4) 0-5.9 when should ec be used? aft er any sexual int ercourse 7 (8.2) 1.8-15.8 aft er every unprot ect ed sexual int ercourse 74 (87.1) 76.5-95.3 unanswered 4 (4.7) 1.2-9.9 unt il when can ec be used? 10 days aft er sexual int ercourse 1 (1.2) 0-4.7 3-5 days aft er sexual int ercourse 17 (20.0) 11.8-29.4 1 day aft er sexual int ercourse 57 (67.0) 54.4-76.5 1 day before sexual int ercourse 1 (1.2) 0-3.5 don’t know 9 (10.6) 3.7-17.6 have you ever used ec? no 60 (70.6) 61.2-80 yes 25 (29.4) 20-38.8 which of ec met hods is more efficient ? hormonal oral cont racept ive pill 56 (65.9) 56.6-75.3 iud 17 (20.0) 11.3-28.2 unanswered 12 (14.1) 7.1-20.5 does ec prot ect against st i? no 79 (92.9) 86-97.6 yes 4 (4.7) 0-9.9 unanswered 2 (2.4) 0-5.9 ci, confidence interval; iud, intrauterine device; ec, emergency contraception; sti, sexually transmitted infections. copper-releasing intrauterine device (iud) the copper-releasing iud can be safely used for ec up to 5 days after upsi or within 5 days from the earliest estimated date of ovulation, reducing the risk of pregnancy by more than 99% [8, 11-14]. the copper-releasing iud can be inserted up to the time of implantation to prevent pregnancy. however, most providers limit insertion to http://www.ajecr.org/ 216 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org within 5 days of intercourse rather than ovulation because it is frequently difficult to estimate the day of ovulation. besides higher efficacy it also works as long-term contraception (up to 10 years). unlike oral forms of ec, the copper-releasing iud requires a physical examination and a physician comfortable in its insertion. for women at risk of sti administration of prophylactic antibiotics at least to cover chlamydia trachomatis should be considered [14]. copper is toxic to the ovum and sperm and therefore this method is effective immediately after insertion and works primarily by inhibiting fertilization. if fertilization has occurred there is an anti-implantation effect [8]. there has not been established a single mechanism of action for ec since it depends on the day of the menstrual cycle on which upsi occurs and ec is administered. 2 pregnancy is the only contraindication for its use, however some methods may be preferred in specific cases. although highly effective, available data demonstrates that women are still not using ec as often as needed. lack of knowledge among women or their physicians, difficulty in acquiring ec, lack of perception of the risk of pregnancy, or feeling of shame and fear if others knew about it may contribute to the underutilization of this method. emergency contraception should be discussed with women previous of their need and it should also be reaffirmed that a regular method of contraception is always more effective as long as properly used. the aim of our study was to evaluate knowledge and personal experience with emergency contraception among a sample of portuguese women at childbearing age through a survey. materials and methods we conducted a prospective survey among women seeking care in centro hospitalar de lisboa central (maternidade dr. alfredo da costa), a tertiary maternity center in portugal, during four months. subjects were included if they were in reproductive age (between 18 and 49 years of age) and were recruited from family planning, gynecology or unwanted first trimester pregnancy appointments and postpartum ward. participants were informed that the survey was anonymous and voluntary and that its purpose was for medical research. verbal consent was obtained and women were not compensated for their participation. after completing the survey they were offered an informative handout addressing different topics of the survey. the survey included 13 questions regarding sociodemographic characteristics, obstetrical history, knowledge of ec methods and timing to use it, personal experience and efficacy of available methods. women were asked about their age, marital status, parity and previous abortions. next, they were questioned if they had ever heard about ec. if the answer was negative, the survey was complete; otherwise it would continue. the following items addressed methods of ec known (“hormonal oral contraceptive pill” and/or “iud”), efficacy of ec in preventing pregnancy (“yes” or “no”), appropriate circumstance (“after any sexual intercourse” or “after every unprotected sexual intercourse”) and proper time limit to use it (“10 days after the sexual intercourse”, “3-5 days after the sexual intercourse” or “1 day after the sexual intercourse”, “1 day before the sexual intercourse”, “don’t know”). previous experience with this method of contraception was evaluated and participants were also asked about the person who counseled them in taking ec (“friend”, “doctor”, “nurse”, “other”, “no one”). the two last items regarded the most efficient method of ec (“hormonal oral contraceptive pill” or “iud”) and if it was effective in preventing sti (“yes” or “no”). sociodemographic characteristics of the participants were described by standard descriptive statistics (using means and standard deviations (sd) for continuous variables) and proportions and 95% confidence intervals (ci) for categorical variables. the remaining items of the survey were determined by using proportions and 95% ci. results a total of 92 women answered the survey but three were excluded due to incorrect filling. consequently, 89 participants were included in the study. the mean age of participants was 30 years and most were single (48.3%), multiparous (47.2%) and with no history of previous abortions (71.9%) (table 1). with respect to specific knowledge of ec (table 2), 95.5% of women answered “yes” to the question “have you ever heard of ec?” therefore, the remaining of the survey was only completed by 85 women. the majority of women (82.3%) knew about hormonal oral contraceptive pill as an ec method but only 14 (16.5%) stated that both the hormonal oral method and the copper-releasing iud were in fact the available methods. when asked if ec was efficient in preventing pregnancy just over half of them (56.4%) said “yes.” as for the appropriate circumstance to use it 87.1% answered “after every unprotected sexual intercourse”. when addressed the proper time limit, through a multiple-choice question, only one fifth of participants knew that ec could be effective within 3-5 days of the unprotected sexual intercourse and about two thirds considered the 1-day after unprotected sexual intercourse option as the correct one. regarding previous personal experience with this method of contraception the majority of participants had never used it before. for those who had used it (25 women), a friend or their doctor were the ones who had counseled them the most (in 7 and 8 women, respectively). as for the most efficient method of ec, the hormonal oral method was the one considered by most participants (65.9%) with only 20% considering the copper-releasing iud as the choice. the last question of the survey addressed knowledge of women on the efficacy of ec in protecting against sti and over 90% said “no.” discussion forty years have passed since the first regimen of ec was established. since then, new and more efficient methods have been developed presenting nowadays rates of 75-99% efficacy on pregnancy prevention. http://www.ajecr.org/ 217 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org since 2007 abortion, by women’s choice, is legal in portugal until the 10 th week of pregnancy. according to the yearly report on pregnancy termination in portugal, during the year of 2013 there were 17964 pregnancy interruptions and of these 17414 were by women’s choice [15]. the authors agree that aside from these numbers of pregnancy interruptions, what really matters is the number of women that would benefit from ec if they were aware of it and if they would consider it to be safe. awareness of ec has always been a key point in preventing its use. however in our study a significant number of women (85 in 89, representing 95%) were aware of ec. previous studies have also evaluated this issue. mollen et al. [16] in 2013 reported a 63.7% rate of awareness of ec among female adolescents, abbott et al. [17] in 2004 reported a 77% rate of awareness in adult women and chuang et al. [18] in 2005 reported an 85% rate of awareness of ec among adult women. despite a higher awareness rate, in our study, participants had significant limitations concerning knowledge of available methods of ec and their proper use. as for the correct timing to use ec about two-thirds of participants considered that it would only be effective 24 hours after upsi. most women still do not consider the copper-releasing iud as a form of ec which is not only the most efficient method but also the only one that has the potential to offer long-term contraception. another misconception in our population concerned the purpose of ec. just over half of participants knew that ec worked by preventing pregnancy but 92.9% knew that it does not protect against sti. indeed, if women do not recognize the aim of ec they will not consider it after upsi. the non-recognition of the risk of pregnancy (either because women are unaware of their ovulatory period or because they do not believe that a single missed pill would be enough), the cost or the difficulty in obtaining ec or the myths associated with ec (induces abortion, contains massive levels of hormones and is only used by irresponsible women) could also contribute to its underutilization. regarding previous personal experience with this method of contraception most participants claimed to have never used it before. forty percent (10 cases) of the 25 women who had previously used ec were counseled either by their doctor or nurse. although health care providers counseling is a positive feature the authors agree that this rate is far too low since every woman attending a gynecology or family planning appointment should be aware of this last option of contraception. previous studies have indicated that women’s health care providers do not routinely discuss ec with them [5]. reasons like lack of knowledge in their use and efficacy have been appointed. surely this study has its limitations: the reduced sample could explain the higher rate of awareness of ec when compared to other studies merely by a casual association and the study population included only women who sought medical care and therefore more prone to have previously discussed it with a health care provider. despite the high rate of awareness of ec our study population had some limitations concerning the purpose and different forms of ec as well as the correct timing to use it. however, the authors believe that it is comforting to know that women are conscious that it does not offer protection against sti. the authors hope that the informative handout that was given to every participant will make a difference by providing accurate information on this matter. ec remains forgotten by many health care providers either by lack of knowledge about this method of contraception or by fearing that women will give up their regular method of contraception. ec should be considered a topic to debate in every medical or nursing appointment and promoting publicity campaigns or other forms of divulgation is crucial. ec is safe and effective to every woman and should not replace a regular method of contraception. this study provides further information on knowledge and personal experience with ec stating that despite a high awareness rate most women believe that it is only effective up to one day after unprotected sexual intercourse. counseling on ec should emphasize the correct time limit to use it. conflict of interest the authors declare that there is no conflict of interests regarding the publication of this article and no sources of funding. references 1. devine k. the underutilization of emergency contraception. am j nurs 112:44-50, 2012. 2. american college of obstetricians and gynecologists. acog practice bulletin no. 112: emergency contraception. obstet gynecol 112:1100-1108, 2010. 3. upadhya k, breuner c, trent m. emergency contraception: committee on adolescence. pediatrics 130:1174-1182, 2012. 4. dovey s, sanfilippo j. emergency contraception: current options, challenges, and future directions. open access j contraception 2:107-117, 2011. 5. lazorwitz a, guiahi m. emergency contraception: focus on options, efficacy, and access. postgrad obstet gynecol 34:1-8, 2014. 6. trussell j, raymond e, cleland k. emergency contraception: a last chance to prevent unintended pregnancy. 2014. accessed 1 july 2014. 7. trussell j; ellertson c, stewart f, raymond e, shochet t. the role of emergency contraception. am j obstet gynecol 190:s30-38, 2004. 8. royal college of obstetricians & gynecologists. faculty of sexual and reproductive healthcare clinical effectiveness unit. fsrh guidance (august 2011) emergency contraception. ceu guidance. accessed 7 july 2014. 9. yuzpe a, thurlow h, ramzy i, leyshon j. postcoital contraception: a pilot study. j reprod med 13:53-58, 1974. 10. london new drugs group apc/dtc briefing. ulipristal acetate (ellaone®). 2011. accessed 1 july 2014. http://www.ajecr.org/ 218 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org 11. allen r, goldberg a. emergency contraception: a clinical review. clin obstet gynecol 4:927-936, 2007. 12. sociedade portuguesa de ginecologia, sociedade portuguesa de contracepçāo. consenso sobre contracepçāo. 2011. 13. harper c, speidel j, drey e, trussel j, blum m, darney p. copper intrauterine device for emergency contraception: clinical practice among contraceptive providers. obstet gynecol 119:220-226, 2012. 14. royal college of obstetricians & gynecologists, faculty of sexual and reproductive healthcare clinical guidance. emergency contraception: clinical effectiveness unit (updated january 2012). accessed 1 july 2014. 15. direçāo geral de saúde. relatório dos registos das interrupções da gravidez ao abrigo da lei 16/2007 de 17 de abril. divisāo de saúde sexual, reprodutiva, infantil e juvenil. 2014. 16. mollen c, miller m, hayes k, barg f. knowledge, attitudes, and beliefs about emergency contraception: a survey of female adolescents seeking care in the emergency department. ped emerg care 29:469-474, 2013. 17. abbott j, feldhaus k, houry d, lowenstein s. emergency contraception: what do our patients know? ann emerg med 43:376-381, 2004. 18. chuang c, freund k. emergency contraception knowledge among women in a boston community. contraception 71:157-160, 2005. http://www.ajecr.org/ 107 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2015;2(3):107-112 review article l-tryptophan depletion bioreactor: a possible cancer therapy rolf bambauer* formerly institute for blood purification, homburg, saar, germany abstract. the cancer therapeutic strategies known to date are not adequate for all cancer patients. most of them are followed by a high rate of side effects and complications. the l-tryptophan depletion bioreactor is described as a possible new method of cancer therapy. ltryptophan is an essential amino acid which has been recognized as an important cancer nutrient and its removal can lead to destruction of the tumour. normal human cells or tumor cells cannot synthesize l-tryptophan and therefore tumor resistance is unlikely to develop. ltryptophan is also a constituent for different bio-molecules such as serotonin, melatonin, and is needed for other synthesis processes in the cell growth. l-tryptophan degrading enzymes with 3 iso-enzymes called tryptophan side chain oxydase (tso) i, ii, iii were isolated. the 3 iso-enzymes can be differentiated by tryptic digestion. they have different molecular weights with different effectivenesses. all the tso enzymes have heme that can catalyze essentially similar reactions involving l-tryptophan as a substrate. the most effective tso is the type tso iii. a column which contained tso as a bioreactor was integrated in a plasmapheresis unit and tested it in different animals. in sheep and rabbits l-tryptophan depletion in plasma was shown at 95% and 100% rates respectively by a single pass through the bioreactor. the results in immune supprimized rats with tumors were impressive, too. in 20 different tumor cell lines there were different efficacies. brest cancer and medulloblastoma showed the greatest efficacy of l-tryptophan degrading. the gene technology of tso production from pseudomonas is associated with formation of endotoxins. this disadvantage can be prevented by different washing procedures or by using fungal sources for the tso production. tso iii is developed to treat cancer diseases successfully, and has low side effects. a combination of l-tryptophan depletion with all available cancer therapies is possible. keywords: l-tryptophan, l-tryptophan side chain oxidase (tso), tso bioreactor introduction approximately10 % of all malignant diseases in a progressive stage can be cured. a great problem of the most administered chemotherapy regimens is often development of resistance against different cancers [1, 2]. in many cases the resistance exists primarily before the chemotherapy is administered, or the oncogenes of cancer cells can be mutated during the chemotherapy. the end result is a resistance against the administered chemotherapy [1]. a comparable mechanism is observed for the new kinase inhibitors. the cancer cells can change their oncogenes by mutations resulting in resistance against the kinase inhibitors. in these cases new drugs and therapeutic concepts must be developed continuously. in the last years various new sophisticated therapeutic strategies were developed of which some are summarized in table 1. new knowledge in the pathology of various cancer diseases have shown that the primary oncogenetic defect shall be acquired resulting in genetic aberration which, independent of the cancer, leads to qualitative and quantitative changes in the production of special proteins. these special proteins have a key function in the regulation system of cell growth and differentiation. different proteins such as growth factors, receptors, cytoplasmatic proteins belong to these substances which by dysregulation can induce a malignant disease. all the previous cancer therapeutic strategies are not effective in all patients and they are often associated with a high rate of side effects [2]. a further problem is the primary or acquired resistance to different chemotherapeutic drugs [1). the high rate of side effects and low effectiveness need the development of new drugs and new therapeutic methods constantly. some new possibilities for cancer therapy for example with regard to modulation of the dysregulation of the cell growth are shown in table 1. various authors reported possibilities of treatment of different cancers with so-called anti-tumor enzymes, bioreactors, as an extracorporeal tumor treatment [13-15]. one possibility is the influence of the protein synthesis by depletion of essential amino acids such as l-tryptophan [15-18]. certain amino acids such as l-asparagine, l-glutamine and l-tryptophan have been recognized as important cancer nutrients, and the removal of these amino acids can lead to decrease and destruction of the tumor. since these ___________________________________________________________ * corresponding author: rolf bambauer, md (rolf.bambauer@t-online.de). http://www.ajecr.org/ mailto:rolf.bambauer@t-online.de 108 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org table 1 cancer therapy strategies new endocrine and cytotoxic therapy like antioestrogene, aromatase inhibitors and cytotoxic drugs like taxane, camptothecin analogs, etc. high dose chemotherapy and stem cell transplantation in leukemia and solide tumours (3) cancer vaccines and specific immunotherapy (4) antibodies as specific cancer therapy with monoclonal antibodies (5) immunotoxins (6) human gene therapy (7) tyrosine kinase inhibitors (8) detection of tumor cell dissemination by immunocytology (9) neoangiogenesis and tumour growth (10) ia* with polyclonal ab against stnfr (11) transforming the ta** into immunologic therapy (12) *ia; immunoadsorption, **ta; therapeutic apheresis, stnfr; tumor necrosis factor receptor. so called anti-tumor enzymes are derived from bacterial or fungal sources immunological responses are observed after parenteral administration [19]. the use of serum amino depletion as an effective anticancer agent was first published by kidd in 1953 [20, 21]. he reported that serum of normal guinea pig could induce regression in certain types of animal lymphomas. subsequently, in 1961 broome showed that the enzyme lasparaginase was the anti-neoplastic substance in normal guinea pig serum which depleted the serum of the nonessential amino acid l-asparaginase [22). the principle of removing amino acids from blood as a form of cancer therapy has proven to be beneficial in cases of acute lymphoblastic leukemia using l-asparaginase to degrade the nonessential amino acid l-asparaginase, constituting an important tumor nutrient. however, lasparaginase sensitive tumors can eventually become lasparaginase resistant. this is usually due to the increased denovo synthesis of l-asparaginase by the tumor cells. lasparaginase is a non-essential amino acid and can be synthesized by the human organism. roberts et al. described the isolation of the ltryptophan degrading enzyme, indolyl-3-alkane-αhydroxylase [14, 23], later shown to consist of 2 isoenzymes and called tryptophan side chain oxidase (tso). blood tryptophan depletion by tso resulted in a significant anti-neoplastic activity against mouse tumors in vivo. methods treatment of certain tumors by deprivation of the essential amino acid l-tryptophan has the advantage over non-essential amino acid deprivation, because tumor cells cannot synthesize l-tryptophan [24]. this offers the potential advantage over non-essential amino acid deprivation because host and tumor cells cannot synthesize l-tryptophan, and tumor resistance is therefore unlikely to develop. l-tryptophan cannot be produced in the organism itself [19]. l-tryptophan is an essential amino acid. l-tryptophan is an important amino acid for the cellular integrity. l-tryptophan is needed for a lot of different metabolic processes. the availability of tryptophan is essential for the protein synthesis and reduction, the genome replication and the growth of cell organelles etc. l-tryptophan is a constituent for different bio-molecules such as serotonin, melatonin and is needed for other synthesis processes in the cell growth. a lack of l-tryptophan is associated with different side effects and is followed by a destruction of cells, especially of cells with a high division rate. with the extracorporeal l-tryptophan depletion the growth of cancer cells can be interrupted and the growth of the cancer can be stopped. after the study of toxic side effects and immunologic reactions in animal experiments using tso, an extracorporeal bioreactor system which contains tso was developed by schmer [25]. the bioreactor for removing the potential cancer nutrient ltryptophan from blood was used in tumor bearing animals. the isolated l-tryptophan degrading enzymes (indolyl3-alkane-α-hydroxylase, indh) has 3 iso-enzymes and called tryptophan side chain oxydase (tso) i, ii, iii. the first iso-enzyme tso i has a molecular weight of about 60,000 daltons, the second iso-enzyme tso ii has a molecular weight of about 44,000 daltons, and the third iso-enzyme has a molecular weight of about 42,000 daltons as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis [26]. these isoenzymes can be differentiated by tryptic digestion. all tso enzymes have been characterized as multienzyme complexes containing heme that catalyze essentially similar reactions involving l-tryptophan as a substrate. however tso i and tso ii are distinguishable by their subunit structure, antigenecity and by their reactivity and specificity for various substrates, indicating that tso i and tso ii are distinct enzymes. in 1978, schmer, at the sloan kettering institute for cancer research, new york, isolated another tso enzyme which he named tso iii, which is more effective in degrading ltryptophan than tso i or ii [17]. schmer tested the isolated tso type iii, the most effective of the 3 types, in animals (sheep, rabbits, and rats), nocked immunosuppremised rats and in 20 different human cell lines. enzymatic removal of l-tryptophan from blood of a patient by plasmapheresis and extracorporeal treatment by enzymatic degradation of l-tryptophan in the pheresed blood has long been perceived to have therapeutic benefits [26]. for example, blood levels of l-tryptophan modulate synthesis and synaptic release of the neurotransmitter serotonin. varying l-tryptophan blood levels provides a means to affect brain serotonin levels. the metabolites which are producing by the l-tryptophan degrading enzymes will be eliminated by the human kidneys. the extracorporeal bioreactor system containing tso type i was developed by schmer et al. [24]. the bioreactor is based on silica. the amino groups containing silica beads were activated with 25 % glutaraldehyde. the activated aminosilane beads were washed with distilled water and finally equilibrated with 0.2 m sodium acetate ph 5.5. the activated silica beads can be stored in this buffer at 4°c and remain fully active for more than 6 weeks. a solution of tso in 0.2 m sodium acetate ph 5.5 was passed over the reactor column until the red colored enzyme solution appeared at the outlet. after different http://www.ajecr.org/ 109 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org washing procedures the pre-activated micro-reactors, consisting of a polyacrylic-cellulose copolymer were equilibrated with 0.2 m sodium acetate with a ph 5.5 and filled with 1 % tso solution in the same buffer. the reaction conditions, wash procedures and sterilization were identical to the procedure described for silica beads derived bioreactor. the enzyme then was eliminated from endotoxin by different washing procedures. the silica based enzyme reactor was filled in columns, washed and sterilized. the amount of tso bound to the matrix was determined by pumping sodium phosphate through the bioreactor. the increase in absorbance at 333nm was then expressed in enzyme unit bounds per ml reactor bed. in vitro leakage was determined by pumping sodium phosphate solution through the bioreactor for 2 hours in a circuit. one ml of the solution was then mixed and the increase in absorbance at 333nm within one hour was observed as a sign of leakage. table 2 blood level of l-tryptophan (trp) treatment days pre-trp post-trp* percent decrease* (μg/ml) (μg/ml) 1 8 0.7 91.3 2 5.3 0 100 3 5 1.4 72 4 2.8 0 100 5 6 0.7 88.3 6 1.1 nd nd 7 1.3 nd nd 8 3.3 0 100 9 1.1 1 9.1 *nd indicates no data. results in a rigorous experiment one could show that ltryptophan can be degraded by the enzyme reactor. one liter of human plasma was perfused at 10 ml/min through the column. the concentration of l-tryptophan was significantly lower after the bioreactor column than the concentration of l-tryptophan before the bioreactor column. the tso-bioreactor was tested in different animals [16, 24, 25, 27]. in sheep and rabbits the tso-bioreactor was tested by schmer with a closed circuit mini plasmapheresis unit. he could show that the l-tryptophan depletion in plasma was 100 % in sheep and 95 % in rabbits by a single pass through the bioreactor. these results are very excellent, because the l-tryptophan was effectively eliminated [25]. the investigations in immune supprimized rats with tumors like medulloblastoma were impressive, too. in 9/10 animals a strong regression of the tumor was observed in comparison to the control animals. in histopathological investigations it could be observed that the destruction of the tumor cells was not only in the center of the tumor but in the periphery of the tumor. this suggests that the treatment with tso-bioreactor can be combined with vascular inhibiting substances [28]. in 20 different tumor cell lines there were some different results. brest cancer and medulloblastoma showed the greatest efficacy of l-tryptophan degrading. with gamma-interferon all cell lines showed a higher ltryptophan use and therefore a rapid destruction of all cells. limitation of l-tryptophan in the culture medium of murine leukemia cells caused a decrease in dna and histone synthesis followed by complete growth arrest. the efficacy can be improved with the vascular inhibitors and/or gamma interferon. the anti-neoplastic effect of gamma-interferon is most recently thought to be caused by intracellular l-tryptophan depletion via activation of indoleamine 2.3-dioxygenase [16, 29]. for instance, when used as an agent for reducing blood l-tryptophan levels in a human patient displaying the symptoms of a malignancy, a tso enzyme composition is administered in an amount sufficient to achieve a dosage of 0.1 to 200 iu/kg body weight/day, and preferable 70 to 120 iu/kg body weight/day, and more preferable 75 to 95 iu/kg body weight/day when given either as a single dose per course or in incremental doses [26]. l-tryptophan cannot be produced by human or animal cells [30]. l-tryptophan is an essential amino acid. removal of this nutrient from blood cannot be overcome by a higher production in the cells, therefore making it possible to treat cancer cells over and over again without the disadvantage of the cancer being able to overcome the “bottle neck” situation of nutrient deprivation. to design a so-called bioreactor for removing the potential cancer nutrient l-tryptophan from blood, the ltryptophan degrading enzyme tryptophan side chain oxydase (tso iii) was chemically bound to glutaraldehyde activated gamma amino silane silica and to zetaffinity micro-columns consisting of a glutaraldehyde activated polyacrylic-cellulose copolymer [31]. the silica beads activated polyacrylic-cellulose copolymers (25-30 ml) are packed in a column. after the sterilization and elimination of endotoxins, one column is integrated in the plasma line of a plasmapheresis unit. the patient blood is separated by a hollow fiber membrane separator in blood cells and plasma, the plasma is then perfused through the tso-bioreactor in which ltryptophan is splitted in metabolites. the blood cells and the plasma with metabolites of l-tryptophan are then pumped back to the patient. the metabolites will be eliminated by the human kidney. the treatment with the tso-bioreactor will be daily 4 to 5 hours and 5 days per week over 3 to maximum of 4 weeks. this is one treatment cycle. the cycle can be repeated every 2 to 3 months until a remission is reached or the cancer can be effectively treated with surgery or radiation or both. important is that l-tryptophan blood level will be kept on a very low level over some hours during the treatment. in this situation l-tryptophan could leave the cells and could invade into the blood, and could split by the tso-bioreactor in metabolites which results in a very low l-tryptophan blood level. in the next treatment the blood level of l-tryptophan increased. l-tryptophan is probably removed from cells to http://www.ajecr.org/ 110 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org increase the blood levels. this can be found in an example from the united states patent for tso i and ii in 1993 as shown in table 2 [26]. discussion a new bioreactor for degrading l-tryptophan (tso iii) created by schmer and his group showed a high effectiveness in anti-neoplastic effect with no resistance possibilities. in animal experiments a closed circuit bioreactor in a single pass was used. zetaffinity bioreactors degraded l-tryptophan in animals to more than 95 % in a single pass [25]. whole blood l-tryptophan levels changed little throughout the experiment indicating a vast extravascular tryptophan pool. the procedures were tolerated well by the animals without any change in vital signs [32]. a new cancer therapy method consisting of the ltryptophan degrading enzyme, which will be produced by gene technology from bacterial or fungal sources, ltryptophan side chain oxydase iii (tso iii), is developed to treat cancer disease successfully. the bioreactor based on silica. the amino groups containing silica beads were activated with glutaraldehyde. the activated aminosilane beads were then washed after different procedures. the activated silica beads can be stored in buffer solution at 4° c and remain fully active for more than 6 weeks. the activated beads (20 to 30 ml) are filled in a column, sterilized and inserted in the filtrate line of an apheresis unit. advantages of the l-tryptophan degrading enzyme tso are the excellent stability, no development of a resistance by tumor cells and the combination of this therapy with all other therapeutic measures, especially with vascular inhibiting substances and/or gamma interferon [24). one column with tso beads will be sufficient for the treatment for 3 to 4 weeks in one patient (5 treatments per week for a maximum of 4 weeks). a serious disadvantage of the tso is the formation of endotoxins during the tso production from pseudomonas by gene technology. the toxicity of endotoxin is high for patients and depends of on its level. therefore intensive washing procedures and treatments to eliminate endotoxin are necessary before administration in patients [33]. ltryptophan degrading enzyme tso can be produced by gene technology from bacterial or fungal sources. the advantage of the fungal sources is to receive a tso without endotoxin. an important benefit of tso enzyme having the indicated minimum specific activity is that the patient is exposed to less enzyme mass per unit of tso enzyme activity and thereby is exposed to less endotoxin in a given treatment. reduced exposure to endotoxin results in because of the disclosed tso enzyme activity. thus the patient can be exposed to greater amounts of tso enzyme activity without risking endotoxin-induced histamineresponse type side effects such as allergy, fever, sweating, bronchospasm, hypotension, sickness, severe shaking and anaphylaxis. the side effects of a potential treatment with tsobioreactor can be the same, such as by serotonin deficiency such as anxiety, fatigue, cognitive impairment, negative thoughts, agitation, chronic pain, feeling worse etc. in summary the side effects and the complications during an l-tryptophan degrading treatment could be by: 1) endotoxin, which can be minimized or stopped by a less amount of enzymes or by production of tso from fungal sources , 2) degrading l-tryptophan in the blood to a very low level which have the influence of the serotonin metabolism. this can be prevented by treatments of a maximum of 3 to 4 weeks with daily sessions of 4 hours (5 treatments per week). this is one treatment cycle, and can be repeated every 2 to 3 months later. 3) treatment cycles over a longer time may lead to an antibody development. these antibodies can be eliminated by plasmapheresis. in a first step, the production of tso iii by gene technology from bacterial or fungal sources must be established then the sterilization of the bioreactor material and the sterile production of the columns with all necessary tests like sterilization or stability tests. all description of these measures is available. at the end of this first step is the industrial production of the tso iii-bioreactor. a second step will be the clinical studies after the revised declaration of helsinki in different countries. after production of the tso iii enzyme by gene technology, sterilization and production of the sterile columns (first step), in the second step the new cancer therapy could be started in a clinical trial. vascular access could be achieved by peripheral veins or by implementing a large bore catheter in the vena cava superior. after evaluation of the laboratory, clinical and other data of 30 to 50 cancer patients with 2 or more different cancers the bioreactor can be distributed. the third step can be the distribution of the bioreactor tso iii, the implementation of the tso iii bioreactor after the study protocol and to summarize and evaluate all clinical and laboratory data to develop a more effective therapy concept. future aspects are the production of tso iii by gene technology from fungal sources. the advantage is that by fungal sources there is no endotoxin production, after which the chemical sterilization and animal tests if necessary may be performed to prove the lack of toxicity. another point will be the investigation if the application of tso iii intravenously is possible, and if possible to find the amount of the tso dose, the preparation, toxicity, and a dosage protocol. the next point will be the investigation to produce tso enzyme as an oral drug. the advantages of the tso bioreactor are that no resistance of the tso iii-bioreactor is possible because human or animal cells cannot synthesize l-tryptophan. the possible cancer nutrient tso iii has a high anti neoplastic effect in breast cancer, medulloblastoma and other metastatic cancers. tso iii enzyme has low side effects, which are not comparable with those of chemoor radiation therapy. side effects could be aggression, tiredness, or somnolence, etc. therefore a limitation of the treatments of 15-20 daily treatments over 3 to 4 weeks is necessary. an l-tryptophan free diet is not necessary. this new therapy concept can be http://www.ajecr.org/ 111 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org combined with all other cancer therapies such as surgery, radiation, chemotherapy etc. a further point is the toxicity of tso. this point must be clarified with different washing procedures before the introduction of this therapy in humans. endotoxins are available only won by pseudomonas sources. they can be eliminated by different washing procedures. an antibody production against tso enzyme is possible but not clarified and is only possible in longer therapy procedure. the antibodies can be eliminated by plasmapheresis. last but not least is the commercial aspect; for example in germany alone 350,000 to 400,000 women and men afflict by different cancers per year. of these patients, 20% to 30% die in the first year after diagnosing the cancer. the therapeutic measures to date have very different results in view point of healing or quality of life, etc. the treatment costs for one therapeutic cycle (5-6 treatments per week, duration 3-4 weeks, daily treatment 4-5 hours) depend on the production costs of the column. the costs for 15-20 primary separation of the blood and the perfusion of plasma through the bioreactor column are lower than the plasmapheresis costs. the costs can be reduced by producing tso from fungal sources and a treatment set of one column for 3-4 weeks per patient and one treatment cycle. if only 0.1%-1% of the new patients who afflict the disease every year will be treated, this would be a tremendous achievement. the treatment could be repeated after 2 to 3 months or more, if no remission is reached by the first treatment cycle. between the cycles a staging of the cancer is necessary. the gene technology for production of the tso enzyme (tso iii) and the producing of tso-bioreactor columns are also patentable, provided that first works be performed. conflict of interest the author declares no conflicts of interest. references 1. reichle a, diddens h, rastetter j, berdel we. resistenzmechanismen maligner zellen gegenüber zytostatika. dtsch med wschr 116:186-191, 1991. 2. volm m, mattern j, samsel b. häufung von zytostatica-resistenten lungentumoren bei rauchern. dtsch med wschr 116:1303 -1306, 1991. 3. kanz l. hochdosistherapie und stammzell transplantation. internist 38:1045, 1997. 4. schirmacher v. tumorvakzine und aktiv spezifische immuntherapie. internist 38:1050, 1997. 5. gramatzki m, valerius t. antikörper als spezifische tumortherapie. internist 38:1055, 1997. 6. barth s, winkler u, diehl v et al. immunotoxin. internist 38:1063, 1997. 7. lindemann a, mertelsmann r. gentherapie. internist38:107 0, 1997. 8. alves p, hiddemann w. thyroxinaseinhibitoren. internist 38:1074, 1997. 9. wörmann b, wulf gg, griesinger f et al. sensitiver nachweis disseminierter tumorzellen. prognostische bedeutung und therapieansätze. internist 38:1083, 1997. 10. fiedler w, gehling u, mende t et al. neoangiogenese und tumorwachstum. dtsch ärztebl 98: a 1392, 2001. 11. lentz mr. the role of therapeutic apheresis in the treatment of cancer. a review. ther apher 3:40, 1993. 12. porrata lf, markowic sn. therapeutic apheresis immunologic graft engineering for the treatment of cancer. transplantationsmedizin 22:379-382, 2010. 13. rosenfeld hj, watanabe ak, roberts j. mechanism of action of indolyl-3-alkane-α-hydroxylase. biol chem 252:6970-6973, 1977. 14. roberts j schmid fa, rosenfeld hj. biologic and antineoplastic effects of enzyme-mediated in vivo depletion of l-glutamine, l-tryptophan and l.-histidine. cancer treat rep 63:1045-1054, 1979. 15. cook sj, pogson ci, smith sa. indoleamine 2,3dioxygenase. biochem j 189:461-466, 1980. 16. schmer g, chandler wl. enzyme reactors: achievements, problems, future perspectives. in: bambauer r, malchesky ps, falkenhagen d (eds.). therapeutic plasma exchange and selective plasma separation. schattauer verlag, new york, pp 437-443, 1987. 17. schmer g, roberts j. purification of indolyl-3alkane-α-hydroxylase by affinity chromatography on indolyl-agarose columns. biochim biophy acta 527:264271, 1978. 18. yoshida r, park sw, yasul h et al. tryptophan degration in transplanted tumor cells undergoing rejection. j immunol 141:12819-2823, 1988. 19. elmada i, leitzmann c. ernährung des menschen. 3. aufl verlag eugen ulmer, stuttgart, 1998. 20. kidd jg. regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum: i. cause of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum. j exp med 98:565, 1953. 21. kidd jg. regression of transplanted lymphoma induced in vive by means of normal guinea pig serum: ii. studies on the nature of the active serum constituent: histological mechanism of regression: tests for effects of guinea pig serum on lymphoma cells in vitro: discussion. j exp med 98:583, 1953. 22. broome jd. evidence that the l-asparaginase activity in guinea pig serum is responsible for its antilymphoma effects. nature 191:114, 1961. 23. roberts j, rosenfeld hj. isolation, crystallization and properties of indolyl-3-alkane-α-hydroxylase. j biol chem 252:2640-2647, 1977. 24. schmer g, roberts j. molecular engineering of the l-tryptophan-depleting enzyme indolyl-3-alkane-αhydroxylase. canc treat rep 63:1123-1126, 1979. 25. schmer g, dennis mb, hsueh s, hou kc. the synthesis of l-trytpophan degrading bioreactors. int j artif org 13:316 -320, 1990. 26. united states patent no. 5,244,807 “pndh enzyme composition and their methods of use,” 14.9.1993. 27. schmer g, roberts j. induction of hypothermia in mice by semi-artificial cell containing indolyl-3-alkane-α http://www.ajecr.org/ 112 am j exp clin res, vol. 2, no. 3, 2015 http://www.ajecr.org hydroxylase. trans am soc artif int org 25:39-43, 1979. 28. schmer g. überblick der forschung betreffend die behandlung bösartiger tumoren durch entfernung des plasma l-tryptophans. manuskript. 29. farin fm, chandler wl, hsueh s, bambauer r, schmer g. l-tryptophan depletion in rats using a ltryptophan free diet and administration of tryptophan side chain oxidase. manuskript. 30. wooley pv, dion ri, bono vh. effects of tryptophan deprivation on l1210 cells in culture. cancer res 34:1010-1014, 1974. 31. dennis mb, jensen wh, bauermeister u vienken j, schmer g. successful use of a miniturized plasmapheresis circuit for long term use in rabbits. trans am soc artif int org 34:651-654, 1988. 32. schmer g, bambauer r. die extrakorporale entfernung von l-tryptophan mit einem plasmapheresebioreaktorsystem. dial j 19:67-69, 2000. 33. schmer g, kruegger m, cole jj. gel-bound resealed red cell membranes: a new type of semi-artificial organs. j biol chem 252:2640-2647, 1977. http://www.ajecr.org/ 180 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2016;3(4):180-183 original article chronic hypertension with gestational diabetes mellitus: what about complications? ana campos 1 *, ana catarina massa 1 , ricardo rangel 2 , manuela cardoso 3 , maria augusta borges 4 1department of maternal-fetal medicine service, alfredo costa maternity, central lisbon hospital center, lisbon, portugal 2department of endocrinology service, curry cabral hospital, central lisbon hospital center, lisbon, portugal 3nutrition services, alfredo costa maternity, central lisbon hospital center, lisbon, portugal 4internal medicine consultation, alfredo costa maternity, central lisbon hospital center, lisbon, portugal abstract. the aim of this study was to evaluate the impact of chronic hypertension and gestational diabetes on pregnancy outcomes. we conducted a historical cohort study of 334 women undergoing singleton births in a portuguese tertiary care center in lisbon during 2012. women were categorized into gestational diabetes mellitus with or without chronic hypertension. pregnancy outcomes were compared using nonparametric tests. multivariable analysis was used to control for potential confounders. the rate of preeclampsia in women with both chronic hypertension and gestational diabetes was 26.8% versus 3.8% in women with only gestational diabetes (p<0.05). preterm birth was significantly more frequent in women with diabetes and chronic hypertension, 22.9% versus 9.7%, compared with women who only had gestational diabetes (p<0.05). the rate of newborns small for gestational age in women with the two conditions was 19.1% versus 7.6% in women with only gestational diabetes (p<0.05), but the rate of large for gestational age newborns in women of chronic hypertension and gestational diabetes was 9.6% versus 3.8% in gestational diabetes (p<0.05). the impact of having both chronic hypertension and gestational diabetes in pregnancy leads to poor pregnancy and perinatal outcomes, represented by more maternal, obstetrical and neonatal morbidity. keywords: chronic hypertension; perinatal outcomes; gestational diabetes introduction gestational diabetes is defined as an abnormal glucose tolerance at any moment of pregnancy 1. it is believed that 6 to 7% of all pregnancies are complicated by diabetes and that gestational diabetes comprises 90% of all these cases. the prevalence has been growing with the sedentary lifestyle, excess of weight and obesity, which are risk factors, when women decide to delay maternity. gestational hypertension and preeclampsia are frequently associated with diabetes in pregnancy, with poor results in terms of maternal and perinatal morbidity 2. hyperglycemia may contribute to fetal macrosomia, increasing the risk of delivery complications, shoulder dystocia and, hence, leads to a higher rate of cesarean sections. neonatal complications are essentially associated to respiratory distress and to metabolic disorder, but can also be linked to traumatic delivery, essentially when there is macrosomia. the maternal long-term risks are type 2 diabetes, metabolic syndrome and cardiovascular disease. lifestyle changes and weight loss are important measures for a risk reduction 3. in a portuguese population study that took place in 2012, the prevalence of gestational diabetes in national health service users was 4.8%, and between 30-39 years old it was 5.9% and 13.5% for women over 40 years old. the international association of diabetes and pregnancy study group (iadpsg) recommendations are implemented in portugal since 2011 4, 5. the factors determining the higher prevalence of diabetes in pregnancy are the same as those observed in chronic hypertension, defined by values of arterial pressure ≥140/90 mm hg measured before or during the first half of the pregnancy 6. chronic hypertension and gestational diabetes are independent risk factors for pregnancy complications and when in association, they can contribute to more maternal and perinatal severe outcomes 7. the prevalence of chronic hypertension in pregnancy is 2 to 8% varying with age, ethnicity and body mass index 8, 9. the rate of hypertensive disorder in pregnancy and puerperium in portuguese women is 6%: 1.5% with chronic hypertension, 2.5% with gestational hypertension, 1.4% with preeclampsia, 0.2% with superimposed preeclampsia, ___________________________________________________________ * corresponding author: ana campos, md (anacampos.campos87@gmail.com). http://www.ajecr.org/ mailto:anacampos.campos87@gmail.com 181 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org 0.1% with eclampsia and 0.1% with hellp syndrome. these data are the result of a first study about the prevalence of this pathology in portugal 10. we have no comparative studies in portugal addressing the impact of chronic hypertension on gestational diabetes or vice versa. the objective of this study was to compare maternal and perinatal results in two cohorts of pregnant women with gestational diabetes: one with chronic hypertension and the other without chronic hypertension. materials and methods this observational study contained two historical cohorts of singleton pregnant women followed in a tertiary care center in lisbon, during 2012: one cohort of 237 pregnancies with gestational diabetes (gd) and the other with 97 pregnant women with gestational diabetes and chronic hypertension (ch). this study was approved by the ethical committee of central hospital of lisbon and all the participants gave their approved consent. data were collected from the service database and from the clinical process. both cohorts had the same surveillance guidelines. socio demographic variables and pregnancy results were compared in both groups: gestational age at delivery, type of delivery, hypertensive complications or preeclampsia, prematurity, weight and percentile for gestational age of newborns. chronic hypertension was defined according to the international society for the study of hypertension in pregnancy (isshp) as an arterial systolic and diastolic pressure (equal or greater than 140/90 mm hg) before pregnancy or in the first 20 weeks; when elevation of arterial pressure occurred after the 20th week, it was classified as gestational 6. preeclampsia was defined according to isshp criteria, by the presence of hypertension and proteinuria after the 20th week of pregnancy and superimposed preeclampsia when the criteria of preeclampsia were applied to chronic hypertension 6, 9. proteinuria was quantified by a dipstick reading of 1+ or ≥30 mg/dl, confirmed by values of 300mg per 24-hour urine collection. if preeclampsia occurred before the 34th week, it was classified as of earlyonset 11. indicators of clinical deterioration were: the need for anti-hypertensive drugs during pregnancy in chronic hypertension; the need to terminate pregnancy if there was a clinical worsening of maternal or fetal status, or a diagnosis of preeclampsia. the definition of gestational diabetes was the presence of a glucose intolerance at any moment of the pregnancy and criteria are defined by international association of diabetes and pregnancy study group (iadpsg) since 2011 3. it is classified as gestational diabetes, a fast glucose value equal or greater than 92 mg/dl in the first trimester or after an ogtt with 75 g oral glucose between the 24th-28th, by the presence of one or more values equal or greater than (0h ≥ 92mg/dl, 1h ≥ 180mg/dl, 2h ≥ 153mg/dl) 5. the gestational results were considered in terms gestational age at the time of delivery, preeclampsia, early onset and severe preeclampsia, fetal growth restriction, table 1 sociodemographic and anthropometric variables bmi: body mass index. table 2 pregnancy and delivery data map: median arterial pressure; ga: gestational age; oad: oral antidiabetics; sga: small for gestational age; aga: adequate for gestational age; gga: great for gestational age; nb: newborn. birth weight, small for gestational age (birth weight <10th percentile for gestational age) on a hadlock growth curve and stillbirth. statistical analysis the data were registered in an informatics database. to compare categorical variables between the study groups, chi-square test or extension to exact fisher test were used; gest at ional diabet es chronic hypert ension + gest at ional diabet es p (n = 237) (n = 97) median age (range) 33 (17-44) 36 (23-49) 0.001 age ≥ 35 yrs (%) 98 (41.4) 63 (55.8) p re-concept ion bmi median (range) 26 (17-50) 31(20-55) 0.001 ≥ 30 yrs: no. (%) 57 (25.6) 56 (58.9) 0.007 ≥ 35 yrs: no. (%) 24 (10.5) 21 (21.9) median weight gain (kg) 9 3 0.001 et hnicit y: no. (%) caucasian 166 (70.0) 74 (76.2) 0.026 african 42 (17.8) 22 (22.8) asian 29 (12.2) 1 (1.0) p arit y: no. (%) nulliparous 114 (48.1) 27 (31.0) 0.001 variable gest at ional diabet es chronic hypert ension + gest at ional diabet es (n = 237) (n = 97) map >85 0 41 (41.2) 0.001 therapy diet n (%) 145 (61.2) 61 (62.9) 0.383 insulin 57 (24.1) 32 (33.3) 0.177 oad 22 (9.3) 3 (3.1) oad + insulin 13 (5.5) 1 (1.0) 28 (11.8) 44 (45.4) 0.001 preeclampsia; no. (%) 9 (3.8) 26 (26.8) 0.001 median delivery ga 38.2 37.5 0.352 <37 weeks; no. (%) 23(9.7) 22 (22.9) 0.001 newborn weight >2500g 217 (91.6) 75 (78.9) <2500g 20 (8.4) 20 (21.1) 0.001 sga ; no. (%) 18 (7.6) 18 (19.1) 0.001 aga 210 (88.6) 79 (68.1) gga 9 (3.8) 9 (9.6) 0.03 mean nb weight 3270g 3180g 0.352 delivery term; no. (%) 214 (90.3) 74 (77.1) preterm 23 (9.7) 22 (22.9) 0.001 cesarean section 88 (37.1) 59 (60.8) 0.001 variable hypertensive complications; no. (%) p http://www.ajecr.org/ 182 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org table 3 pregnancy hypertensive complications gd: gestational diabetes; ch: chronic hypertension; pe: preeclampsia. for the analysis of continuous variables the non-parametric test of mann-whitney was used. univariate analysis was used to assess differences in baseline demographic and pregnancy characteristics and in the results between the 2 groups. unadjusted odds ratios (ors) with 95% confidence intervals (cis) were calculated to quantify the risk of hypertensive complications, preeclampsia and preterm birth in women with superimposed preeclampsia and preeclampsia. multivariate logistic regression was used to calculate adjusted odds ratios (aors) for preeclampsia and hypertensive complications in the groups. all statistical analysis was performed with statistical package for social sciences (spss) version 20.0. tests with probability values of p<.05 were considered significant. results the analysis of socio demographic and anthropometric variables shows that the mean maternal age was superior in the cohort of chronic hypertension with gestational diabetes (35.5 vs. 32.9; p<0.001) with more women being over 35 years old (55.7% vs. 41.4%). the body mass index (bmi) was superior in this cohort (30.9 vs. 27.1; p<0.001). in the cohort of chronic hypertensive disease associated to gestational diabetes, caucasian women were more frequent, but asian women were more present in the cohort of gestational diabetes without chronic hypertension, corresponding to a growing prevalence of diabetes in this group of immigrants. gestational diabetes without chronic hypertension had more nulliparous (48.1% vs. 31.0 %; p=0.001) (table 1). therapeutic measures had no differences in the two cohorts (table 2). the cohort of chronic hypertension used insulin therapy more frequently, but without significance; however, weight gain during pregnancy was superior in the cohort of gestational diabetes without chronic hypertension (table 1). the cohort of chronic hypertension and gestational diabetes had a higher incidence of preterm birth (22.9% vs. 9.7%; p=0.001), more hypertensive complications (43.3% vs. 11.8%) (p<0.001), more preeclampsia (22.7% vs. 3.8; p<0.001), a higher rate of cesarean sections (62.5% vs. 37.1%; p=0.001), more small for gestational age infants (sga) (19.1% vs. 7.6%; p<0.001), but also more large for gestational age infants lga) (9.6% vs. 3.8%) (table 2). multivariate regression analysis confirmed that hypertensive complications are associated with obesity (58% vs. 25%; aor 4,2; ic 95%, 1.38-12.70); african ethnicity (22.8% vs. 17.8%; aor: 28.7; ic 95%, 2.3-34), preterm birth ( 22.9% vs. 9.7%; aor 6,1; ic 95%, 1.4425.70) and higher cesarean section rates (60.8% vs. 37.1%; aor 2.36; ic95%, 1.22-4.57). in the presence of gestational diabetes and chronic hypertension there is a greater risk to have preeclampsia (26.8% vs. 3.8%; or: 9.4; ic 95%, 2.4-37.3) and cesarean section (60.8% vs. 37.1%; aor: 7.2; ic 95%, 1.9-27.1) than when only gestational diabetes is present (table 3). discussion in this study we intended to address the question: in a pregnancy complicated by gestational diabetes and chronic hypertension what can be expected? in gestational diabetes, the presence of chronic hypertension contributed to a worst pregnancy result. chronic hypertension is usually present in older women and in the present study we have confirmed that chronic hypertensive women have larger body mass index and obesity. in women with chronic hypertension and gestational diabetes it was necessary to prescribe a greater insulin dosage in order to achieve metabolic control, which suggests that there is an insulin resistance in gestational diabetes with chronic hypertension; we think that the number of cases enabled us to find a statistical significance. the reason is not only the larger body mass index, but also probably the chronic hypertension, by unknown reasons. in this study, we found a lower weight gain in women with chronic hypertension than in women with gestational hypertension, probably due to a more intense role of dietary measures. in women with chronic hypertension and gestational diabetes there were more cases complicated by preterm birth, and of small for gestational age newborns, owing to more preeclampsia complications with maternal or fetal deterioration status; but we had also in this group more newborns large for gestational age, probably due to a more difficult metabolic control. african ethnicity has an increased risk to have chronic hypertension and this is also present in this study 15, 16. we had no cases of maternal or perinatal mortality. the relationship with carbohydrate metabolism in gestational diabetes when there is simultaneously chronic hypertension is under investigation 17. some authors have concluded that carbohydrate metabolism is different, and there is probably a link between insulin resistance and chronic hypertension 18. when both pathologies are present the metabolic control is more difficult and greater doses of insulin are needed. this group has more adiposity, complication no. (%) aor 95% ci hypertensive gd 57 (25) gd+ch 56 (58) gd 114 (48.1) gd+ch 27 (31) gd 23 (9.7) gd+ch 22 (22.9) gd 88 (37.1) gd+ch 59 60.8) gd 24 (10.5) gd+ch 21 (21.9) preeclampsia gd 0 (3.8) gd+ch 97 (26.8) gd 59 (37.1) gd+ch 88 (60.8) 1.44-25.70 2.36 1.22-4.57 1.9-27.1 nulliparity preterm birth cesarean section age ≥35 yrs 1.792 0.90-3.56 2.4-37.39.4 4.2 1.38-12.70 1.89 0.94-3.72 6.08 bmi ≥ 30 cesarean section chronic hypertension 7.2 http://www.ajecr.org/ 183 am j exp clin res, vol. 3, no. 4, 2016 http://www.ajecr.org but this is possibly not the only factor, and median arterial pressure (map) in the first trimester may be a good predictor of insulin resistance. in our study we confirmed this predictive role of first trimester median arterial pressure; 41% of women with chronic hypertension had an elevated map and in this group we had more therapy for arterial pressure control and also more insulin use. for the explanation of the worst metabolic control in women with chronic hypertension with gestational diabetes some theoretical hypothesis were tried. gestational diabetes is the result of an exaggerated insulin resistance characteristic of the pregnancy; if there is the association with obesity and dyslipidemia, endothelial dysfunction, oxidative stress and inflammatory response can be more expressive and can contribute to worsening the chronic hypertension pathological findings 19. after delivery, it is important to do the diabetesscreening test to diagnose type 2 diabetes, a fast abnormal glucose or a glucose tolerance reduction. lifestyle changes are also important to prevent metabolic syndrome, type 2 diabetes, vascular dysfunction and atherogenesis, which are characteristics of cardiovascular diseases. the prevention and correction of obesity is a major measure that should begin in childhood. conclusion this is an observational study introducing iadpsg criteria for the diagnosis of gestational diabetes. these results show the negative influence of chronic hypertensive disease and obesity in gestational diabetes and pregnancy. it is the first study of diabetes and chronic hypertension in pregnant portuguese women with the new diagnosis criteria. our conclusions are similar to other studies and prove the importance of obesity prevention for cardiovascular risk prevention. conflict of interest the authors declare no conflicts of interest. references 1. american college of obstetrics and gynecology. national institutes of health consensus development conference statement – diagnosing gestational diabetes mellitus, march 4-6, 2013. obstet gynecol 122:358-369, 2013. 2. loguercio v, mattei l, trappolini m, festa c, stoppo m, napoli a. hypertension in diabetic pregnancy: impact and long-term outlook. best pract res clin endocrinol metab 24:635-651, 2010. 3. nih conference statement gestational diabetes mellitus. diagnosing gestational diabetes mellitus. obstet gynecol 122:358-369, 2013. 4. gardete cl, boavida jm, almeida jpf, cardoso sm, dores j, diabetes: factos e números 2013. relatório anual do observatório nacional da diabetes. sociedade portuguesa de diabetologia. letra solúvel publicidade e marketing lda. lisboa 2013. 5. macedo me, lima mj, silva ao, alcântara p, ramalhinho v, macedo a. prevalência, conhecimento, tratamento e controlo da hipertensão em portugal. estudo pap. rev cardiol port 26: 21-39c, 2007. 6. report of the national high blood pressure education program working group on high blood pressure in pregnancy. am j obstet gynecol 183:s1-22, 2000. 7. the hapo study cooperative research group. hyperglycemia and adverse pregnancy outcomes. new engl j med 358:1991-2002, 2008. 8. duckitt k, harrington d. risk factors for preeclampsia at antenatal booking: systematic review of controlled studies. brit med j 330:565-567, 2005. 9. august p, lindheimer md. chronic hypertension and pregnancy. in: lindheimer md, roberts jm, cunningham fg, editors. chesley’s hypertensive disorders in pregnancy. 2nd ed. stamford (ct): appleton & lange 1999, pp. 605-633. 10. póvoa a m, costa f, rodrigues t, patrício b, cardoso f. prevalence of hypertension during pregnancy in portugal. hypertens pregnancy 27:279-284, 2008. 11. american college of obstetricians and gynecologists. intrauterine growth restriction. acog practice bulletin no. 12. washington, dc: american college of obstetricians and gynecologists, 2000. 12. american college of obstetricians and gynecologists. fetal macrosomia. acog practice bulletin no. 22. washington, dc: american college of obstetricians and gynecologists, 2000. 13. sibai bm. chronic hypertension in pregnancy. obstet gynec 100:369-776, 2002. 14. o’sullivan jb, mahan cb. criteria for the oral glucose tolerance test in pregnancy. diabetes 13:278-285, 1964. 15. lawrence jm, contreras r, chen w, sacks da. trends in the prevalence of preexisting diabetes and gestational diabetes mellitus among a racially/ethnically diverse population of pregnant women, 1999-2005. diabetes care 31:899-904, 2008. 16. martin ja, hamilton be, sutton pd, et al. births: final data for 2007. natl vital stat rep 58:1-85, 2010. 17. yanit ke, snowden jm, cheng yw, et al. the impact of chronic hypertension and pregestational diabetes on pregnancy outcomes. am j obstet gynecol 207:333.e16, 2012. 18. caruso a, ferrazzini s, carolis s, lucchese a, lanzone a, giancarlo p. carbohydrate metabolism in gestational diabetes: effect of chronic hypertension. obstet gynecol 94:556-561, 1999. 19. carpenter mw. gestational diabetes, pregnancy hypertension, and late vascular disease. diabetes care 30s:246-250, 2007. http://www.ajecr.org/ 247 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(4):247-249 original article effect of cerebrolysin in patients with ischemic stroke: a double-blind randomized control study sayed ali mousavi1 * , azam moradi1, omid mirmosayyeb1, 3, mansour salehi2, fariborz khorvash1 1 department of neurology, faculty of medicine, isfahan university of medical sciences, isfahan, iran 2 department of cell and molecular biology, faculty of medicine, isfahan university of medical sciences, isfahan, iran 3medical research committee, isfahan university of medical sciences, isfahan, iran abstract. we investigated the effect of cerebrolysin compared with placebo efficacy in patients with ischemic stroke. a total of 50 patients with ischemic stroke participate in this randomized double-blind placebo-controlled study. patients were randomly divided into two groups; main group (n=25) was treated by 30cc iv cerebrolysin daily for 5 days. control group (n=25) administrated 30 cc iv normal saline as a placebo daily during first five days of stroke attack. three scoring system was used in the present study: national institutes of health stroke scale (nihss) on admission and after five days, and barthel and rankin’s scale after the 90 days. data were analyzed by spss 16 using t-test. p-value less than 0.05 considered as significant level. the nihss score showed no significantly difference after 5 days (independent t-test, p = 0.195). there was a significant difference between barthel and rankin’s scale after 90 days (p = 0.039 and p = 0.008 respectively). in conclusion, cerebrolysin prevented the development of ischemic stroke’s sign and symptoms through 90 days. keywords: cerebrolysin, ischemic stroke, neuroprotection introduction acute stroke leading to cause of adult disability and has poor prognosis of survival than most forms of cancer. in recent years, considerable efforts have been devoted to the development of new therapeutic strategies for acute stroke victims [1, 2]. substantial effort has been expended to the development of neuroprotective therapies that intervene at various stages of the ischemic cascade and which potentially have a wider clinical application. several classes of compound have been tested, including calcium channel blockers, n-methyl-d-aspartate (nmda) antagonists, glutamate release inhibitors, anti-adhesion antibodies (anti-icam 1), gm-1 gangliosides, gammabutyric acid antagonists, sodium channel blockers, glycine antagonists, free oxygen radical scavengers, and potassium channel agonists [3, 4]. numerous neuroprotective agents have shown promising results in animal experiments. however, clinical trials have thus far failed to confirm benefits [5-8]. cerebrolysin is a peptide preparation for iv infusion which mimics the action of neurotrophic factors. the compound is produced by a biotechnological process, a controlled hydrolysis of purified brain proteins, and consists of low-molecular-weight neuropeptides and free amino acids. cerebrolysin has been shown to exert neurotrophic as well as neuroprotective effects in vitro and in vivo. it induces neuritis out growth and reduces apoptosis triggered by growth factor withdrawal in cultivated neurons. in animal models of stroke, intravenous cerebrolysin reduced mortality by about 50% after bilateral carotid artery occlusion in rats and reduced infarct size as well as the loss of map-2 immunoreactivity in a model of middle cerebral artery occlusion [9]. ladurner et al. reported a significantly better regression of motor disturbances by the day 21 and 3 month after administration of 50 ml/day cerebrolysin [9]. hence, the aim of present study was to investigate the efficacy of cerebrolysin management of ischemic stroke. materials and methods ethical approval this randomized double-blind placebo-controlled study conducted in department of neurology, al zahra hospital during 2014-2015. this study was submitted to and approved by the ethical committee for research in isfahan university of medical sciences, isfahan, iran and had no conflict with declaration of helsinki. all patients signed the inform consent from. ___________________________________________________________ * corresponding author: f. khorvash, m.d (fkhorvash@gmail.com). http://www.ajecr.org/ mailto:fkhorvash@gmail.com 248 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org table 1 demographic characteristics and questionnaire score of study subjects inclusion criteria patients who referred to emergency room by diagnosis of acute ischemic stroke (within 6-24 hours after attack) in middle cerebral artery territory of both genders, more than 45 year old, and the stroke deficit of moderate degree (nihss score more than 7, less than 17) selected for this study. patients were examined by a single physician on admission, after neuroimaging and confirmation of diagnosis; routine stroke management was started including anti-platelet therapy, statins and etc. according to mentioned criteria fifty patients were selected. exclusion criteria patients with ich, sdh, edh, sah, cerebrodegenerative or demyelinating disease, brain tumor, previous stroke, metabolic disorders, and patients with stroke in other artery distributions and patients with lacunar infarct were excluded. study groups patients were randomly divided into two groups; first group (n = 25) treated by 30 cc iv cerebrolysin daily for 5 days. second group (n = 25) received 30 cc iv normal saline as a placebo daily during first to five days of stroke attack. neurological score three scoring systems were used in this study: national institutes of health stroke scale (nihss) [10], barthel’s scale [11], and rankin’s scale 12. each patient examined by nihss scoring system on admission and after five days. the barthel activities of daily living index and rankin’s score were checked after 90 days. the study end point is after completion of the 3 months follow up period. also all subjects were visited every 4 weeks up to 3 months. statistical analysis data were analyzed with spss 16 using independent ttest for study groups. differences between mean score of nihss on admission and after 5 days were determined using paired t-test for each group. pearson correlation was implemented to compare barthel and rankin’s scales with each other. p-value less than 0.05 considered as significant level. results table 1 shows the demographic feature of subjects and the mean score of nihss, barthel and rankin’s scales. seven patients out of 50 were died (4 in cerebylosin group and 3 in control group). the mean nihss’s score of cerebylosin group on admission and five days after was 10.64±2.6 and 8.92±4.5 respectively (paired t-test, p = 0.026). the mean nihss’s score of placebo group on admission and five days after was 11.56±2.3 and 9.92±3.7 respectively (paired t-test, p = 0.011). there was no statistically significant difference between the mean score of nihss on admission and after five days through the groups (independent t-test, p > 0.05). there were significant differences between study groups after 90 days by barthel and rankin’s scales evaluation (independent t-test, p= 0.039 and chi-square, p= 0.027 respectively). pearson correlation was used to determine the correlation of barthel and rankin’s scales with each other after 90 days. the result showed a significant correlation of these questionnaire (r = -0.928, p = 0.000). discussion the authors conducted that cerebrylosin therapy can reduce cerebral infraction signs and symptoms volume after 3 months. the results of this study confirmed an improvement of activity in patients who treated with cerebrylosin. the term “neuroprotection” is used to describe the putative effect of interventions protecting the brain from pathological damage. in ischemic stroke, the concept of neuroprotection includes inhibition of pathological molecular events leading to calcium influx, activation of free radical reactions and cell death. knowledge of pathophysiology in acute ischemic stroke stimulated development of a number of potential neuroprotective agents [13, 14]. in a preliminary randomized controlled trial (saint i) with 1722 patients, nxy-059 treatment of acute ischemic stroke was associated with a small but significant reduction in the primary outcome of disability at 90 days compared with placebo, but there was no significant improvement in co-primary outcome of neurologic function or any of the secondary outcome measures [15]. in the saint ii trial involving over 3000 patients, nxy-059 treatment compared with placebo did not result in a statistically significant reduction in the primary outcome of strokerelated disability or improvement on any of the secondary outcome measures [16]. similarly, a pooled analysis of the saint i and ii trials confirmed the lack of benefit for nxy-059 on all primary and secondary end points in the overall population and in the pre-specified subgroups [17]. several methods have been introduced for the management of ischemic stroke patients such as; the use of neurotrophic factor and stem cell therapy. for this respect, main group cont rol group (cerebylosin) (p lacebo) male 11 10 female 14 15 age 70.96±9.1 69.84±11.8 mean nihss score (admission) 10.64±2.6 11.56±2.3 mean nihss score (aft er 5 days) 8.92±4.5 9.92±3.7 mean bart hel score aft er 90 days 78.80±27.6 62.27±16.0 variable http://www.ajecr.org/ 249 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org basic fibroblast growth factor (bfgf), brain derived neurotrophic factor (bdnf), insulin-like growth factor (igf) and osteogenic protein 1 have been studied. the therapeutic potential has convincingly been shown for bfgf and osteogenic protein 1. both factors achieved improvement of behavioral outcome and a reduction of infarct size in animal model [13, 18]. cerebrolysin is a peptide which induces neurite out growth and reduces apoptosis triggered by growth factor withdrawal in cultivated neurons. in a recent study in rats, cerebrolysin has been shown to increase the number of neuronal progenitor and to enhance neurogenesis in the dentate gyrus of adult animals which correlated with improved spatial memory performance in these animals. in other hand, on a molecular level, an inhibitory effect of cerebrolysin on calpain has been demonstrated. in animal models of stroke, intravenous cerebrolysin reduced mortality by about 50% after bilateral carotid artery and reduced infarct [9]. ladurner et al. [9] evaluated the safety and preliminary outcome of cerebrolysin treatment in patients with acute stroke. the results of their study showed that neurotrophic treatment with cerebrolysin is safe and well tolerated by patients with acute stroke. in conclusion, cerebrolysin may be considering as a potent therapeutic approach cerebral ischemic stroke. conflict of interest the authors declare no conflicts of interest. references 1. clark wm, wechsler lr, sabounjian la, schwiderski ue. a phase iii randomized efficacy trial of 2000 mg citicoline in acute ischemic stroke patients. neurology 57:1595-1602, 2001. 2. de keyser j, sulter g, luiten pg. clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing? trends neurosci. dec ２２:535540, 1999. 3. martinez-vila e, sieira pi. current status and perspectives of neuroprotection in ischemic stroke treatment. cerebrovasc dis 11:60-70, 2001. 4. sacco rl, derosa jt, haley ec, jr., et al. glycine antagonist in neuroprotection for patients with acute stroke: gain americas: a randomized controlled trial. jama 285:1719-1728, 2001. 5. bath pm, sprigg n. colony stimulating factors (including erythropoietin, granulocyte colony stimulating factor and analogues) for stroke. cochrane database syst rev 2:cd005207, 2007. 6. gladstone dj, black se, hakim am. toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. stroke 33:2123-2136, 2002. 7. muir kw, lees kr. excitatory amino acid antagonists for acute stroke. cochrane database syst rev 3:cd001244, 2003. 8. savitz si, fisher m. future of neuroprotection for acute stroke: in the aftermath of the saint trials. ann neurol 61:396-402, 2007. 9. ladurner g, kalvach p, moessler h. neuroprotective treatment with cerebrolysin in patients with acute stroke: a randomised controlled trial. j neural transm 112:415-428, 2005. 10. goldstein lb, samsa gp. reliability of the national institutes of health stroke scale. extension to non-neurologists in the context of a clinical trial. stroke 28:307-310, 1997. 11. mahoney fi, barthel dw. functional evaluation: the barthel index. md state med j. feb 1965;14:61-65. 12. van swieten jc, koudstaal pj, visser mc, schouten hj, van gijn j. interobserver agreement for the assessment of handicap in stroke patients. stroke 19:604607, 1988. 13. lin sz, hoffer bj, kaplan p, wang y. osteogenic protein-1 protects against cerebral infarction induced by mca ligation in adult rats. stroke 30:126-133, 1999. 14. schabitz wr, schwab s, spranger m, hacke w. intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. j cereb blood flow metab 17:500-506, 1997. 15. lees kr, zivin ja, ashwood t, et al. nxy-059 for acute ischemic stroke. n engl j med 354:588-600, 2006. 16. shuaib a, lees kr, lyden p, et al. nxy-059 for the treatment of acute ischemic stroke. n engl j med 357:562-571, 2007. 17. diener hc, lees kr, lyden p, et al. nxy-059 for the treatment of acute stroke: pooled analysis of the saint i and ii trials. stroke 39:1751-1758, 2008. 18. kawamata t, dietrich wd, schallert t, et al. intracisternal basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a molecular marker of neuronal sprouting following focal cerebral infarction. proc natl acad sci u s a 94:81798184, 1997. http://www.ajecr.org/ 242 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(4):242-246 original article evaluation of history taking status and diagnostic and therapeutic measures before applying spine mri mohammad bagher owlia1, fatemeh jaferi1 * 1 department of medicine, shahid sadoughi university of medical sciences, yazd, iran 2 shahid sadoughi university of medical sciences, yazd, iran abstract. according to increasing use of clinical facilities such as mri to diagnose diseases around the world, it is important to know that to what extent these facilities are used based on the existing indications. accurate history taking and physical examination are essential and first step in requesting para-clinical measures. it is also very important in interpreting the spine mri findings in patients with back pain. therefore, it seems necessary to examine physicians' attention to history taking and physical examination before applying mri to enact rules for optimal use of existing facilities. a total of 195 outpatients referred to the imaging center of shahid sadoughi hospital in yazd for spine mri were selected by easily and convenience sampling method. the data were collected using a structured interview. of 195 patients referred, 17.5% (n = 31) and 8.2% (n = 16) reported that the physician was not aware of the main complaint duration and the exact pain localization, respectively, and 31% of patients had not been examined by the physician. based on the results of this research, inattention of some physicians to accurate history taking and physical examination is one of the reasons for aberrant spine mri. accordingly, lack of attention to the absence of clinical signs consistent with the results of mri can create a chain of futile diagnostic and therapeutic measures as well as financial and psychological burden on the patients. the results showed that solving the problem of aberrant requests for mri requires fundamental and comprehensive planning to develop national guidelines, inclusion of these guidelines in educational system of medical students and doctors, encouraging patients to ask physicians about the reason for applying mri and its impact on diagnostic and therapeutic processes, and finally national legislation and insurance to monitor and reduce requests without indications. keywords: medical history taking, physical examination, magnetic resonance imaging, indications introduction more than 84% of adults experience back pain in their lifetime [1, 2]; the back pain is the second leading cause of referral to the physicians [1]. fortunately, most back pain is self-limiting, but there are some conditions defined for patients with back pain that a physician accordingly should request specific clinical measures for the patient. diagnostic tests without clear indications among the population with low pretest probability lead to a series of other tests to assess false-positive outcomes, unnecessary interventions, anxiety in patients and increased costs. the results of imaging requests for back pain are usually misleading or have low sensitivity and much lower value than the accurate history taking and physical examination [3]; hence, these imaging are required to be requested appropriately and based on the indications. currently, aberrant imaging requests are on the rise around the world. according to a study in america, 30%40% of diagnostic imaging is aberrant in this country [8]. in a study by salari et al. in iran in 2012, 56% of spine mri requests were reported aberrant [9]. the number of existing mri devices has been increasing in iran since 2005 [10], and this indicates that the annual volume of the costs associated with the health system is spent for aberrant clinical requests. numerous programs have been implemented and reviewed to solve this problem in different countries, such as training guidelines to physicians on imaging requests [11]. these guidelines are effective when are used properly. therefore, it seems necessary to evaluate history taking status and physical examinations as well as diagnostic and therapeutic measures by physicians before applying mri for future planning purposes. materials and methods in this cross sectional study, 195 outpatients referred to imaging center of shahid sadoughi hospital in yazd, iran for spine mri were selected by easily and convenience sampling method from october 2014 until the completion of the sample members. the data were collected using a ___________________________________________________________ * corresponding author: fatemeh jaferi, m.d f.jaferi.med88@gmail.com). http://www.ajecr.org/ 243 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org table 1 frequency of specialists requesting mri table 2 frequency of patients in terms of pain localization table 3 relationship between doctor specialties and knowledge of the duration of main complaints p-value: 0.708 structured interview. consequently, information was obtained from patients who had minimal memory and verbal ability to answer questions, and the aim of the study was explained to the patients. data were recorded on a checklist prepared based on american college of radiology (acr) guidelines [12]. the patients were asked for information required in history taking (according to the acr guidelines) to request spine mri. the patients were asked about whether or not to bring up these issues with their doctor visit. the examinations performed in different modes easily to remember were asked separately. data were analyzed using the spss 21 software with regard to frequency tables and chi-square test. results of 195 patients referred for performing spine mri, 90 cases (46.2%) were male and 105 (53.8%) were female, 1.2% (n=4) below 20 years, 81% (n=158) aged between 20 and 60 years, and 16.9% (n=33) over 60 years. in terms of education, 37.4% (n = 73) were uneducated, 25.6% (n = 50) were under diploma, 21.5% (n=42) had high school graduate, and 15.4% (n = 30) had university education. in terms of occupation, 47.2% (n = 92) of patients were housekeeper, 6.7% (n = 13) of patients mentioned that their job requires lifting heavy objects and physical activity is high, 23.1% (n = 45) had a job that requires prolonged sitting on chair, and 23.1% (n = 45) had varied and light job; among them, 96.4% (n=188) were insured patients and only 3.6% (n = 7) were uninsured, 88.7% (n=173) were living in urban areas, and 11.3% (n=22)in rural areas. frequency of physicians requesting mri in terms of specialization is shown in table 1. other specialists included neurologists, internists, physical medicine specialists, general surgeons and pain specialists. of the physicians requesting mri, 22.1% (n = 43) were faculty members at the university of medical sciences. of patients, 30.3% (n=59) had duration of complaint less than six weeks and 69.7% (n=136) had duration of complaint over 6 weeks. among the patients with history of main complaint over 6 weeks, 22.1% (n = 43) reported that their pain has intensified over the past six weeks. of patients, 82.5% (n=146) mentioned that the physician was informed during the main complaint, 17.5% (n=31) noted that the doctor did not ask about the duration of complaints and the patient did not also raise this issue, and 9.2% (n=18) did not remember about whether or not to record these issues in history. the frequency of patients according to pain localization is listed in table 2. of the patients, 836% (163) reported that the physician was informed on the exact location of the pain, 8.2% (16) stated that the physician did not ask about the exact location of pain and the patient did not also raise this issue, and 8.2% (16) did not remember about whether or not to record these issues in history. six patients (3.07%) had requested from their doctors to prescribe mri. chi-square test was used to examine the relationship between doctor specialties and knowledge of the duration of main complaints (table 3). according to the results presented in table 3, there was no significant difference in knowledge of the duration of main complaints among different specialists. chi-square test was recruited to study the relationship between doctor specialties and knowledge of the pain localization (table 3). based on the results shown in table 4, there was no significant difference in knowledge of the pain localization among different specialists. the frequency of therapeutic measures of the patients before applying mri is listed in table 5. medication therapy mentioned by patients included the arbitrary or prescribed use of painkillers. of patients, 23.6% (n = 46) mentioned that there were paper or electronic records for them in the visit resulted in request for mri. among the patients, 68.2% (n=133) were undergone mri for the first time, 12.8% (n=25) had once mri earlier because of the same problem and between the two mris were just under conservative therapy. ten patients (4.2%) had the mri between two and four specialist frequency p ercent neurosurgeon 66 33.8 ort hopedist 88 45.6 rhaumat ologist 15 7.7 ot hers 26 12.9 t ot al 195 100 pain location frequency percent back pain 26 13.3 radicular 28 14.4 mix 141 72.3 total 195 100 yes, n (%) no, n (%) t ot al, n (%) neurosurgen 40(80/3%) 12(19/7%) 61(100%) ort hopedist 65(81/3%) 15(18/8%) 80(100%) rhaumat ologist 13(86.7%) 2(13.3%) 15(100%) ot hers 19(90/5%) 2(9.5%) 21(100%) specialist knowledge http://www.ajecr.org/ 244 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org table 4 relationship between doctor specialties and knowledge of the pain localization p-value: 0.879 table 5 frequency of previous therapeutic measures table 6 frequency of important information missing in the history and physical examination table 7 frequency of patients with any of the components of the physical examination performed for them times, and 27 patients (13.8%) had the mri because of same chronic pain several years ago. in terms of other diagnostic measures of the patients before applying mri, plain radiography of the spine had been requested for 7.2% (n=14), for 3.1% (n=6) of laboratory tests, for 2% (n=4) of ncv. more than a laboratory test had been requested for 2% (n=4) and no other diagnostic measures of the patients before applying mri had been performed for 85.1% (n=166). of patients, 5.6% (n=11) had more diagnostic measures based on the history that had not been raised doctor visits and had not been examined (table 6). the frequency of patients with any of the components of the physical examination performed for them by asking the patients are listed in table 7. maximum 6.6% (13) did not remember well examinations conducted for them, which were not included in the result. in total, 31% of patients had not been examined at all. overall, the mean duration of history taking status resulted in the request for mri was 4.05 minutes for each patient and the mean duration of clinical examination was 2.20 minutes (with std deviation of 3.9 and 2.5, respectively). discussion the results of this research showed that 31% of patients had not been examined at all by doctor prior to applying mri. the main complaint duration and the exact pain localization are two fundamental questions in any history that 15.9% and 8.2% of patients had not been asked respectively according to results of this research. however, these figures do not show presence or absence of the indication for mri, but certainly indicate the underlying gap of aberrant requests for mri. although the history and physical examination alone sometimes have shortcomings that do not lead to the correct diagnosis [12, 13], but the significance of the history and physical examination to determine disease is no secret [15-17]. in addition, the lack of history taking and clinical examination may cause the loss of important information, which may affect the prognosis of patients so that 11% of the patients in this study had main complaints that had not raised them with the physician. another challenge ahead is how to deal with mri incidental findings that can also be seen in healthy subjects. judging solely based on these findings without matching with history leads to a chain of other futile diagnostic and therapeutic measures. another reason for excessive requests to mri is its successive repetitions in people who suffer from chronic low back pain and no specific effective treatments is performed on most of them as well as are only treated with painkillers; so that 12.8% of the patients in this study were in this group. mri was the first steps in diagnosis in 85.1% of patients; and this statistic has become thinkable due to its higher cost compared to other methods. of the patients, 3.07% had requested from their doctors to prescribe mri. this rate was 9% in a similar study that was conducted in shiraz, iran [9]. given the high cost of mri, these requests are causing great financial burden on yes, n (%) no, n (%) t ot al, n (%) neurosurgeon 56(90/3%) 6(9/7%) 62(100%) ort hopedist 73(90/1%) 9/9(8%) 81(100%) rhaumat ologist 14(93.3%) 1(6.7%) 15(100%) ot hers 20(95.2%) 1(4.8%) 21(100%) knowledge specialist t reat ment frequency p ercent surgical 7 3.6 medical 165 84.6 p hysiot herapy 12 6.2 epidural inject ion 1 0.5 supplement al med 1 0.5 no t herapy 9 4.6 t ot al 195 100 missed informat ion n (%) urinary incont inence 2 (1.02%) loss of sensat ion and mot ion progressive 5 (2.5%) weakened immune syst em 1 (0.51%) st art ed t raumat ic pain 2 (1.02%) overnight exacerbat ed pain 1 (0.51%) t ot al 11 (5.06%) p hysical examinat ion non-performed n (%) t ot al n (%) walking 181 (97.8%) 185 (100%) evaluat ion of muscle st rengt h, or walk on t he heel and t oe 127 (69.4%) 183 (100%) bending from side t o side, forward and backward 146 (80.2%) 182 (100%) sit t ing down and up 163 (88.1%) 185 (100%) examinat ion in supine posit ion 81 (43.8%) 185 (100%) examinat ion in prone posit ion 141 (76.2%) 185 (100%) examinat ion in sit t ing posit ion on chair 160 (87%) 184 (100%) sensory examinat ion 178 (96.2%) 185 (100%) examinat ion of reflexes 142 (77.6%) 183 (100%) http://www.ajecr.org/ 245 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org insurance companies. it seems necessary to remedy the cultural dimension of undue interference of patients to request for diagnostic and therapeutic measures. the number of existing mri devices has been increasing in iran since 2005 [10]. many of these devices are unnecessary; the question that arises is that whether the large number of facilities encourages physicians to increase request for mri, or vice versa, the large number of requests for mri makes false sense of need for these devices. according to the survey results, the most specialists requesting mri were orthopedists and neurosurgeon. in a similar study, the most requests were from neurosurgeons and orthopedic neurosurgeons [18]. in this study, there were no significant differences among the various specialists in the knowledge of the pain localization and the duration of main complaints. in a similar study, the results were different in the relationship between doctor specialties and the rate of aberrant requests for mri, so that the highest timely request was by the rheumatologists in the research conducted by salari et al. in shiraz [9]. in another study by emery et al., the mris requested by neurosurgeons had the most indications [19]. it seems that further researches are needed to determine the role of specialization in aberrant requests. the mean duration of history taking was 3.9 minutes and the mean duration of clinical examination was 2.5 minutes. however, this rate was recorded approximately through the patients; this rate seems to be much less than the standard duration of history taking and physical examination. numerous programs have been implemented and reviewed to solve this problem in different countries, such as training guidelines to physicians on imaging requests by jayak et al. in 2005, which had no effect in reducing aberrant requests [11]. the other strategy was the refusal of insurance companies to pay for aberrant imaging requests, which had no effect in reducing aberrant requests in other performed studies [20]. at present, the most effective considered method is the use of imaging clinical decision support system that is one of new decisions in the field of healthcare in some countries. many studies have been conducted to evaluate this method, such as a study by black more et al. in 2011 who reported significant reduction in aberrant requests [21]. given that these requests are higher in iran than in other countries, practical and appropriate strategy is more essential to solve this problem. conclusion the results of this study suggest that the large percentage of the spine mri are requested without complete history taking and physical examination, causing enhanced aberrant requests as well as increased financial and emotional burden on health systems, insurance companies and patients. fixing this problem requires a set of actions, including developing national guidelines, inclusion of these guidelines in educational system of medical students and doctors, informing amongst the general public that aberrant requests for para-clinical measures can also be harmful as much as no request, encouraging patients to ask physicians about the impact of these measures on diagnostic and therapeutic processes and finally national legislation and insurance to monitor of reducing requests without indications. conflict of interest the authors declare no conflicts of interest. references 1. deyo ra, tsui-wu y-j. descriptive epidemiology of low-back pain and its related medical care in the united states. spine 12:264-268, 1987. 2. david cj, carroll lj, côté p. the saskatchewan health and back pain survey: the prevalence of low back pain and related disability in saskatchewan adults. spine 23:1860-1866, 1998. 3. us department of health and human services. acute low back problems in adults: assessment and treatment. 1994. 4. use of imaging studies for low back pain: percentage of members with a primary diagnosis of low back pain who did not have an imaging study (plain x-ray, mri, ct scan) within 28 days of the diagnosis. national committee for quality assurance (ncqa). hedis 2014: healthcare effectiveness data and information set. vol. 1. washington (dc): national committee for quality assurance (ncqa); 2013. 5. lehnert be, bree rl. analysis of appropriateness of outpatient ct and mri referred from primary care clinics at an academic medical center: how critical is the need for improved decision support? j am coll radiol 7:192-197, 2010. 6. saadat s, ghodsi sm, firouznia k, etminan m, goudarzi k, naieni kh. overuse or underuse of mri scanners in private radiology centers in tehran. int j technol assess health care 24:277-281, 2008. 7. mayo jr, peter lm. towards clarity: what does inappropriate imaging really mean? canadian assoc radiologists j 5:250-251, 2010. 8. pennsylvania health care cost containment council. the growth in diagnostic imaging utilization. accessed 2007 july. 9. salari h, ostovar r, esfandiari a, et al. evidence for policy making: clinical appropriateness study of lumbar spine mri prescriptions using rand appropriateness method. int j health policy manag 1:17-21, 2013. 10. palesh m, fredrikson s, jamshidi h, jonsson pm, tomson g. diffusion of magnetic resonance imaging in iran. int j technol assess health care 23:278-285, 2007. 11) rao jk, kroenke k, mihaliak ka, eckert gj, weinberger m. can guidelines impact the ordering of magnetic resonance imaging studies by primary care providers for low back pain? am j managed care 8.1:2736, 2002. 12. davis pc(1), wippold fj 2nd, brunberg ja, et al. acr appropriateness criteria on low back pain. j am coll radiol 6:401-407, 2009. 13. ramirez n, flynn jm, hill bw, et al. evaluation of a systematic approach to pediatric back pain: the utility of magnetic resonance imaging. j pediatric orthoped 35: http://www.ajecr.org/ 246 am j exp clin res, vol. 4, no. 4, 2017 http://www.ajecr.org 28-32, 2005. 14. kim hj, suh bg, lee db, et al. the influence of pain sensitivity on the symptom severity in patients with lumbar spinal stenosis. pain physician 16: 135-144, 2013. 15. hall h. effective spine triage: patterns of pain. ochsner j 14: 88-95, 2014. 16. spanjer j, krol b, popping r, groothoff jw, brouwer s. disability assessment interview: the role of detailed information on functioning in addition to medical history-taking. j rehab med 41:267-272, 2009. 17. avoundjian t, gidwani r, yao d et al. evaluating two measures of lumbar spine mri overuse: administrative data versus chart review. j american col radiol 13:1057-1066, 2016. 18. palesh m(1), fredrikson s, jamshidi h, tomson g, petzold m. how is magnetic resonance imaging used in iran? int j technol assess health care 24:452-458, 2008. 19. emery dj, shojania kg, forster aj, mojaverian n, feasby te. overuse of magnetic resonance imaging. jama int med 25:1-3, 2013 20) robinson jd, hippe ds, hiatt md. the effect of a no-denial policy on imaging utilization. j am col radiol 10:501-506, 2013. 21. blackmore cc, mecklenburg rs, kaplan gs. effectiveness of clinical decision support in controlling inappropriate imaging. j am col radiol 8: 19-25, 2011. http://www.ajecr.org/ 223 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(3):223-228 original article hemodynamic changes in exodontia patients using 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine renuka makani 1 , kumar nilesh 2 * 1 school of dental sciences, krishna institute of medical sciences deemed university, karad, india 2 department of oral and maxillofacial surgery, school of dental sciences, krishna hospital, krishna institute of medical sciences, deemed university, karad, india abstract. this study was conducted to evaluate and compare the hemodynamic changes associated with intraoral injection of 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine in patients undergoing dental extraction. a prospective observational study was carried out in patients undergoing tooth extraction. local anesthesia was administered using 4% articaine with 1:100,000 epinephrine to one group (a), while other group (b) received 2% lidocaine with 1:200,000 epinephrine. the parameters that were monitored at four different points of time (in waiting room, immediately after local anesthesia injection, during tooth extraction, and 15 minutes after tooth extraction) included systolic and diastolic blood pressures, pulse rate and oxygen saturation. no statistically significant differences were observed in systolic blood pressure, diastolic blood pressure and pulse rate at any evaluation time between both groups. however, measurement of oxygen saturation showed statistically significant differences at the time of local anesthesia administration and 15 minutes after tooth extraction, with group b showing higher values (p < 0.05). administration of 2% lidocaine with 1:200,000 epinephrine and 4% articaine with 1:100000 epinephrine was safe during tooth extraction procedure with no statistically difference in hemodynamic status between the two groups. only oxygen saturation showed statistically significant difference at the time of local anesthesia administration and 15 minutes after tooth extraction in group b. keywords: exodontia, vital signs, local anesthesia, articaine, lidocaine introduction local anesthesia causes loss of painful sensation from a localized area of body without inducing a loss of consciousness, by reversibly blocking nerve conduction. tooth extraction is one of the most feared dental procedures by patients. use of safe and efficient local anesthetic agent and local anesthesia technique has evolved with time making the procedure more patient friendly. various local anesthetic agents are used to attain local anesthesia such as lidocaine, bupivacaine and mepivacaine. use of lidocaine has been a gold standard for pain control in dentistry. it is an amide local anesthetic first prepared in 1943 by nils lofgren, and was subsequently approved for clinical use by 1948 [1]. articaine is an intermediate duration local anesthetic which was first introduced in 1976 in germany and switzerland, 1983 in canada and in 2000 in united states. use of articaine has become more popular in the recent past. it possesses the ability to diffuse through hard and soft tissues more reliably than lidocaine, thus providing more profound anesthesia [2]. hemodynamic changes during tooth extraction can be attributed to both stresses during the procedure and presence of the vasoconstrictor, epinephrine in the local anesthetic solution. epinephrine is added to the local anesthesia to slow the systemic absorption of local anesthetic thus prolonging the action and intensity of the block. also, the use of vasoconstrictor increases safety by lowering the required anesthetic dose. monitoring hemodynamic changes during extraction allows the dentist to immediately identify situations of increased risk, establish an early diagnosis, prevent possible complications and operate with increased safety. the present study evaluates the hemodynamic changes associated with 4% articaine with 1:100,000 epinephrine, a relatively new anesthetic agent and compares it with 2% lidocaine with 1:200,000 epinephrine. materials and methods the present study was undertaken at exodontia clinics of department of oral and maxillofacial surgery, school ___________________________________________________________ * corresponding author: dr. kumar nilesh (drkumarnilesh@yahoo.com). http://www.ajecr.org/ mailto:drkumarnilesh@yahoo.com 224 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org table 1 data measuring the haemodynamic values and statistical analysis using unpaired t test for equality of means of parameters in group a and group b of dental sciences, karad, india after due approval of the institutional ethical committee. both male and female patients reporting to the department for routine dental extraction and willing to participate in the study were included. hypertensive patients and patients with known cardiovascular disorders were excluded from the study. patients were randomly divided into two groups: group a included patients undergoing tooth extraction using local anesthetic injection of 4% articaine with 1:100,000 epinephrine. group b included patients undergoing tooth extraction under local anesthetic injection of 2% lidocaine with 1:200,000 epinephrine. dental extractions were carried out in a relaxed atmosphere, with no anxiolytic premedication. on the day of the extraction the patients were asked to have a light breakfast. the indications for dental extraction were caries, periodontitis and combined caries and periodontitis. the patients were monitored for diastolic blood pressure (dbp) and systolic blood pressure (sbp), pulse rate and oxygen saturation (spo2). blood pressure (bp) was measured with sphygmomanometer (diamond, india). the cuff was placed on the right arm with the patient sitting in the dental chair and bp recorded. oxygen saturation was measured using pulse oxymeter (skanray healthcare pvt. ltd, india ® ), applied to left index finger, that was made clean and free of nail varnish. these hemodynamic parameters were recorded in the waiting room before the injection (t1), immediately after local anesthetic injection (t2), p aramet ers t ime group no. mean sd t st at ist ic p value a 25 125.28 7.591 b 25 125.48 8.559 a 25 120.72 7.808 b 25 124.32 9.945 a 25 122.88 7.44 b 25 124.56 9.408 a 25 122 8.145 b 25 124.72 8.6 a 25 81.04 6.113 b 25 84.28 6.779 a 25 80.8 7.979 b 25 84.16 9.45 a 25 80.72 7.231 b 25 83.52 8.471 a 25 79.68 9.141 b 25 83.44 6.893 a 25 79 11.993 b 25 80 9.613 a 25 80.8 14.79 b 25 80.72 12.857 a 25 79.16 13.594 b 25 78.84 10.459 a 25 79.6 13.766 b 25 78.2 9.704 a 25 97.88 1.269 b 25 98 1.354 a 25 97.64 1.35 b 25 98.44 1.446 a 25 97.92 1.115 b 25 98.24 1.508 a 25 97.36 1.35 b 25 98.44 1.53 oxygen st urat ion in wait ing room -0.323 0.748 at t ime of local anaest het ic inject ion -2.022 0.049 during t oot h ext ract ion -0.853 0.398 15 mins aft er t oot h ext ract ion -2.647 0.011 p ulse rat e in wait ing room -0.325 0.746 at t ime of local anaest het ic inject ion 0.02 0.984 during t oot h ext ract ion 0.093 0.926 15 mins aft er t oot h ext ract ion 0.416 0.68 diast olic blood p ressure in wait ing room -1.775 0.082 at t ime of local anaest het ic inject ion -1.358 0.181 during t oot h ext ract ion -1.257 0.215 15 mins aft er t oot h ext ract ion -1.642 0.108 syst olic blood p ressure in wait ing room -0.087 0.931 at t ime of local anaest het ic inject ion -1.424 0.161 during t oot h ext ract ion -0.7 0.487 15 mins aft er t oot h ext ract ion -1.148 0.257 http://www.ajecr.org/ 225 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org figure 1. mean systolic blood pressure (in mm hg) in patients under 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine at 4 different evaluation times; in waiting room (t1), at time of local anesthetic injection (t2), during tooth extraction (t3) and 15 minutes after tooth extraction (t4). bars demonstrate mean in each group. figure 2. mean diastolic blood pressure (in mm hg) in patients under 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine. during tooth extraction (t3), and 15 minutes after tooth extraction (t4). regional local anesthesia was provided using 4% articaine with 1:100,000 epinephrine (group a) and 2% lidocaine with 1:200,000 epinephrine (group b). aspiration in two planes was done and then the solution was deposited slowly, to confirm that the anesthetic solution was not directly injected into the bloodstream. a maximum of 3 ml solution was injected. the data collected was subjected to statistical analysis. all the values were analyzed for mean, standard deviation, errors and range. unpaired t test, anova and tukey’s multiple comparison post hoc test were used for evaluation of statistical significance between the two groups. results these patients were studied for hemodynamic changes after the administration of local anesthesia at various points of time. group a included 25 patients in age range of 20-84 (mean 56.24), with 15 males and 10 females. group b included 25 patients in age range of 25-75 (mean 51.20), with 15 males and 10 females. measurement of hemodynamic parameters showed no hypertensive peaks in the measurement of sbp and dbp at figure 3. mean pulse rate in patients under 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine. figure 4. mean oxygen saturation in patients under 4% articaine with 1: 100,000 epinephrine and 2% lidocaine with 1:200,000 epinephrine. table 2 repeated measure anova test for group a table 3 tukey’s post hoc test for systolic blood pressure syst olic blood pressure 10.627 <0.0001 diast olic blood pressure 1.076 0.3649 p ulse rat e 61.053 0.2384 oxygen sat urat ion 2.143 0.1023 p aramet ers f p value time 1 time 2 mean difference p value at time of local anaesthetic injection 7.729 <0.001 during tooth extraction 4.068 <0.05 15 mins after tooth extraction 5.559 <0.01 in waiting room http://www.ajecr.org/ 226 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org table 4 repeated measure anova for group b any evaluation time. the type of local anesthetic slightly affected diastolic and systolic bp during different measurement periods. however, this variation did not achieve statistical significance (p > 0.05; figs. 1 and 2, table 1). in both groups, pulse rate varied slightly during different measurement periods, however, this variation did not achieve statistical significance (p > 0.05; fig. 3, table 1). measurement of oxygen saturation showed statistically significant differences at time of local anesthesia administration and 15 minutes after tooth extraction, with group b showing higher values (p < 0.05; fig. 4, table 1). repeated measure anova test was done to study the equality of means of four parameters for group a and group b. as with any anova, a repeated measure anova, tests the equality of means. however, a repeated measure anova is used when all members of a random sample are measured under a number of different conditions. as the sample is exposed to each condition in turn, the measurement of the dependent variable is repeated. for group a, it revealed that, there was significant difference for systolic blood pressure (table 2). tukey’s multiple comparison post hoc test was used to find out in which measurement period, difference was there. results showed that systolic bp at t2, t3, & t4 was higher, than at t1 (table 3). there was no significant difference for diastolic blood pressures, pulse rate and oxygen saturation. for group b, it revealed that, there was no significant difference for sbp, dbp, pulse rate, and oxygen saturation (table 4). alternatively all these four parameters passed the test of equality of means. discussion given the number of local anesthetic agents available in market, it has becomes necessary to investigate the efficacy of these drugs in order to develop a favorable clinical practice. properties of these anesthetic drugs vary depending on their molecular structure. increased diffusiblity of articaine is attributed to its liposolubility and greater plasma protein binding, which can be due to the thiophenic ring and an additional ester ring when compared to traditionally used lidocaine. this property of articaine has led to its increased popularity in past few years. monitoring hemodynamic changes while administering local anesthesia, is essential so as to ensure safety of the patient. this will provide a continuous evaluation of patient’s condition on the chair so that in case of emergency, immediate action can be taken. these fluctuations apart from the vasoconstrictor’s effect can also be attributed to other patient related factors like gender, age, anxiety level, systemic condition. no significant adverse reactions have been previously reported during the usage of these local anesthetics. although, certain adverse events associated with the use of articaine with epinephrine were reported such as edema of lips, headache, soreness, trismus, paraesthesia, swelling [3, 4]. some researchers have also indicated a higher risk of neurosensory disturbance with the use of 4% articaine than with other drugs in use (mainly in mandibular block anesthesia) [5-9]. morais et al. [10] in 2012 conducted a study to analyze hemodynamic changes following the administration of either 2% lidocaine or 4% articaine (both with epinephrine 1:100,000) in the surgical removal of lower third molars. the results showed no hypertensive peak in systolic blood pressure, diastolic blood pressure and mean blood pressure. a study by perusse r et al. [11] suggested that hemodynamic changes can also depend on the dose of injected vasoconstrictor. therefore, variations should be expected if injected technique is not performed carefully and in case the solution gets accidentally injected into a blood vessel [12]. according to silvestre fj et al. [13], no significant hemodynamic changes were observed in well controlled hypertensive patients that can be attributed to the presence of vasoconstrictor in the local anesthesia. a study conducted by aurelia am et al. [14] on the hemodynamic changes during the surgical removal of lower 3rd molars showed that the fluctuations can also be related to anxiety which was found to be higher in females but these changes were not statistically significant. also, no significant changes were observed with the parameters consideredsystolic bp, diastolic bp, oxygen saturation. in this study, group a (articaine) and group b (lidocaine) patients did not show statistically significant changes in blood pressure and pulse rate at various point of time during tooth extraction. while for oxygen saturation, there was significant difference between group a and group b at time of local anesthetic injection and 15 minutes after tooth extraction. no hypertensive peak was observed in the measurement of systolic and diastolic at any evaluation time. moreover, the type of local anesthetic slightly affected diastolic or systolic blood pressure during different measurement periods. however, this variation did not achieve statistical significance. in both groups, pulse rate varied slightly during different measurement periods, however, this variation did not achieve statistical significance. according to some authors, oral surgical procedures are very likely to induce patient stress, resulting in the release of endogenous catecholamines, hence giving rise to small hemodynamic fluctuations rather than epinephrine associated with local anaesthesia [15-17]. but, a study conducted by meilleret al. [18] on blood pressure fluctuations during oral surgical procedures in hypertensive patients, showed no such correlation between patient stress and the changes associated in hemodynamic variables. some authors 19-22 have also stated that the amount of epinephrine that is used in local anesthetic formulation exerts a cumulative effect with plasma catecholamine levels but this phenomenon would not be sufficient to induce any major hemodynamic changes in parameters f p value systolic blood pressure 0.5699 0.6366 diastolic blood pressure 0.7106 0.7106 pulse rate 0.0973 0.0973 oxygen saturation 0.2274 0.2274 http://www.ajecr.org/ 227 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org young and healthy individuals. however, risk of complications increases due to this mechanism in patients suffering from cardiovascular disease. this necessitates systemic monitoring of such patients [23-26]. conclusion administration of 2% lidocaine with 1:200,000 epinephrine and 4% articaine with 1:100,000 epinephrine was safe during tooth extraction procedure with no statistically difference in haemodynamic status between the two groups. only oxygen saturation at time of local anesthesia administration and 15 minutes after tooth extraction was higher in group b. with statistically significant difference. conflict of interest the authors declare no conflicts of interest. references 1. malamed sf, gagnon s, leblanc d. efficacy of articaine: a new amide local anesthetic. j am dent assoc 131:635-642, 2000. 2. mikesell p, nusstein j, reader a, beck m, weaver j. a comparison of articaine and lidocaine for inferior alveolar nerve blocks. j endod 31:265-270, 2005. 3.silva lc 1 , santos td, santos ja, maia mc, mendonça cg. articaine versus lidocaine for third molar surgery: a randomized clinical study. med oral patol oral cir bucal 17:e140-145, 2012. 4. adewumi a, hall m, guelmann m, riley j. the incidence of adverse reactions following 4% septocaine (articaine) in children. pediatr 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https://www.ncbi.nlm.nih.gov/pubmed/?term=stanton-hicks%20m%5bauthor%5d&cauthor=true&cauthor_uid=8448101 228 am j exp clin res, vol. 4, no. 3, 2017 http://www.ajecr.org 25. sack u, kleemann pp. intraoral conduction anesthesia with epinephrine-containing local anesthetics and arterial epinephrine plasma concentration. anesth pain control dent 1:77-80, 1992. 26. tolas ag, pflug ae, halter jb. arterial plasma epinephrine concentrations and hemodynamic responses after dental injection of local anesthetic with epinephrine. j am dent assoc 104:41-43, 1982. http://www.ajecr.org/ 440 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2022;9(1):440-446 review article migraine headaches as a preliminary symptom for cognitive decline ahad hasan syed hasani*, melika faani shahid beheshti university of medical sciences, student research committee, iran abstract. migraine headaches are amongst the most prevalent neurological disorders. dementia on the other hand, is classified as a neurodegenerative disease with symptoms of slow progressive memory loss and cognitive decline. identifying the preliminary signs and symptoms of such neurological diseases are crucial in preventing the onset of such disease. in this review article, we analyzed several studies signifying and or contradicting the claim of migraine headaches as a preliminary symptom of dementia. this can ultimately aid in focusing specific treatments to reduce the risk of patients developing dementia. databases including google scholar, pubmed, and ncbi were searched by using keywords including migraine, dementia, association, symptom, etc. this study analyzed articles that were sought through various databases. several studies signified a positive correlation between the two diseases. studies that contradicted this claim, were vastly outnumbered by studies that depicted the claim to be true. it was eminent that preventing and treating migraine headaches in patients with a high-risk profile of dementia should be considered and studied more extensively. keywords: migraine, dementia, cognitive decline, neurodegenerative disease, headache introduction headaches, affect 46% of the world population [1]. they are considered as the leading cause for outpatient and emergency department visits [2]. migraine headaches are defined as headaches lasting four to 72 hours with symptoms including unilateral location; pulsating quality; moderate to severe intensity; intensified by physical activity; occurrences of nausea, vomiting, phonophobia, and or photophobia [3]. the epidemiological burden of this disease has been observed to have a greater portion allocated to women. in a study conducted by rebeca et al. a significant 2-fold difference was present when comparing females and males that showed symptoms consistent with migraine [4]. migraine headaches were also observed to have a maximum 33% prevalence in asian countries as compared to 9% in western pacific countries such as china [3]. the variable prevalence in certain areas can be denotive of the cultural and dietary attitudes effective in causing migraine headaches. the third most prevalent disorder that was suggested by the global burden of disease study conducted in 2010 was migraine headaches. currently, it is suggested that 35 million american are victims of the mental disorder and suffer from migraines in a variety of ways [2]. migraine headaches the pathophysiology of migraine headaches has advanced over the previous decades. current studies now point out peripheral nerve compression and or traction as the etiology of the disease. these compressed nerves can be found throughout the head and even the neck region. migraine, can be considered an inherited, episodic disorder that is assisted with sensitivity of the ear and light receptors. the common symptoms and complaints include a throbbing headache, nausea, and vomiting. dizziness and light headedness can also be noted in patients as many complain having a feeling of being drunk or have stepped off a boat. the nervous pathways allocated to cerebral pain include the ophthalmic branch of the trigeminal nerve (cn v), a sensory nerve that receives input from the orbital region. the facial nerve also consists of sensory fibers that perceive pain from the maxillary area [5]. dementia dementia, a general term which incorporates several diseases that cause a loss of function or cellular death in the central nervous system (cns).as studies suggest, the behavior, memory and even personality of the patient are susceptible to change. finding the main etiology and or the reason for such neurodegeneration can aid in treating the patient in a proper and rapid manner, limiting the adverse effects it could have on the patient. the chances of reversing the neurodegeneration and trauma in dementia is minimal and can be allocated to 9-11% of cases [6]. there are several forms of dementia, most of which are based on their primary cause, which include alzheimer’s disease, vascular dementia, frontotemporal dementia, dementia with lewy bodies, and mixed dementia [7, 8]. all ___________________________________________________________ * corresponding author: dr. ahad hasan syed hasani (ahad.sbmu@gmail.com) http://www.ajecr.org/ 441 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org types of dementia ultimately lead to neurodegeneration and an inability to fully use one’s cognition. memory loss, personality change and an inability to properly process environmental phenomena are severe symptoms of dementia which if not treated can possibly lead to a shorter life time [9-11]. the association between migraine headaches and dementia has been focused on in many studies. however, a vague conclusion for migraine headaches as a preliminary symptom of dementia still exists as many factors must be taken into consideration [12]. in this study, we review the existing literature for commonalities and relations between both mental discrepancies. materials and methods search strategy included identifying mesh terms and keywords (i.e., migraine, dementia, association, symptom, etc.) and conducting an advanced search in databases such as google scholar, pubmed, and ncbi. results were screened based on criteria and were selected for data extraction. results and discussion migraine is considered to be a recurrent primary headache disease [93]. several studies have demonstrated that migraine prevalence can range from 6 % to 13%. this headache disorder affects nearly 18% of the women population as well as 6% of the male population [41]. furthermore, it has a pulsating attribute and usually lasts from a couple hours to 3 days [94]. migraine can cause vomiting, nausea, sensitivity to smell and light as well as aura [95, 96]. also, physical activity has been observed to worsen the situated pain [97]. this as a result, leads to limitations in a vast variety of activities, ranging from everyday chores to profession related tasks. moreover, approximately 12% of individuals suffer from migraine, including 6% of men as well as 18% of women [98]. in other words, it is the third most common and prevalent global disease [108]. nevertheless, both environmental and genetic factors trigger migraine [99]. it can be induced by hormonal changes in the body, for instance, girls tend to have more migraines after puberty as significant hormonal changes are eminent [107]. it is estimated that migraine is seen more in women as compared to men [100, 101]. nonetheless, medications such as beta blockers, topiramate and sodium valproate are efficient in decreasing the pain [102]. the tension type headache (tth) is considered the most prevalent kind of primary headache which comes along with mental tension and pain in the cerebral area. also, this type of headache is not correlated to physical activity as it does not change when heart rate, blood pressure and or endocrine stimulation occurs [13]. this is true whilst the intensity of migraine is more severe than tth [14]. on the other side recent evidence demonstrate an association between headache and dementia, while most studies are circulated around migraine [15-22]. dementia is considered to be one of the most prevalent types of neurodegenerative diseases that has a huge effect on the quality of life [23]. its symptoms are variable and include slow memory impairment, headache, and change in personality etc. [24]. nearly 24.3 million people have been diagnosed with dementia globally, this number is expected to jump to 81.1 million people by the year 2040 [25]. there are several kinds of dementia that have been identified. amongst them, non-vascular dementia (such as alzheimer's disease (ad) is known to be the most common type, which makes up 80–90% of present cases [26, 27]. the risk of dementia increases with age, a variety of comorbidities, including hypertension, diabetes, dyslipidemia, heart failure (hf), ischemic heart disease (ihd), chronic obstructive pulmonary disease (copd), head injury, depression, parkinson’s disease and stroke [2832]. the association between somatic symptoms, like headaches, and clinical symptoms, is assumed to be more than a simplistic coincidence [33]. several cohort studies have previously suggested a connection between migraine and dementia [34, 35]. nonetheless, the results of other studies on migraine and cognitive disorders were controversial and mixed, with some of them failing to find any direct association. in most countries, alzheimer is assumed to be the most common cause of dementia in senior citizens [36]. women tend to contract alzheimer’s disease approximately two times as much as men do. which is induced by the fact that women tend to have much more life expectancy than men [37]. numerous analyses imply that incidence rates are higher in older patients. neuroprotective precautions along with estrogen effect on b-amyloid formation and basal forebrain cholinergic neurons appear relevant to the treatment as well as alzheimer’s disease prevention. there is insignificant clinical support concerning whether estrogen has a part in treating alzheimer or not, such as a year-long research conducted over 120 hysterectomized ladies in united states, where the interventions were unopposed conjugated equine estrogen [38] and or a 7-month research of 117 females in france, where the intervention applied was transdermal estradiol along with, oral micronized progesterone [39]. primary results of whims published in 2003 signified dementia as primary cause [40]. nevertheless, migraines increase the risk of brain lesions statistically [42]. more recent research has associated brain lesions to incidence of cognitive decline and dementia. as a result, migraines might signify a positive correlation and a progressive brain disease [43]. several studies have also contradicted such, and have shown no significant link between migraine and dementia [44, 45, 46]. this is, while others, proved cognitive function to decline amongst individuals with migraine [47, 48, 49]. a study from a valid national database showed that migraines end up in a higher risk of developing dementia in future by adjusting for hypertension, diabetes, depression and head injury, cad, all of which, are able to boost the risk of dementia in patients suffering from migraine [50]. in asia, migraine headaches are the most common kind of headache that is diagnosed in walk in clinics [51]. the prevalence of migraine headache is 6.5% in men and 18.2 % in women in the usa, as well as 7.5% and 16.8 %, respectively in europe [52, 53]. people with migraine exhibit a high risk of vascular diseases due to the fact that migraine headaches, in nature, are a neurovascular dysregulation disorder [54, 55]. nevertheless, http://www.ajecr.org/ 442 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org a theory about migraine mechanism demonstrates that the abnormality in electrophysiological activities of the cortex is of great importance in migraineurs [56, 57]. epilepsy is another disease associated with the brain function, which is more severe than migraine, with a 0.5-1% prevalence rate globally [58, 59]. seemingly, epilepsy and migraine have been assumed to be comorbid diseases for decades [60, 61]. it is estimated that 46 million individuals have dementia worldwide [62]. as the population ages, incidence rates of dementia may continue to rise [63]. however, there is no effective treatment for this condition. investigations have shown that migraine affected approximately 1.04 billion people worldwide [64]. the overlap of biological mechanisms between dementia and migraine, including brain microstructural changes, neuro-inflammation, and subcortical white matter disorders [65-68]. this shows that migraine might elaborate the risk of dementia. on the other hand, recent studies that claim a link between dementia and migraine demonstrated inconsistent connections [69, 70]. although many researches have suggested a possible association between dementia and migraine, there are concerns that these links be due to unmeasured, confounding or diagnostic bias. although, the mgrs was significantly related to migraine (p < 0.0001), there was not any association between migraine and dementia [71]. nevertheless, many studies found variable results such as chabriat et al. [72] in a case study, where 8 cases of dementia were collected as case reports and were analyzed to signify various symptoms. all of 8 patients were related as primary, secondary, or tertiary family. each patient had separate diagnosis at various times and were cared for separately. case reports from all 8 patients were summarized and compared in terms of symptoms such as headaches, fever, neurological disorders and etc. this study specifically focused on the outcome of common symptom findings amongst many patients. the migraine headache was observed in 6 of 8 cases, which was included using ihs classification criteria for the disease. the patients were remarkable for the great frequency of throbbing headaches. in all but one patient focal neurological symptoms were present preceding headaches, gradually developing over a course of 5 to 15 minutes. 7 of 8 patients were found to have nausea and vomiting alongside their headaches. thus, such symptoms were seen in almost all of 8 patients with diagnosis of dementia. migraine headaches were noted as a cause for many of their symptoms including nausea, vomiting as well as neurological defects [72]. finsterer [73], in a review article sought to observe cognitive decline alongside mitochondrial disorders. respiratory chain disorder (rcd) was the prominent subject of discussion and was analyzed in different aspects. various symptoms including dementia, ataxia, migraine headaches and etc. were observed in most cases. rcd was prominent alongside other diseases such as leigh syndrome, ks syndrome, and melas. as discussed in the review article, rcd diagnosis was based off symptoms such as cognitive impairment, neurological impairments, as well as dementia. a major tip off symptom for rcd diagnosis was considered to be migraine headaches with improper and short stature. a vague but present bond was noted between dementia and migraine headaches [73]. the most common cause for dementia is alzheimer’s disease [74]. thus, the comorbidity profile in dementia with lewy bodies (dlb) compared to alzheimer’s disease was studied by fereshtehnejad et al. in a detailed format [17]. information and data were sourced from the swedish dementia registry as well as the national patient registry. this linkage study over saw 634 patient profiles with dlb and 9161 patient profiles diagnosed with ad between the years 2007-2021. migraine diagnosis as a symptom was defined according to icd version 10 codes. taking into factor the timing of events, migraine and depression were more commonly significant preliminary to the diagnosis of dlb. migraine headaches were seen more prominently in the dlb group as compared to the ad group. however, a minimal amount of 6 patients of the 634 were seen to have such headaches. alongside migraine headaches, depression, stroke, and cerebrovascular infarctions were noted simultaneously. consecutively, post diagnosis onset of migraine headaches was noted in none of the 634 patients. the correlation between migraine and dementia was observed in the dbl group. however, the strength of correlation can be debated as only 1 in 100 patients were considered to have migraine headaches before dementia onset [75]. similarly, lee et al. conducted a case-control study on 11,438 dementia patients. diagnostic criteria were based off international classification of disease-10 (icd10f00). patients aged 60> were considered for the study due to ethical research purposes. the results signified that the average duration from significance of migraine until dementia diagnosis was 58.6 months compared to 60.8 months in the control group. respectively, the rate of occurrence for migraine diagnosis preliminary to dementia was higher in the cases (7.7%) as compared to the control (6.3%) group. like other studies conducted, it was also seen that dementia diagnosis was greater in women than in men. it was observed that the risk of dementia was higher in patients that were diagnosed with migraine headaches after matching for age, sex, income, region of residence and past medical history. women above the age of 70 showed the greatest association between migraine and dementia. it was concluded that dementia rates could possibly be decreased by controlling migraine headaches before onset of dementia symptoms [76]. in a meta-analysis between 1966 and 2004, migraine headaches were proven to be a risk factor for ischemic stroke [77]. white matter hyperintensities were seen to be more prevalent in patients diagnosed with migraine headaches as compared to the general population [78-81]. various studies showed migraine to have a negative effect on cognitive skills including attention, verbal ability, memory and psychomotor ability [81-87]. on the other hand, certain studies proved no correlation between migraine and cognitive decline and thus contradicting the argument [81, 87-92, 103]. another population-based study that was noted by koen was significant of no difference in cognitive tests on 536 migraineurs [81, 103]. as seen in most studies, dementia and migraine can be allocated to cadasil as an intermediating factor. the http://www.ajecr.org/ 443 am j exp clin res, vol. 9, no. 1, 2022 http://www.ajecr.org thickening of arteries can cause migraines, leukoencephalopathy, small deep and subcortical infarcts and ultimately lead to dementia [104, 105]. recurrent migraines can be seen in mitochondrial myopathies, encephalopathy, lactic acidosis and stroke like episodes. mitochondrial disorders such as leber’s hereditary optic neuropathy, myoclonic epilepsy are also significant for migraine headaches [105, 106]. research by le pira et al. [105] signified that migraineurs had a decrease in shortand long-term memory recall. the migraine groups self-reported problems with concentration, comprehension, and communication shortly after an onset of their migraine headaches. signifying mild symptoms of dementia [105]. in a population-based study using data from the nordtrondelag health surveys conducted between the years 1995-1997 (hunt2) and 2006-2008 (hunt3) the relation between migraine and dementia was analyzed by røttereng et al. in the study conducted years ago, questionnaires consisting of more than 200 health-related items were presented to citizens of nord-trondelag county in norway. using the dementia registry, 1332 dementia patients were distinguished and identified. diagnostic criteria for dementia were the same as national and international standards. after adjusting for age, gender, education, smoking, hospital anxiety and depression scale (hads) and severe comorbid conditions, headaches were more persistent and more likely to be reported in hunt2 from those who later on were associated with the dementia registry. this signified a direct and positive correlation between dementia and preceding headaches. it was also discussed that it was less likely for someone to report headaches that later were confirmed non-demented. in their other studies including 378 cases of dementia, patients with headaches proved to have higher risks for dementia onset [107, 108]. multiple silent or sub-clinical lacunar strokes are amongst the findings in patients diagnosed with dementia. a hypothesis presented by arnold eggers, signified that a similar presence of silent cryptogenic strokes were seen in migraine patients. thus, pointing out another relationship between the 2 diseases [109]. in a similar review article, the relationship between dementia and migraine was sought. the researchers deduced that migraines with or without aura can ultimately lead to cognitive decline. it was noted that the frequency of 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"1. migraine". ichd-3 the international classification of headache disorders 3rd edition. retrieved 22 october 2020. http://www.ajecr.org/ https://doi.org/10.1111%2fj.1468-1331.2006.01184.x 396 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2020;7(2):396-404 review article viral respiratory infection in people with obesity – a review mohammad ali saghiri1, 2 *, chun kai tang3, ali mohammad saghiri4 1department of restorative dentistry, rutgers school of dental medicine, nj, usa 2department of endodontics, university of the pacific, arthur a. dugoni school of dentistry, san francisco, ca, usa 3rutgers biomedical and health sciences, nj, usa 4sector of angiogenesis regenerative medicine, dr. hajar afsar lajevardi dental material and devices group (admd), hackensack, nj, usa abstract. this review article focused on a statistical analysis of the obesity population’s viral infection rate and compared the effects from different viral respiratory infections, such as abnormal immune responses and hyperglycemia in the population with obesity. obesity has become more prevalent in developed countries while still less in developing countries. people with obesity have higher adipose fat accumulation underneath the organ membrane and in-between tissues, which can lead to abnormal organ function and respiratory difficulties. respiratory problems can exist in people with obesity that usually related to hypertension and dyslipidemia. meanwhile, obesity downregulates many immune and metabolic responses in the body. viruses like h1n1, sars-cov, and sars-cov-2 have pathological damages in the lung shared in common, in which a higher severity was observed in people with obesity. the review was done by an online article search from pubmed, google scholar, and nemj database to allocate potential case studies and review articles of our keyword criteria. the initial article search layout 206 articles related to obesity, respiratory, and virus in human studies carried out from 2000 to 2020. further analysis has shown that 15 articles pointed out a clear relationship between obesity and viral infection. influenza, mers, and sars coronaviruses reported a significantly higher infection rate in the population with obesity condition. however, there were inconsistent results regarding the relationship between obesity and pneumonia, in which pneumonia was observed in most viral respiratory infections. comparing pathological evidence from obesity and sars viral infections, both share in common on hyperglycemia and increase of ace engagement, whether it is to increase blood pressure or increase virus entry. obesity condition itself makes consequent treatments even more difficult. in suggestion, there is a need to conduct further extensive clinical studies on the people with obesity that are covid-19 positive. keywords: influenza, obesity, respiratory, sars, virus introduction in 2016, the center for disease control and prevention (cdc) reported more than 39.8% of adults in the u.s. to have an obesity problem [1]. in addition, people with obesity are highly correlated with cardiovascular disease, type-ii diabetes, and metabolic syndrome [2]. metabolism is highly related to the quantity of energy consumed and how much the body required [3]. when the body’s metabolism function is impaired, individuals may have a higher risk of getting chronic diseases due to leukocyte distribution problems and increase the production of leptin hormone that triggers abnormal innate and adaptive immune responses [4]. therefore, an abnormal increase in immune responses promotes inflammation and sclerosis of cells from bacterial and viral infections by promoting immune cell production [4]. as for severe viral infections in the respiratory tract, higher body weight profiles have led to a higher risk of systemic failure and further require a mechanical ventilator to survive [5]. metabolic syndrome (mets) occurs when obesity disrupts the functions of metabolic tissues, such as adipose tissue, liver, and pancreas [3]. as the oxidative stress and dysfunction of the metabolic tissue progress, lymphoid tissues were also altered by the abnormal cytokine production and further cause problems in leukocyte distribution that can affect immune surveillance. this disruption in the immune system will re-occur in a cycle and eventually leads to chronic diseases such as type ii diabetes and vasoconstrictions [3]. metabolism must be in balance; otherwise, the body will induce abnormal adaptive and innate immunity, which leads to pro-inflammation and cause systemic failures that are likely to get external infections. leptin acts as the crucial hormone that controls the metabolism of energy source into lipids and cytokine production in the immune system [4]. as in people with obesity, leptin functions were resistive to be controlled by brain signals and continue making lipids, which increase the ___________________________________________________________ * corresponding author: mohammad ali saghiri d.eng., ms, phd (mohammadali.saghiri@rutgers.edu) http://www.ajecr.org/ mailto:mohammadali.saghiri@rutgers.edu 397 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org feeling of starvation, upregulate food consumption, and eventually lead to hyperglycemia [4]. the overproduction of fatty acids was stored in adipocytes and distributed around the lung, heart, and other cardiovascular organs. furthermore, higher lipid content in the lungs and bronchioles occupy the lung chamber, which further restricts the respiratory volume [6]. besides, people with obesity may show signs of hard breathing before the actual viral infection due to their systemic oxygen volume requirement is higher than healthy people. the change in the lung environment affects the viral pathogenesis and immune response, which makes people with obesity problem prone to more extensive viral spreads [7]. due to the reduction of immune surveillance in the human body, the respiratory tract as the first line of the immune barrier gets the most immediate symptoms after infection [8]. the range of symptoms includes coughing, sore throat, fever, hard breathing, and even diarrhea. furthermore, patients with obesity have a slower response to fight viral infection and weak recovery from the disease. in the study from melo l. et al. [9], individuals with obesity show reduced lung volume compared to individuals with normal weight. since there is a restriction in respiratory volume, individuals with obesity show symptoms listed above. according to a recent study by green et al. [10], people with obesity have less of a response to the flu vaccine. this issue is due to changes in the metabolic profile of t cells that impairs the activation and function of adaptive immunity [10]. with chronic illnesses, such as cardiovascular disease, respiratory difficulties, and heart disorders, individuals with obesity need specific health guidelines to prevent them from getting severe bacterial and viral infections. in addition, frontline physicians and nurses can rely on the medical information of the patient’s weight profile to categorize the risk severity and perform the most effective treatment for patients attend for health care with symptoms shown. during the h1n1 pandemic in 2009, obesity was an independent risk factor for disease severity [11]. however, obesity did change the individual’s interferon (ifn) response, which then causes a lower response rate when infection of the virus enters the human body. therefore, people with obesity shows the potential risk of respiratory viral infection due to its attenuation in ifn property. for the infection of middle east respiratory syndrome, the viral engaging point utilized dpp-4 on the epithelial tissues, similar functionality as sars-cov’s engagement with ace-2 on the epithelial cell surface [12]. when comparing sars-cov to sars-cov-2, they shared similar receptor binding motifs and bind to ace-2, which is release in high amount by the obesity’s effect [13]. both dpp-4 and ace-2 increased in amount while an individual’s bmi increases, which is highly related to obesity condition [14]. currently, the number of populations globally who tested positive on covid-19 surpassed 3 million by may 2020 [15]. it’s an ongoing global health emergency, and now, not enough information on covid-19 infected population has a conclusive answer for the relationship between obesity and covid-19 in each country. besides, if we look at the data of hospitalized patients, most of the reported data suggest people with obesity might be at higher risk of getting severe symptoms and requires mechanical ventilators to survive [16, 17]. the review was performed and discussed the potential relationship between individuals with obesity and respiratory issues from different viral infections. further analysis focused on checking for the prevalence of severe viral respiratory diseases and immune system alterations from the people with obesity that were diagnosed and hospitalized with covid-19, mers, or h1n1 viral infections. materials and methods purpose of review the review was conducted to compare different viruses’ effects from statistical view and pathological evidence in people with obesity to provide potential disease information for clinical studies to deal with the unknowns of covid19. the goal of the review was to find relevant pathological evidence that speaks to a definite cause of higher infection rates for covid-19 in people with obesity. in addition, people with obesity might have a higher risk of immune dysfunction and potentially get infected with severe respiratory diseases. furthermore, successfully categorizing risks can save future diagnosis time and prescribe more accurate treatment of the illness inclusion and exclusion criteria inclusion criteria: 1) the study used keyword combinations of obesity, respiratory, and virus to conduct an extensive search for articles in online databases. 2) selected studies are human studies carried out between the years 2000 to 2020. 3) include reviews on other factors for infection of covid-19. 4) selected articles in the english language only. exclusion criteria: 1) exclude articles that are not in the time range. 2) exclude articles of non-human studies. 3) exclude articles that are not in the keyword of selection. search methodology the search for articles must meet our specific criteria, including using mesh and searching on pubmed to allocate correlated articles to our goal correctly. by inputting obesity, respiratory, and virus into mesh, the initial result provides 206 articles within our search criteria. search strategy further analysis of article choice was performed to pick out 15 articles with firm supporting details to our goal. our search has narrow down to focus on the effects of viral infection in population with or without obesity conditions and compare the immune responses between people that are healthy and people with obesity. as for covid-19, the reported data collected from hospitalized patients were also analyzed in this review and provide crucial information for future studies. table 1 http://www.ajecr.org/ 398 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org relationship of obesity to viral respiratory infection in each study title of article type of study types of viral infection relationship between obesity and viral infection investigating obesity as a risk factor for influenza like illness during the 2009 h1n1 influenza pandemic using the health survey for england case study h1n1 influenza no significant relationship obesity as a risk factor for severe influenzalike illness. influenza other respir viruses case study h1n1 influenza little to moderate relationship obesity and respiratory infections: does excess adiposity weigh down host defense? review article h1n1 influenza some articles reviewed show independent risk but some articles show high influences between obesity and influenza [18] underweight, overweight, and obesity as independent risk factors for hospitalization in adults and children from influenza and other respiratory viruses. case study influenza and other respiratory viruses adults that have obesity with h1n1 got a six-fold increase in chance of hospitalization over h3n2 and influenza type b [19]. relationship between community prevalence of obesity and associated behavioral factors and community rates of influenza‐related hospitalizations in the united states case study influenza viruses obesity were more likely to have high influenza related hospitalization rates [20]. obesity impairs the adaptive immune response to influenza virus review article influenza virus (seasonal) obesity impairs cellular response and further induce higher risk of viral infection [10]. impact of obesity on influenza a virus pathogenesis, immune response, and evolution case study influenza type a virus obesity generate lean environment for viral infection [7]. an international perspective on hospitalized patients with viral community-acquired pneumonia case study influenza and viral pneumonia obesity and need for invasive ventilation represent independent risk factors for viral community acquired pneumonia [21]. epidemiology of severe influenza outcomes among adult patients with obesity in detroit, michigan, 2011 case study h1n1 influenza patients with obesity were more likely to require hospital admission as well as a lengthy hospital stay (>7 days) [22]. obesity and pro-inflammatory mediators are associated with acute kidney injury in patients with a/h1n1 influenza and acute respiratory distress syndrome. case study h1n1 influenza high levels of c-peptide and bmi were associated with the development of acute respiratory distress syndrome patients due to a/h1n1 infection [23]. factors associated with hospitalization and critical illness among 4,103 patients with covid-19 disease in new york city. case study sars-cov-2 6.2% of the 1,999 hospitalized covid-19 patients have bmi over 40 [24]. hospitalization rates and characteristics of patients hospitalized with laboratoryconfirmed coronavirus disease 2019 — covid-net, 14 states case study sars-cov-2 over 48% of the hospitalized covid-19 patients have obesity issues [25]. high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sarscov-2) requiring invasive mechanical ventilation. case study sars-cov-2 in the collection of 124 patients, obesity (bmi >30 kg/m2) and severe obesity (bmi >35 kg/m2) were present in 47.6% and 28.2% of the total cases respectively. sars-cov-2 infection and obesity: common inflammatory and metabolic aspects review article sars-cov-2 sars-cov-2 share the same metabolic pathway with obesity and engage ace as a primary player. http://www.ajecr.org/ 399 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org table 1 continued two important controversial risk factors in sars-cov-2 infection: obesity and smoking review article sars-cov-2 angiotensinogen ii are highly produced from obesity condition, which ace is upregulated. sarscov-2 can utilize the excess ace-2 to enter host cells. results according to the comparison shown in table 1, people with obesity were highly studied in influenza virus infection and sars viral infection. when comparing h1n1’s viral respiratory infection to sars infections, most of the sars infection showed patients with obesity are in a chronic state of inflammation that leads to decreased and compromised immunity. however, according to some studies, mixed results were discovered. some findings show little to no relationship between obesity and h1n1 infection, while some show twice or higher prevalence in patients with obesity. in addition, from some early findings in the covid-19 patients’ profile, close to half of the hospitalized patients have bmi over 30, which is a factor that indicates obesity. in addition, findings from sars-cov-2 and the ace-2 engagement gave a potential connection between obesity and viral infection. statistical importance according to center of disease control and prevention (cdc), the classification of obesity is as follow: class i obesity (bmi ≥ 30-34.9 kg/m2); class ii obesity (bmi ≥ 3539.9 kg/m2); class iii obesity (bmi ≥ 40kg/m2) [26]. this classification of data is used to determine the state of obesity to further organized data of viral infection rates. as for the result, h1n1 infection reported 50% of the hospitalized patients to have bmi ≥ 30 kg/m2 [22]; sarscov-2 disease reported 68.5% of the hospitalized patients to have an average bmi of 31.1 kg/m2 [27], while data from new york university hospital reported 6.2% of the hospitalized patients to have bmi ≥ 40kg/m2 [24]. discussion viral infection in respiratory tract respiratory systems are lined with epithelial cells composed of adipocytes and columnar epithelial cells that form a lining that surveillance the area [28]. in a viral respiratory infection, the upper and mild respiratory tract is damaged and causes severe illness like pharyngitis, rhinitis, otitis media, and sinusitis [29]. most of the lower respiratory tract infections were associated with bacteria, respiratory syncytial virus (rsv), and influenza virus, including symptoms like pneumonia and bronchiolitis [30]. therefore, the members of the beta coronavirus family, including mers, sars, and sars-cov-2, are more likely to cause upper and mild respiratory tract infections. in addition, most of the pathological data collected from the recent covid-19 outbreak reported 80% of the symptoms in infected patients restricted to upper respiratory tract infections, such as fever, fatigue, cough, sore throat, runny nose, and sneezing [31]. moreover, h1n1 infections reported more cases of pneumonia and bronchiolitis during the 2009 pandemic [32]. as shown in figure 1, when the respiratory tract is abnormally lined with fatty acids on the epithelial cells, the more likely the receptors for virus were express and allow viral entry. effects in the body caused by obesity obesity is a severe overweight health disorder that can lead to cardiovascular diseases, respiratory diseases, stroke, diabetes, and cancers. individuals with obesity issues usually correlate with excessive accumulation of body fat, which further causes cardiovascular diseases, diabetes, musculoskeletal disorders, and cancers [33]. according to a recent report from brethauer s. [34], most of the patients with obesity have type ii diabetes and cardiovascular diseases as the most common disorders. furthermore, the classification in bmi index number shows how severe the obesity issues individuals got, which bmi number higher than 30 km/m2 accounts for obesity. obesity’s effects started when the pancreas’ production of insulin is resistive to ingested food glucose and further cause hyperglycemia in peripheral tissues. leptin and resistin hormone increased production as a response to increasing metabolism in the body. as metabolism increases, lipid substances increase in output due to the high amount of free fatty acids. obesity also triggers the production of angiotensinogen hormone, which is used to regulate blood constrictions and pressure. a high amount of angiotensinogen will induce the production of angiotensinconverting enzyme (ace). ace can convert angiotensinogen to angiotensinogen type ii (at-ii) [35]. a high amount of at-ii will increase the blood pressure and further cause vasoconstrictions at the lungs, heart, and cardio-vasculatures. these issues can further cause a reduction in immune response and increase the risk of type ii diabetes [8]. influenza effects in people with obesity h1n1 pandemic in 2009 has caused more than 1.6 million people worldwide diagnosed with virus infection [36]. the epidemic of h1n1 has a typical infection route via the upper and lower respiratory tract as well as infecting cells in the lungs [32]. the relationship between obesity and influenza discovered from studies have different results. some research studies have pointed out that influenza has little to no relationship between flu-like illness in the population that have obesity [37, 38]. according to a study from murphy r. et al. [37], the obesity factors are independent of the flu-like illness. their research focused on 8,407 individuals in the u.k that had developed the flu http://www.ajecr.org/ 400 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org figure 1. respiratory function comparison between normal and obesity individual [48]. like disease from may 2009 to december of 2009. they categorized these individuals into groups of ages, bmi, and prior disorders to find out that only 12.8% of the individuals with obesity in the u.k during the pandemic period exists with flu-like illness [37]. another study conducted by cocoros et al. [38] pointed out that their results show little to moderate correlation between obesity and flu-like illness during the h1n1 outbreak. their discussion explained the reason why not getting a significant substantial relationship between obesity and flu-like symptoms might be due to their small sample size used in the study. on the other hand, several studies pointed out that people with obesity poses a higher risk of getting flu-like illness as much as twice the chance when compared to healthy people. a study conducted by green w. et al. [10] measures two different influenza seasons (2013-14 and 2014-15) and allocate 1022 participants to study their illness responses. their results show that vaccinated people with obesity have twice the chance of getting flu-like illnesses than the people that are vaccinated and healthy. furthermore, their findings pointed out that people with obesity have impaired cd4 t-cells for immune response, which then poses a higher risk of getting the illness. besides, people with obesity can produce inflammatory cytokines that promote more inflammation responses. another study carried out by martin e. et al. [22] used data from influenza patients admitted to detroit medical center system, during the period january of 2011 to the end of march 2011, to collect medical records to access the risk of getting hospitalized for influenza infection. in the 161 patients studied, 81% had influenza a, while more than half of the inpatients have obesity class i to class iii [22]. since the h1n1 influenza infection is mostly associated with lower respiratory tract infection, the relevance to covid-19 is less suitable to compare. however, the prevalence of h1n1 viral infection in people with obesity does prove the effects of abnormal metabolism somehow disrupt the immune system. middle east respiratory syndrome’s effects in people with obesity middle east respiratory syndrome (mers) is caused by one of the β coronavirus family members (mers-cov), which induces severe respiratory diseases in human subjects. in addition, the recent covid-19 has been reported shown using similar receptors from the upper and lower respiratory tract to enter host cells, which is also used in sars-cov and mers-cov infections [39]. however, the significant difference between mers-cov and sars covs are the receptor-binding motifs (rbm) in their core structure of receptor-binding domain (rbd), mers-cov utilized the dipeptidyl peptidase 4 (dpp-4) while sarscov and sars-cov-2 utilized angiotensin-converting enzyme 2 (ace-2) [13]. the adipokine, dpp-4, has been reported by studies showing that it is the docking receptor for mers-cov into host cells [12, 40]. in addition, dpp-4 has demonstrated a high influence on the obesity condition in humans [14]. the study by sell et al. [14], shows a higher release of dpp-4 from the visceral and subcutaneous adipose tissues from insulin-resistant patients and women higher than men. as the amount of lipid being metabolized increased, the amount of dpp-4 accumulate in the adipose tissue increases. dpp-4 is responsible for keeping the balance between glucose metabolism dpp-4 is the receptor for mers-cov entering host cells, expressed highly in epithelial cells at the http://www.ajecr.org/ 401 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org figure 2. diagram showing the relationship between obesity and coronavirus viral infection. upper and lower respiratory tract, which makes people get mers infected [40]. mers infection is pathologically related to obesity in that people with obesity express more dpp-4 in their adipose tissue. the overexpressed dpp-4 at the human upper and lower respiratory tract can further on, causing these people with obesity conditions more likely to get mers infected [41]. severe acute respiratory syndrome’s effects in people with obesity according to a study reported by yang j. et al. [42], the severe acute respiratory syndrome (sars) can induce transient islets damage and cause acute insulin-dependent diabetes mellitus. in addition, the sars-cov virus was found to use ace-2 protein as the entry receptor to enter host cells. in further examination, the sars-cov and the sars-cov-2 shared similar receptor-binding motifs, ace-2. ace-2 is highly expressed in epithelial cells that surround the upper and lower respiratory tract. due to pancreatic islets expressed more ace-2 protein than the exocrine cells, sars-cov can cause more damage to the pancreatic islets. then, the damage to islet cells can cause hyperglycemia to occur temporally, in which hyperglycemia is commonly observed in people with obesity [42]. furthermore, people with obesity have an even higher risk of sars-cov infection due to enlarged hyperglycemia from both the islets and leptin hormone malfunction [42]. pre-existing symptoms in people with obesity can become worse when sars-cov binds to ace2 in the pancreatic islet, which can induce a larger hyperglycemia event, and lead to severe diabetes mellitus condition. in figure 2, the relationship of obesity and coronaviruses infection, such as mers, sars, and covid-19, have been sketched out with direct links to increase the chance consequent damage in host cells. covid-19’s effects in people with obesity the function of epithelial cells’ angiotensin-converting enzyme 2 (ace-2) this particular enzyme attached to the outer membrane of human cells, especially epithelial cells located at the mucosal organs such as salivary glands, lung, heart, esophagus, kidney, bladder, and ileum [43]. ace-2 has a specific receptor-binding domain (rbd) that allows sarscov-2 to bind and dock on host cells. when sars-cov2’s spike proteins attached to the rbd on ace-2, viral rna is released from the viral envelope and enters human cells for replication. the process allows sars-cov-2 to rapidly produce and increase its chance of survival on the human host to overcome the immune system’s attack. there are two waves of inflammatory response when sars-cov2 binds ace-2. the primary inflammatory response increases host immune attack and increases cytokine production. this change in immune response will then cause viral cell apoptosis. in the secondary response, the neutralizing antibodies are formed and trigger a macrophagic mechanism. however, these neutralizing antibodies are not optimized to kill the sars-cov-2, which later on might cause leakage of virus and induce symptoms such as coughing and lung injury [44]. currently, there is no definite route of immunological suppression, as many subtypes of sars-cov-2 have been discovered and http://www.ajecr.org/ 402 am j exp clin res, vol. 7, no. 2, 2020 http://www.ajecr.org manifest human cells in a slightly different pathway. current knowledge of covid-19’s relationship with obesity as of june 2020, the covid-19 disease has no cure and vaccine yet. the chances of getting the infection, especially in people with obesity, might be higher than other groups of the population. physicians and health authorities are eager to find a health and safety guideline to help prevent further transmission and reduce the number of cases infected with sars-cov-2. sars-cov-2 from covid-19 utilizes its sglycoprotein to bind tightly to human angiotensinconverting enzyme 2 (ace-2) receptors on adipose tissue, specifically in the respiratory tract. in the recent findings from jia x. et al. [45], the ace-2 receptors are highly expressed in the lung adipose tissue, which shows more immediate respiratory symptoms such as coughing and restricted breathing after the infection of sars-cov-2. in their further findings, jia x. et al. [45] also pointed out that since the population with obesity has higher adipose fat contents in their lung tissue, more ace-2 receptors are expressed to allow more sars-cov-2 to get attached. on april 8, 2020, the center for disease control and prevention (cdc) [25] has published an early report of multiple hospitals’ non-peer reviewed data collection on hospitalized covid-19 patients. within the data collected, more than 48.3% of the covid-19 patients have obesity. in the covid-19 patients with obesity conditions, age from 18 to 40 years old account for more than 59%, which is higher than the total percentage of the population with obesity in the u.s. [25]. therefore, a clear indication of obesity increases the risk of severe respiratory illness can acknowledge from the statistics; however, we still need more samples to cross-reference data to get a conclusive answer to the relationship between obesity and covid-19. meanwhile, another study from doctors at new york university grossman school of medicine collected patients’ profiles and medical records that are covid-19 positive. in the study, petrilli c. et al. [24] pointed out that in the 4,103 covid-19 positive patients, 1,999 were hospitalized, and 6.2% of the hospitalized patients have a bmi over 40, which is count as obesity. the goal is to find out who is at risk for hospitalized after the sars-cov-2 infection and to help clinicians to assess the severity to give the best treatment immediately. according to the study from simonnet et al. [27], the clinical data collected from 124 patients for sars-cov-2 infection reported that 85 patients (68.6%) required invasive mechanical ventilation (imv). the bmi data collected was higher than in the 39 patients (31.4%) who did not require imv: 31.1 (27.3-37.5) kg/m2 vs 27 (25.330.8) kg/m2, respectively (p<0.001, t-test) [27]. furthermore, using the medical data collected from recent hospitalized covid-19 patients to conduct crossreferencing researches and clinical studies to find the specific cause of covid-19 in the population with obesity. declined in immune surveillance or t-cells response might be the cause. clinical evidence does show ace-2 engagement are highly related to the upregulation of ace in obesity condition. obesity upregulates ace to overcome a high amount of angiotensinogen, which virus-like sarscov-2 can use ace to dock to host cells to induce viral replication. furthermore, it is suggested to conduct an extensive study of immune factors to clarify the cause of hyperglycemia from sars-cov-2. conclusion the current pandemic of covid-19 has been reported to infect over 2.2 million people globally, 150 thousand of which resulted in death [46]. in discovery, countries that have a high percentage of populations with obesity have clinical data reporting a higher prevalence of covid-19 infection and higher chances of getting hospitalized with a mechanical ventilator required. studies on previous h1n1 viral respiratory pandemic, some clues on immune system downregulation, and distress could be a similar cause in the covid-19 cases. however, the h1n1 influenza virus is not as closely related to sars-cov-2. the receptors ace-2 in sars coronavirus and dpp-4 in mers-cov were released in high amounts from the obesity condition when looking at viral infections of mers and sars coronaviruses. there was pathological evidence in terms of showing a positive relationship between abnormal hormone responses in obesity affecting the receptors that bind viruses, 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portugal abstract. the objective of this study was to compare clinical, laboratorial, maternal and perinatal results between hellp syndrome and severe preeclampsia. an observational study comparing women with hellp syndrome (n=71) to women with severe preeclampsia (n=253) was done. the authors analyzed the early course of the pathologies and the outcomes in both groups. hellp syndrome occurred in 28% of all the cases and was more frequent at gestational age before 32 weeks (n=39 – 55%) than severe preeclampsia (n=108 42%), with more newborns weighting less than 1500g (27 – 38.6% vs 65 – 25.6%; p=0.036). thrombocytopenia below 100 000/µl (aor, 2.14; 95% ci, 1.49 – 3.06) and ldh>1 000 ui/l (aor: 5.17; 95% ci 2.19 – 12.16) were risk factors for hellp. maternal morbidity (eclampsia, abruptio placentae, and acute renal failure) was similar in both cohorts; eight stillbirths (6 in severe preeclampsia and 2 in hellp syndrome) occurred. there were no maternal deaths. in conclusion, in this study the authors confirmed that hellp syndrome is a severe form of preeclampsia with an earlier presentation in pregnancy, worst laboratorial findings and more prematurity rates. keywords: preeclampsia, thrombocytopenia, maternal and perinatal morbidity, hellp syndrome introduction although being a leading cause of great maternal and perinatal morbidity and mortality, even in developed countries, preeclampsia is present in only 2 to 8% of all pregnancies [1]. it has higher prevalence in nulliparous and is present as a maternal syndrome, characterized by arterial hypertension generally with proteinuria, and a fetal syndrome, with fetal growth restriction and amniotic fluid reduction. it may have several systemic manifestations, variable degrees of severity and either an early onset with maternal and fetal morbidity, or a late onset, near term, with less severity and reduced fetal compromise. preeclampsia is characterized by hypertension (systolic and diastolic blood pressure of ≥ 140 and 90 mm hg, respectively, on two occasions, at least 6 hours apart) and proteinuria (protein excretion of ≥ 300 mg in a 24 h urine collection, or a dipstick of ≥ 1+), developing after 20 weeks of gestation in previously normotensive women[1] . the analysis of the physio-pathological process shows that its origins are related to an immunological process associated with a defective and insufficient placentation [1, 2]. although the placental origins of the disease are largely accepted maternal or fetal predisposing factors are also considered when an early onset or a high severity are present [3]. the syndrome characterized by micro-angiophatic hemolysis, liver enzymes elevation and thrombocytopenia (hellp syndrome) was individualized in 1982 by weinstein and is considered a severe form of preeclampsia, occurring in 0.5% to 0.9% of all pregnancies and complicating 10% to 20% of severe preeclampsia cases [46]. it may have a sudden aggravation and even though some clinical aspects may be similar to preeclampsia, in hellp syndrome the blood pressure can be normal, the proteinuria is not always present and there is more expression of inflammation markers [6-8]. additional characteristics of hellp syndrome include higher platelet and coagulation activation, with thrombocytopenia, coagulation disorders and disseminated intravascular coagulation (dic) [8]. the tennessee classification defines as characteristics of the disease the presence of hemolysis, with total serum lactic dehydrogenase greater than or equal to 600 iu/l, serum aminotransferases greater than or equal to 70 iu/l and platelets less than or equal to 100 000/µl [6]. the mississippi classification proposes the existence of categories according to platelet count: class 1 stands for less than 50 000/ µl, class 2 for less than 100 000/ µl and class 3 for a platelet count less than 150 000/µl [9]. hemolysis is the main finding of this syndrome and can be documented by an elevation of serum lactic dehydrogenase (ldh), anemia with presence of schistocytes in peripheral smear and low haptoglobin concentration. the elevation of both aspartate aminotrans ___________________________________________________________ * corresponding author: ana campos, md (anacampos.campos87@gmail.com) . http://www.ajecr.org/ mailto:anacampos.campos87@gmail.com 171 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org ferase (ast) and alanine aminotransferase (alt) are mainly due to a hepatic lesion. low platelet count is a result of a higher consumption: activated platelets adhere to endothelial cells with a reduced life-span [9, 10]. perinatal morbidity and mortality are greater in hellp syndrome and are related to gestational age of the onset of the clinical situation [7]. excluding cases with no doubts in the classification of hellp syndrome, when there is an hepatic involvement and coagulation disorders, sometimes the distinction with severe preeclampsia is difficult [8, 9]. the aim of this study was to identify factors that can distinguish preeclampsia from hellp syndrome. we analyzed the early or late onset of the disease, based in clinical features and laboratorial findings, and the results in terms of maternal and fetal morbidity. materials and methods we reviewed the medical records of women diagnosed with severe preeclampsia or hellp syndrome between january 1, 2008 and december 31, 2013 in a tertiary perinatal care center in lisbon, portugal. socio-demographic, clinical and laboratorial data were analyzed. classification criteria for preeclampsia diagnosis were defined according to guidelines of the international society for the study of hypertension in pregnancy (isshp) [11] and from the american college of obstetrics and gynecology (acog) [12]. according to acog criteria (2013) we classified as severe preeclampsia cases with a systolic arterial pressure (sap) greater than or equal to 160 mm hg and a diastolic arterial pressure (dap) greater than or equal to 110mm hg on two occasions 4 hours apart and the occurrence of any of these findings: impaired liver function as indicated by abnormally elevated blood concentrations of liver enzymes, significant proteinuria (≥300 mg/dl) per 24 hour urine collection, severe and persistent right upper quadrant or epigastric pain, headaches or visual disturbances, serum creatinine concentration values greater than 1.1mg/dl or pulmonary edema. fetal compromise due to maternal placental insufficiency may be presented as fetal growth restriction, oligo-hydramnios, disturbed doppler velocimetry with absent or reversed end diastolic flow in umbilical artery or other fetal vessels. if established before 34 weeks, preeclampsia was classified as of early onset and as of late onset if the diagnose was done only after 34 weeks. according to the acog we defined fetal growth restriction as an estimated fetal growth at ultrasound < 10th percentile or an abdominal circumference < the 10th percentile for a given gestational age [12]. we defined low birth weight as a newborn weight lesser than 2,500g and extremely low birth weight as a newborn weight lesser than 1,500g. the management during pregnancy consisted in symptomatic treatment, maternal condition stabilization, and eventually pregnancy termination when the risks of pregnancy continuation outweighed the benefits for either the mother or the fetus. the mississippi criteria were applied for hellp syndrome classification [10,13,14], with the categories’ table 1 preeclampsia and hellp: socio-demographic data and clinical evaluation pe: preeclampsia; bmi: body mass index; ch: chronic hypertension; ah: arterial hypertension; ga: gestational age; uad: umbicial artery doppler; fgr: fetal growth restriction; mgso4: magnesium sulfate; * fisher exact test. division according to the number of platelet count, impaired liver function (ast or alt) and lactate dehydrogenase (ldh) always under 600 iu/l. we also considered this syndrome as early or late onset, if the disease criteria were established before or after 34 weeks. for a complete evaluation, clinical and laboratorial parameters were associated with fetal status study, by ultra-sonographic assessment of fetal growth and doppler velocimetry of uterine, umbilical and middle cerebral arteries and ductus venoso, for evaluation of fetal adaptation to an insufficient placental perfusion. we used anti-hypertensive therapy to stabilize maternal hypertension and to prevent cerebrovascular events, when arterial pressure was equal or greater than 160/100 mm hg or with lower values associated with other symptoms, such as headaches or epigastric pain. magnesium sulfate was used for convulsions prophylaxis. when gestational age was lesser than 34 weeks 6 days, antenatal corticosteroid therapy was used for fetal lung maturation. when maternal platelet count was lesser than 80,000/ µl we started corticosteroid therapy, according to missi p e hellp adjust ed or (n = 253) (n = 71) (95% ci) age median 30.5/6.6 31.3/6.7 1,009 (0.969 – 1.051) 0.65 min-max. 14 – 45 17 – 45 ≥35 years 81(30.9%) 24(33.3%) 0.895 (0.513 – 1.560) 0.696 et hnicit y caucasian 156 (63.0%) 44 (64.7%) afro-american 66 (26.7%) 17 (25.0%) 0.813 (0.548 – 1,206) 0.303 asiat ic 3 (1.2%) p arit y nulliparous 164 (68.6%) 41 (68.3%) 1,088 (0.792-1.496) 0.602 mult iparous 75 (31.4%) 19 (31.7) bmi median/sd 25.7/5.6 25.0/4.5 0.968 (0.888 -1.056) 0.464 ≥25 59 (53.6%) 11 (44%) 0.384 ≥30 25 (22.7%) 5 (20%) 0.767 ch/ht in previous pregnancy 67 (26%) 8 (11.1%) 1.122 (1.008-1.249) 0.034* diabet es 27 (10.7%) 6 (8.4%) 0.993 (0.885 – 1.115) 0.907 ht t herapy labet alol 64(24.5%) 17 (23.6%) nifedipine 41(15.7%) 9 (12.5%) associat ion 78 (29.9%) 17 (23.6%) no t herapy 39(14.9) 22(30.6%) 0.804 (0.672 – 0.962) 0.017 ga at incoming median/sd 33.3/4.1 33.07/ 4.4 ig <32s 81 (32%) 32 (45.1%) 1.988 (1.168-3.384) 0.011 ig <34s 108 (42.2%) 39 (54.9%) 1.670 (0.984-2.835) 0.057 uad alt ered <34s 54 (56%) 17 (51,5%) 0.969 (0.606-1.549) 0.896 fgr 101 (41.1%) 22 (31.4%) 0.741 (0.427 – 1.287) 0.288 t herapy mgso4 153 (58.6%) 50 (70.4%) 0.976 (0.628 – 1.518) 0.915 fet al cort icot herapy 102 (94.4%) 36 (92,3%) 1.417 (0.337 – 5.962) 0.635 mat ernal cort icot herapy 13 (5.2%) 35 (50%) 16.688 (8.196-33.982) >0.001 variable p http://www.ajecr.org/ 172 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org table 2 laboratorial data ast: aspartate aminotransferase; alt: alanine aminotransferase; ldh: lactate dehydrogenase. ssippi protocol [10]. the data was registered in an informatics database. to compare categorical variables between the study groups, qui-square test or extension to exact fisher test were used; to analyze continuous variables the non-parametrical test of mann-whitney was used. univariate analysis was used to assess differences in baseline demographic and pregnancy characteristics and in the outcome among the 2 groups. unadjusted odds ratios (ors) with 95% confidence intervals (cis) were calculated to quantify the risk of severe preeclampsia and hellp syndrome. multivariate logistic regression was used to calculate adjusted odds ratios (aors) and 95% confidence intervals (95% ci) for the outcome of hellp syndrome. all statistical analysis was performed with statistical package for social sciences (spss) version 20.0 (spss inc., chicago, il). tests with probability values of α < 0.05 were considered significant. results in the period in a total of 33 620 deliveries, 324 cases in the period of the study, in a total of 33620 deliveries, 324 cases of severe preeclampsia occurred, representing a prevalence of 0.9%. according to clinical and laboratory parameters the diagnosis of severe preeclampsia was present in 253 cases while hellp syndrome in 71, resulting in a hellp syndrome prevalence of 28% among all cases of severe preeclampsia and of 0.2% among all deliveries. the median maternal age was similar in both groups (30.5 years in severe preeclampsia and 31.3 years in hellp syndrome) (p = 0.65); 30.9% of the women were older than 35 years in severe preeclampsia and 33.3% in hellp syndrome (p = 0.69). most of the women were nulliparous (68.6% in preeclampsia and 68.3% in hellp syndrome), without differences between the two groups (p = 0.60). a history of hypertension was more frequent in preeclampsia group (aor, 1.12; ci, 1.01-1.25); there were no differences in ethnicity and body mass index (bmi) (table 1). gestational age at the diagnosis was the same in both groups (33 weeks); no differences were found in doppler flow velocimetry (p = 0.34) or fetal growth restriction (fgr) (p = 0.29) (table 1). the clinical classification and differences in severe preeclampsia and hellp syndrome were based on laboratory tests results: elevated aspartate aminotransferase (ast) (p < 0.001), elevation in alanine aminotransferase (alt) (p <0.001), lactate dehydrogenase (ldh) >1 000ui/l (p <0.001) and platelet count <100 000/µl (p <.001). hellp syndrome class 1 or 2 was present, according to mississippi criteria when platelet count <100 000/ µl (41% of hellp cases) or <50 000/ µl (24% of hellp cases) (table 2). there were no differences in other laboratorial results, such as hemoglobin (p=0.4), hematocrit (p = 0.57), uric acid (p = 0.37) or proteinuria (p = 0.84), but haptoglobin <20 g/l was significantly more frequent in hellp syndrome (p = 0.04) (table 2). the early onset of the disease (<34 weeks) was more frequent in hellp syndrome cases (55% vs 42%). compared with the late onset cases, arterial pressure was higher when the disease established before 34 weeks, with higher need for anti-hypertensive drugs in association (p = 0.002). fetal growth restriction was more frequent (p< 0.001) as well as elevated pulsatility index (ip) doppler velocimetry (p < .001) (table 3). the newborn median weight was similar in preeclampsia and hellp cases (2027g vs 1861g) (p = 0.104), although the hellp syndrome group had more newborns under 1500g (a or 1.82; ci, 1.10 – 3.18) (p = 0.036) (table 4). antenatal glucocorticoid for fetal lung maturation was used in 94% of preeclampsia and in 92% of hellp syndrome cases (table 1). corticosteroids were administered for maternal severe or moderate thrombocytopenia in 50% of hellp cases and only in 5.2% of preeclampsia cases (p > 0.001) (table 1). no differences were found in severe preeclampsia cases and in class 3 of hellp cases, in pregnancy results or in newborn morbidity (table 3). there were 6 stillbirths (2.5%) in preeclampsia and 2 (3.3%) in hellp syndrome. three occurred between 31 and 35 weeks after placental p reeclampsia hellp syndrome adjust ed or (n = 253) (n = 71) (95% ci) hemat ocrit med/dp 3.,7/4.8 36/4.3 0.572 mín-max 22-49 28-46 hemoglobin ≥13g 68 (27.0%) 18 (25.4%) 0.4 ≥10-<13g 157 (62.3%) 42 (59.2%) <10g 25 (9.9%) 11 (15.5%) uric acid med/dp 6.3/1.6 5.9/1.4 0.368 mín-máx 3.1-11.8 2.0-9.2 ast normal 151 (60.6%) 12 (16.9%) 3.341 (2.371-4.707) <0.001 alt ered 51 (20.5%) 15 (21.1%) duplicat ed 44 (17.7%) 43 (60.6%) alt normal 161 (64.7%) 16 (22.5%) 3.112 (2.253-4.300) <0.001 alt ered 39 (15.7%) 9 (12.7%) duplicat ed 45 (18.1%) 45 (63.4%) ldh normal 120 (47.6%) 11 (15.5%) 3.811 (2.592-5.604) <0.001 alt ered 93 (36.9%) 15 (21.1%) >1000 iu/l 29 (11.5%) 43 (60.6%) p lat elet s ≥100 000 µl 212 (83.8%) 20 (28.2%) 2.885 (2.077-4.009) <0.001 <100 000 µl 22 (8.7%) 29 (40.8%) <50 000 µl 2 (0.8) 17 (23.9%) hapt oglobin <20 g 15 (6.0%) 27 (38.0%) 1.246 (1.0111.536) 0.039 p rot einuria <100 89 (35.4%) 29 (41.4%) 0.844 ≥100-<500 64 (25.4%) 15 (21.4%) ≥500 61 (24.2%) 17 (37.2%) variable p http://www.ajecr.org/ 173 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org table 3 diagnosis before and after 34 weeks pe: preeclampsia; aud: arterial uterine doppler; fgr: fetal growth restriction. abruption; five occurred between 24 and 26 weeks with an early and severe fetal growth restriction (table 4). there were no maternal deaths but there were two cases of eclampsia, six of coagulopathy after placental abruption, two cases of renal acute failure. post-partum hellp syndrome was diagnosed in 21% of the cases (table 4). after using multivariate logistic regression we identified thrombocytopenia <100 000/µl (aor: 2.14; p <.01) and alt >1000 ui/l(aor:5.17; p <.001) as factors influencing hellp syndrome diagnosis(table 5). discussion in this comparative study, we tried to identify specific characteristics of hellp syndrome. in the studied period, in a total of 33 620 deliveries, 324 cases of severe hypertensive disease were verified, with a prevalence of 0.9%. the prevalence of hellp syndrome was 0.2% and complicated 27% of the cases of severe preeclampsia, results similar to other studies [10, 12, 13]. we didn’t find any differences in socio-demographic aspects nor in parity, in contrast to other studies, where multiparous and advanced maternal age are is more frequent in hellp syndrome [6]. these results may be related to a greater prevalence of nulliparity in women with advanced age and with a reduced representation of multiparous women in our studied women, which may be an expression of delaying childbearing age. history of hypertensive disease was more frequent in severe preeclampsia cases than in hellp syndrome. arterial pressure values were greater, with higher need of anti-hypertensive drugs in severe preeclampsia cases, which is also present in other studies [6, 8, 14, 15]. early onset of the disease, and extremely low birth weight, were more frequent in hellp syndrome, supporting a more severe clinical picture in these cases, which is similar to other reports [8, 15]. an expectant attitude before 34 weeks was more frequent in severe preeclampsia than in hellp syndrome cases, because in the latter there was a more frequent deterioration of clinical situation, determining an urgent pregnancy termination. table 4. pregnancy outcome* *univariate analysis; pe: preeclampsia; pa: placental abruption; arf: acute renal failure; ga: gestational age; nb: newborn. table 5. hellp syndrome: multivariate analysis ldh: lactate dehydrogenase. nevertheless, in 92% of hellp syndrome cases, it was possible to administrate antenatal corticosteroid for fetal lung maturation. in the presence of moderate or severe thrombocytopenia dexametasone was used, according to mississippi protocol, for a faster mother recovery [14]. there was an elevation of platelet count, allowing not only hellp 39 (54.9%) p e 108 (42.2%) hellp 32 (45.1%) p e 148 (57.8%) ant ihypert ensive t herapy associat ion 1.78 (1.21-2.31) 0.002 aud alt ered 4.21 (2.92-6.06) <0.001 fgr 2.72 (1.71-4.32) <0.001 cesarean sect ion 3.93 (2.24-6.95) <0.001 fgr 0.698 cesarean sect ion 0.772 aud alt ered 0.876 delivery <34s 0.19 newborn weight <1500g 0.313 p 71 (49.3%) 73 (52.5%) 52 (28.9%) 112 (62.2%) adjust ed or (95% ci) 37 (20.7%) 18 (10.3%) 58 (26.5%) <34w (n = 147) ≥34w (n = 180) 52 (35.4%) 26(17.2%) 93 (42.3%) 14 (56%) 88 (40%) 162 (73.3%) 9 (36%) variable severe p e (n = 226) hellp (n = 25) 9 (36%) 3 (15.8%) 19 (76%) 123 (86.6%) obst et ric out come p lat elet s≥100 000 p e hellp adjust ed or (n = 253) (n = 71) (95% ci) p a 4 (1.7%) 2 (2.8%) eclampsia 2 (0.8%) 1 (1.4%) fet al deat h 6 (2.4%) 2 (2.8%) arf 2 (0.8%) 2 (2.8%) hellp aft er birt h 15 (21.1%) ga at delivery (median) 35weeks 33weeks 0.329 <34weeks 108(42.2%) 39 (54.9%) 0.50 (0.29 – 0.85) 0.057 <32weeks 81 (32%) 32 (45.1%) 0.011 at t it ude delivery 160 (61.1%) 42(58.3%) 0.675 expect ant <34 weeks 61 (56.5%) 19 (48.7%) 0.404 labour induct ion 120 (46.5%) 27 (37.5%) 0.601 vaginal birt h 70(27.2%) 18 (25.0%) 0.92 cesarean 187 (72.5%) 54 (75.0%) nb weight median/dp 2027g /837 1861g/836 1.82 (1.04-3.18) <1500g 65(25.6%) 27 (38.6%) <2500g 182 (71.7%) 50 (71.4%) pvariable variable adjust ed or (ci 95%) p plat elet s <100 000/ µl 2.14 (1.49 – 3.06) <.001 ldh >1000iu/l 5.17 (2.19 – 12.16) <.001 http://www.ajecr.org/ 174 am j exp clin res, vol. 3, no. 3, 2016 http://www.ajecr.org epidural anesthesia, but also a reduction of transfusion needs. the length of hospital staying was similar to severe preeclampsia cases and we maintain this protocol, even if there is not an evidence supporting this attitude. the mississippi classification proposes the existence of classes according to platelet count. the class 3 with a platelets count less than 150 000/ µl is very similar to severe preeclampsia cases, when compared to clinical and laboratorial results. the early and sudden onset, the elevation of liver enzymes and thrombocytopenia are characteristic of hellp syndrome severity. in this study, thrombocytopenia < 100 000/µl, and elevation of the liver function tests, were the identification factors of this syndrome. when multivariate analysis was done, thrombocytopenia < 100 000/µl and lactate dehydrogenase more than 1000ui/l, are the significant risk factors for this syndrome. although this is a retrospective study with known limitations, we think that it has a significant number of cases, with classification and management protocols based on international guidelines, making possible the comparative evaluation between severe preeclampsia and hellp syndrome concerning early onset cases, severity, and the decision of optimizing the correct moment of pregnancy termination. conflict of interest the authors declare no conflicts of interest. references 1. sibai b, dekker g, kupferminc m. pre-eclampsia. lancet 365: 785-99, 2005. 2. redman cw, sacks gp, sargent il. preeclampsia: an excessive maternal inflammatory response to pregnancy. am j obstet. gynecol 180: 499-506, 1999. 3. savvidou md, hingorani ad, tsikas d, frolich jc, vallance p, nicolaides kh. endothelial dysfunction and raised plasma concentrations of asymmetric dimethylarginine in pregnant women who subsequently develop pre-eclampsia. lancet 361: 1511-1517, 2003. 4. weinstein l. syndrome of hemolysis, elevated liver enzymes, and low platelet count: a severe consequence of hypertension in pregnancy. am j obstet. gynecol 142: 159-167, 1982. 5. sibai bm, taslimi mm, el-nazer a, amon e, mabie bc, ryan gm. maternal-perinatal outcome associated with the syndrome of hemolysis, elevated liver enzymes, and low platelets in severe preeclampsiaeclampsia. am j obstet. gynecol 155: 501-509, 1986. 6. barton jr, sibai bm: diagnosis and management of hemolysis, elevated liver enzymes, and low platelets syndrome. clin. perinatol 31: 807-833, 2004. 7. dadelszen p, payne b, li j, ansermino jm, et al. prediction of adverse maternal outcomes in pre-eclampsia: development and validation of the fullpiers model. lancet 377: 219-227, 2011. 8. haram k, svendsen e, abildgaard u. the hellp syndrome: clinical issues and management. a review. bmc pregnancy childbirth 9: 8, 2009. 9. martin jn. milestones in the quest for best management of patients with hellp syndrome (microangiopathic hemolytic anemia, hepatic dysfunction, thrombocytopenia). int j gynecol obstet 121: 202-207, 2013 10. abildgaard u, heimdal k. pathogenesis of the syndrome of hemolysis, elevated liver enzymes, and low platelet count (hellp): a review. eur j obstet gynecol reprod biol 166: 117-123, 2013 11. report of the national high blood pressure education program.working group report on high blood pressure in pregnancy. am j obstet gynecol 183: s1-22, 2000 12. american college of obstetricians and gynecologists.taskforce on hypertension in pregnancy. hypertension in pregnancy/developed by the task force on hypertension in pregnancy (2013) acog. 13. sibai bm. diagnosis, controversies, and management of the syndrome of hemolysis, elevated liver enzymes, and low platelet count. obstet gynecol 103: 981991, 2004. 14. sibai bm, ramadan mk, usta i, salama m, mercer bm, friedman sa. maternal morbidity and mortality in 442 pregnancies with hemolysis, elevated liver enzymes, and low platelets (hellp syndrome). am j obstet gynecol 169(4): 1000-1006, 1993. 15. sep s, verbeek j, koek g, et al. clinical differences between early-onset hellp syndrome and early-onset preeclampsia during pregnancy and at least 6 months postpartum. am j obstet gynecol 202: 271.e1-5, 2010. 16. fitzpatrick k e., hinshaw k, kurinczuk j j., knight m. risk factors, management, and outcomes of hemolysis, elevated liver enzymes, and low platelets syndrome and elevated liver enzymes, low platelets syndrome. obstet gynecol 123: 618-127, 2014. 17. haram k, svendsen e, abildgaard u. the hellp syndrome: clinical issues and management.a review. bmc pregnancy childbirth 9: 8, 2009. 18. wallace k, martin jn jr, tam k, et al. seeking the mechanism(s) of action for corticosteroids in hellp syndrome: smash study. am j obstet gynecol 208: 380.e1-8, 2013. 19. sep s, verbeek j, koek g, et al. clinical differences between early-onset hellp syndrome and early-onset preeclampsia during pregnancy and at least 6 months postpartum. am j obstet gynecol 202: 271.e1-5, 2010. http://www.ajecr.org/ 189 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org american journal of experimental and clinical research am j exp clin res 2017;4(1):189-193 review article approach to pharmacotherapy of botulinum toxin a in the field of urology hamid mazdak 1, 2, zahra tolou-ghamari1* 1isfahan kidney transplantation research center, isfahan university of medical sciences, isfahan, iran 2department of urology, alzahra research centers, isfahan university of medical sciences, isfahan, iran abstract. by altering discharge of neurotransmitter from the end of nerve, the strong toxin that is called botulinum toxin or btx could results in paralysis of different muscles into the human bodies. however there are seven serotype of botulinum toxin, but only botulimum toxin a (btx-a) was recommended as the most commonly prescribed of btx by urologists. when treatment strategy based on use of simultaneous anticholinergics could not meet a satisfactory criterion for pharmacologist, urologist and patients, then it seems that btx-a expressively could improve urinary incontinence symptoms, urodynamic, and quality of life in patients with both neurogenic and nonneurogenic detrusor activity. related to its use in urological disorders, it seems that intravesical btx-a injection is useful in inflammatory bladder disease such as chemical cystitis, radiation cystitis, and ketamine related cystitis. dysuria and urinary retention could be mentioned as the maximum and minimum reported side-effects after injection. it is prescribed in urinary incontinence that could be a result of urethral underactivity (stress) or bladder overactivity (urge), or a combination of two urethral overactivity/bladder underactivity (overflow incontinence). hematoma, pain at injection site, intractable headache, ptosis, diplopia and hyperactivity of the local antagonist muscle could be mentioned as the temporary side effects. dry and red eye, space striving, dry mouth, abdominal turbulences, dysphagia, throatiness and lastly breathing difficulties could be specified as potentially serious events. keywords: botulinum toxin-a, btx-a, urology, bladder, pharmacotherapy introduction the neurotoxic protein that is produced by the bacterium clostridium botulinum was originally discovered by emile van ermengem in the year 1895 and subsequently called botulinum toxin (btx). however, there are seven serotype of botulinum toxin, but only botulimum toxin a (btx-a) was recommended as the most commonly prescribed of btx by urologists. in the year 1989, the food and drug administration (fda) approved botox® for the pharmacotherapy of strabismus, blepharospasm, and cervical dystonias in population of patients with > 12 years old. the neurotoxins produced by the bacterium clostridium botulinum are the mainly strong acute toxins and are the contributing agents of the neuroparalytic disease botulism. the toxins act mainly at peripheral cholinergic synapses by blocking the evoked discharge of the neurotransmitter acetylcholine [1]. the estimated human median lethal dose (ld50) is 1.3-2.1 ng/kg intravenously or intramuscularly and 10-13 ng/kg when inhaled [2]. a dose of 200 u for treatment of neurogenic detrusor overactivity (ndo) was recommended by fda [3]. the general uses of btx in medicine include; upper motor neuron syndrome, severe primary axillary hyperhidrosis, belpharospasm, strabismus, chronic migraine, bruxism, cervical dystonia, neuromuscular disorder of the head and neck, severe primary axillary hyperhidrosis, muscle spasm and overactive bladder (oab). when injected in small amounts, it could weaken a muscle for a period of three to four months. as voiding dysfunctions are common problems in urological practice [4], efficacy and safety of repeated btx-a injections for patients with drug refractory non-neurogenic overactive bladder (noab) seems to be important issue related to its prescription [5, 6]. in the year 2011, it was approved for urinary incontinence due to detrusor overactivity [7]. the efficacy of btx-a in management of oab has been recommended by previous publications. according to the definition by the international continence society, urgency with or without urge incontinence, usually with frequency or nocturia called over active bladder (oab) [8]. related to its use in urological disorders, publications suggested that intra-detrusor injection of botulinum toxin may have beneficial effects in patients with medication refractory detrusor overactivity and may offer a new minimally invasive alternative to patients with severe overactive ___________________________________________________________ * corresponding author: prof. zahra tolou ghamari (toloeghamari@pharm.mui.ac,.ir). http://www.ajecr.org/ mailto:toloeghamari@pharm.mui.ac,.ir 190 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org table 1 pharmacological properies of botulinum toxin a im: intramuscular, sc: subcutaneous. bladder symptoms [1-8]. this study was designed in order to gather talented satisfying evidence, strong review articles and research in order to focus in btx injections associated to urological-disorders. materials and methods united states national library of medicine (pubmed, nlm/ medline®) were searched. the main words relevant to; “1) butilinum toxin a, 2) butilinum toxin a efficacy and safety, 3) butilinum toxin a in clinical practice, 4) butilinum toxin a dosing 5) butilinum toxin a in urology, 6) butilinum toxin a in urological disorders were investigated. a total of: 1) 7649 (18 april 2016 to 17 may 1946), 2), 545 (29 february 2016 to february 1997), 3) 199 (29 january 2016 to 1998), 4) 121(december 2015 to january 1996), 5) 446 (31 march 2016 to september 1998), 6) 735 (20 october 2015 to february 1997), manuscripts were recognized. consequently, research papers appropriated to the pharmacotherapy management of btx-a, in urology were selected and assessed entirely [9-12]. results table 1 shows pharmacological properties of btx-a, which could be categorized the novel treatment associated to a range of therapeutic domains. btx-a has been established for the management of numerous lower urinary tract symptoms (luts), sexual dysfunctions such as oab, detrusor-sphincter dyssynergia (dsd), benign prostatic hyperplasia (bph), interstitial cystitis/painful bladder disorder, long-lasting pelvic pain and more recently early ejaculation. it has expected guiding authorization for the use in ndo and oab, but its' use remains unlicensed in other luts such as non-neurogenic luts in men with bph, bladder pain syndrome and dsd [13]. it could be prescribed in patients with urinary urgency and frequency, urge incontinence and nocturia those suffer from oab [7]. the first randomized, double-blind, placebo-controlled trial to compare the efficacy of btx-a versus placebo in treating patients with refractory ido of either sex was reported by sahai et al. in 2007 [8, 14]. in another study performed by brubaker l. et al., in 2008, 200 u (10 u/ml) was injected at 20 sites with trigone sparing. significant increases in maximum cystometric capacity (mcc) from 182 ml to 313 ml were observed at 4 weeks. btx-a also reduced episodes of frequency (mean change from 15.44 to 7.93 times per day), urgency (mean change from 11.69 to 9.21 times per day) as well as uui (mean change from 4.98 to 1.9 times per day) at 4 weeks, and a significantly better improvement in qol as compared with placebo was noted. the beneficial effects persisted for at least 24 weeks. brubaker et al. also compared 200 u intradetrusor btx-a injection at 15 to 20 sites versus placebo in women with refractory idiopathic uui [8, 15]. a durable efficacy for dose groups of 100 u or greater was reported by dmochowski r, et al., in 2010 that was performed a clinical trial related to doses of 150 to 300 u. in that study doses more than 150 u contributed formula: c6760h1044n1743o2010s32 molar mass: 150 kg/mol (150,000 g/mol) route of administration: im (approved), sc, intradermal, into gland c l i n i cal effi cacy: an inject ion of 200 u (10 u/ml) at 20 sit es wit h t rigone sparing , significant increases in maximum cyst omet ric capacit y (mcc) from 182 ml t o 313 ml were observed at 4 weeks. reduced episodes of frequency (mean change from 15.44 t o 7.93 t imes per day), urgency (mean change from 0.69 t o 9.21 t imes per day) as well as uui (mean change from 4.98 t o 1.9 t imes per day) at 4 weeks, and a significant ly bet t er improvement in qualit y of life as compared wit h placebo was not ed by brubaker l. et al., in 2008 [15]. dose -re l ati on sh i p effe cts: a durable efficacy for dose groups of 100 u or great er was report ed by dmochowski r, et al. in 2010 who performed a clinical t rial relat ed t o doses of 150 t o 300 u. in t hat st udy, doses great er t han 150 u cont ribut ed minimal addit ional or clinically relevant improvement in sympt oms and healt h-relat ed qualit y of life [16]. s i de effe cts: according t he met a-analysis of bt x-a in t reat ing idiopat hic det rusor overact ivit y pat ient s, bt x-a significant ly augment ed post -void residual volume (32.77 vs. 2.01), proport ions of urinary t ract infect ion (19.69% and 5.94), and proport ions of clean int ermit t ent cat het erizat ion (8.41% and 0.46%) versus placebo [29]. as relat ed t o t he age >61 years, low maximum flow rat e, low voiding efficiency (a percent age of t he voided volume compared t o t he prevoid bladder volume <90%), and large post -void residual volume at baseline has been report ed as risk fact ors for t hese adverse event s [30]. hemat oma, inject ion sit e pain, int ract able headache, pt osis, diplopia and hyperact ivit y of local ant agonist muscle could be ment ioned as t he t emporary and benign side effect s. dry and red eye, space st riving, dry mout h, abdominal t urbulences, dysphagia, t hroat iness and last ly breat hing difficult ies could be specified as pot ent ially serious event s [1-30]. http://www.ajecr.org/ 191 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org negligible advance or clinically pertinent improvement in signs and health-related quality of life [16]. in patients with intractable oab non-responsive to anticholinergics drugs, it seems to be trust worthy management. inoculated into the detrusor muscle, btx-a toxin is well tolerated with negligible threat of systemic side effects. a recent study showed that the injections of 100 units of btx-a in the submucosal coating of the bladder are ineffective in those with over active bladder (oab), while additions in the detrusor indicate to an imperative improve in signs of urgent and recurrent urination for six months [8]. btx-a is a strong neurotoxin that could discriminately modulate neurotransmitter discharge from the end of nerve, that could consequences to muscular paralysis. due to its' possible effect on sensory nerve it could have anti-inflammatory effect [18]. study performed in women with medication-resistant, urodynamic-confirmed idiopathic detrusor overactivity showed an improvement in the median scores from baseline to one month on the incontinence impact and urogenital distress inventory [19]. results of metaanalysis performed by duthie et al., in 2011 specified that however, the administered doses of 100 to 150 units of btx-a, seem to demonstrate beneficial effects, but prescription of 300 units of btx-a might have more effective and longer lasting, but with more side-effects. related to the type and doses of btx-a, its' effect might be last after 6-12 months. repeated injections of btx-a, do not seem to become refractory to btx-a [20]. diagnosis and treatment for bladder pain syndrome/interstitial cystitis (bps/ic) seems to be puzzling. neuhaus et al., in 2012 studied intravesical treatments of bps/ic and suggested that as a second line of therapy, btx-a injection, intravesical sodium hyaluronate instillation and dmso instillation suggested to be the best-performing managements [21]. pinto et al., in 2013, studied persistent therapeutic effect of repeated injections of onabotulinum toxin a in refractory bladder pain syndrome/interstitial cystitis. it was mentioned that mean decrease in pain score (compared to baseline 5.9 ± 1.8), o'leary-sant score (associated to baseline 28.8 ± 6.3), and urinary frequency (related to baseline 16.4 ± 5.3) and mean increase in voided volume (likened to baseline 112 ± 42 ml) and superiority of life were comparable after each management. individual symptom relief lasted 6 to 12 months with an average duration of 9.9 ± 2.4 months [22]. study related to repeated injections of btx-a (dysport) for 33 women with intractable detrusor overactivity was performed by abeywickrama et al. in 2014. in this study mean duration between the first and second injections was 15.2 ± 7.2 months, whereas between the second and third was 19.2 ± 10 months (p = 0.025). two women developed uti and required clean intermittent self-catheterization. three women required dose escalation to 750 units. longer duration of subjective quality of life improvement was stated among the second and third btx-a injections paralleled to period among the first and second injections [23]. a comparison between abo-btx-a and control patients at baseline and at 3 months of follow-up performed by manning et al., in 2014. based on their report, ols questionnaires exhibited upgrading at 3 months. only the o'leary-sant questionnaire consists of problem (ols-pi) was improved in the abobtxa group (p = 0.04). at 3 months, no difference was found in either leary-sant questionnaire consisting of problem and symptom (ols-si) or total ols score. twelve patients had urinary tract infection (uti) treated during the follow-up period, which confounded results. in the 38 patients without uti, there was improvement in total ols score (p = 0.02), ols-pi (0.08), and ols-si (p = 0.008) for the abo-btx-a group at 3 months. only five abo-btx-a compared with two control patients had a 50% reduction in ols score [24]. discussion in recent years, there has been an augmented consideration in the use of btx-a to treat medical conditions that were intractable to conservative management. according to publication by dhaked et al. in 2010, botulinum neurotoxin approaches a main bioweapon hazard because of: 1) its dangerous strength and lethality, 2) its easiness of production, 3) carriage and waste, and 4) the need for protracted concentrated maintenance between pretentious individuals. a single gram of crystalline toxin, calmly spread and inhaled, can kill more than one million people. the source of the remarkable power of botulinum toxin is enzymatic. the toxin is a zinc proteinase that slashes neuronal vesicle related proteins accountable for acetylcholine release into the neuromuscular junction. a valuable feature of btx study in modern years has been expansion of the strongest toxin into a molecule of important therapeutic usefulness. it is the first organic poison which is approved for dealing with human diseases [6]. previous published articles suggested that btx-a, could be effectual in cautiously selected group of patients and has negligible side effects profile and usually well accepted by numerous patients. btx-a is the most commonly used for treatment of luts. botox® is the onabotulinum toxin a (available in a 100 u or 200 u vials) that is available in united states. dysport® is the abobotulinum toxin a (available in a 3000 unit or 5000 u vials) that is available in the europe. 1 unit of onabotulinum toxin a is equivalent to approximately 3 to 5 unit of abobotulinum toxin a [2]. under cystoscopic management, an injection of 300 units of botox® (ranged from 100 to 400 units) in 30 injection sites (ranged from 15 to 40) of 10 units/ml (ranged from 6.7 to 25 units/ml) in the bladder, usually sparing the trigone has been reported by karsenty et al, in 2008 [25]. streeper et al. in 2016 based on periureteral injection of btx-a for stone passage in the porcine model, stated that, btx-a may offer a simple, office-based endoscopic management possibility for ureteral stones [26]. investigation performed by shim et al, in 2016, showed no differences in efficacy compared with placebo and also showed no difference in procedurerelated adverse events occurred after btx-a injection for luts/bph (benign prostatic hyperplasia) [27]. el-enen et al., in 2015 noted that btx-a could be used as the more effective option in patients with small prostate and short symptom duration and could be prescribed for http://www.ajecr.org/ 192 am j exp clin res, vol. 4, no. 1, 2017 http://www.ajecr.org management of patients with refractory chronic prostatitisassociated chronic pelvic-pain syndrome [28]. according the meta-analysis of btx-a in treating idiopathic detrusor overactivity patients, btx-a significantly augmented post-void residual volume (32.77 vs. 2.01), proportions of urinary tract infection (19.69% and 5.94), and proportions of clean intermittent catheterization (8.41% and 0.46%) versus placebo [29]. related to the age >61 years, low maximum flow rate, low voiding efficiency (a percentage of the voided volume compared to the pre-void bladder volume <90%), and large post-void residual volume at baseline were risk factors for these adverse events have been reported [30]. finally clarification and recognition of patients signs with over active bladder that not wellmanaged with more traditional treatments need a talented decision to achieve the precise approach for the use of btx-a injections. additional investigations in iranian population with such disorders seem to be advantageous. acknowledgement the authors are grateful to the isfahan university of medical sciences for supporting this study. conflict of interest the authors declare no conflicts of interest. references 1. hambleton p. clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use. j neurol 239:1620, 1992. 2. omprakash hm, rajendran sc. botulinum 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research am j exp clin res 2019;6(4):364-377 original article saxitoxin time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory investigation of vibronic–mode coupling structure in vibrational spectra analysis alireza heidari1,2*, jennifer esposito1, angela caissutti1 1faculty of chemistry, california south university, comet st. irvine, california, usa 2american international standards institute, irvine, california, usa abstract. saxitoxin (stx) is a potent neurotoxin and the best–known paralytic shellfish toxin (pst). ingestion of saxitoxin by humans, usually by consumption of shellfish contaminated by toxic algal blooms, is responsible for the illness known as paralytic shellfish poisoning (psp). parameters such as ft–ir and raman vibrational wavelengths and intensities for single crystal saxitoxin (stx) are calculated using density functional theory and were compared with empirical results. the investigation about vibrational spectrum of cycle dimers in crystal with carboxyl groups from each molecule of acid was shown that it leads to create hydrogen bonds for adjacent molecules. the current study aimed to investigate the possibility of simulating the empirical values. analysis of vibrational spectrum of saxitoxin (stx) is performed based on theoretical simulation and ft–ir empirical spectrum and raman empirical spectrum using density functional theory in levels of hf/6–31g*, hf/6–31++g**, mp2/6–31g, mp2/6–31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6–31g and b3lyp6– 31–heg**. vibration modes of methylene, carboxyl acid and phenyl cycle are separately investigated. the obtained values confirm high accuracy and validity of results obtained from calculations. keywords: vibronic structure, vibrational spectra analysis, density functional theory (dft), saxitoxin (stx), non–focal functions, becke, correlation functions, lee–yang–parr, time–resolved absorption, resonance, ft–ir, raman biospectroscope introduction saxitoxin (stx) is a potent neurotoxin and the best– known paralytic shellfish toxin (pst). ingestion of saxitoxin by humans, usually by consumption of shellfish contaminated by toxic algal blooms, is responsible for the illness known as paralytic shellfish poisoning (psp). density functional theory (dft) is one of the most powerful calculation methods for electronic structures [5– 7]. numerous results have been previously studied and indicate successful use of these methods [8–10]. the theory is one of the most appropriate methods for simulating the vibrational wavenumbers, molecular structure as well as total energy. it may be useful to initially consider the calculated results by density functional theory using hf/6– 31g*, hf/6–31++g**, mp2/6–31g, mp2/6–31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6–31g and b3lyp6–31–heg** approach [11–16]. it should be noted that calculations are performed by considering one degree of quantum interference as well as polarization effects of 2d orbitals in interaction [17–244]. details of calculations all calculations of molecular orbital in the base of ab are performed by gaussian 09. in calculation process, the structure of saxitoxin (stx) molecule (fig. 1) is optimized and ft–ir and raman wavenumbers are calculated using hf/6–31g*, hf/6–31++g**, mp2/6–31g, mp2/6– 31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6– 31g and b3lyp6–31–heg** base. all optimized structures are adjusted with minimum energy. harmonic vibrational wavenumbers are calculated using second degree of derivation to adjust convergence on potential surface as good as possible and to evaluate vibrational energies at zero point. in optimized structures considered in the current study, virtual frequency modes are not observed which indicates that the minimum potential energy surface is correctly chosen. the optimized geometry is calculated by minimizing the energy relative to all geometrical quantities without forcing any constraint on molecular symmetry. calculations were performed by gaussian 09. the current calculation is aimed to maximize structural optimization using density functional theory. the calculations of density functional theory are performed by hf/6–31g*, hf/6–31++g**, mp2/6–31g, mp2/6–31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6–31g and b3lyp6–31–heg** function in which ___________________________________________________________ * corresponding author: prof. alireza heidari (alireza.heidari@calsu.us). http://www.ajecr.org/ mailto:alireza.heidari@calsu.us 365 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org figure 1 different sections of the saxitoxin (stx) [1–93]. non–focal functions of becke and correlation functions of lee–yang–parr beyond the franck–condon approximation are used. after completion of optimization process, the second order derivation of energy is calculated as a function of core coordination and is investigated to evaluate whether the structure is accurately minimized. vibrational frequencies used to simulate spectrums presented in the current study are derived from these second order derivatives. all calculations are performed for room temperature of 317(k). vibration analysis analysis of vibrational spectrum of saxitoxin (stx) is performed based on theoretical simulation and ft–ir empirical spectrum and raman empirical spectrum using density functional theory in levels of hf/6–31g*, hf/6– 31++g**, mp2/6–31g, mp2/6–31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6–31g and b3lyp6–31– heg**. vibration modes of methylene, carboxyl acid and phenyl cycle are separately investigated. c–h stretching vibrations in single replacement of benzene cycles are usually seen in band range of 3500–4000 cm–1. weak raman bands are at 3200 cm–1 and 3210 cm–1. c–c stretching mode is a strong raman mode at 1200 cm– 1. raman weak band is seen at 1673 cm–1, too. bending mode of c–h is emerged as a weak mode at 1400 cm–1 and 1410 cm–1 and a strong band at 1300 cm–1 in raman spectrum. raman is considerably active in the range of 1500–2000 cm–1 which 1220 cm–1 indicates this issue. c–h skew–symmetric stretching mode of methylene group is expected at 3190 cm–1 and its symmetric mode is expected at 3000 cm–1. skew–symmetric stretching mode of ch2 in saxitoxin (stx) has a mode in mid–range of raman spectrum at 3000–3550 cm–1. when this mode is symmetric, it is at 3100 cm–1 and is sharp. the calculated wavenumbers of higher modes are at 3050 cm–1 and 3150 cm–1 for symmetric and skew–symmetric stretching mode of methylene, respectively. scissoring vibrations of ch2 are usually seen at the range of 1550-1600 cm–1 which often includes mid–range bands. weak bands at 1550 cm–1 are scissoring modes of ch2 in raman spectrum. moving vibrations of methylene are usually seen at 1470 cm–1. for the investigated chemical in the current study, these vibrations are at 1340 cm–1 were calculated using density functional theory. twisting and rocking vibrations of ch2 are seen in raman spectrum at 950 cm–1 and 1190 cm–1, respectively, which are in good accordance with the results at 900 cm–1 and 1200 cm–1, respectively. in a non–ionized carboxyl group (cooh), stretching vibrations of carbonyl [c=o] are mainly observed at the range of 1850–1950 cm–1. if dimer is considered as an intact constituent, two stretching vibrations of carbonyl for symmetric stretching are at 1750–1800 cm–1 in raman spectrum. in the current paper, stretching vibration of carbonyl mode is at 1810 cm–1 which is a mid–range value. stretching and bending bands of hydroxyl can be identified by width and band intensity which in turn is dependent on bond length of hydrogen. in dimer form of hydrogen bond, stretching band of o–h is of a strong raman peak at 1370 cm–1 which is due to in–plain metamorphosis mode. out–of–plain mode of o–h group is a very strong mode of peak at 1050 cm–1 of raman spectrum. the stretching mode of c–o (h) emerges as a mid–band of raman spectrum at 1250 cm–1. lattice vibrations are usually seen at the range of 0-850 cm–1. these modes are induced by rotary and transferring vibrations of molecules and vibrations and are including hydrogen bond. bands with low wave numbers of hydrogen bond vibrations in ft–ir and raman spectrum (fig. 2) are frequently weak, width and unsymmetrical. http://www.ajecr.org/ 366 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org figure 2 3d simulation of (a) ft–ir spectrum and (b) raman spectrum of saxitoxin (stx). rotary lattice vibrations are frequently stronger than transferring ones. intra–molecular vibrations with low wavenumbers involving two–bands o–h …o dimer at 90 cm–1, 200 cm–1 and 250 cm–1 are attributed to a rotary moving of two molecules involving in–plain rotation of molecules against each other. summary and conclusion calculations of density functional theory using hf/6– 31g*, hf/6–31++g**, mp2/6–31g, mp2/6–31++g**, blyp/6–31g, blyp/6–31++g**, b3lyp/6–31g and b3lyp6–31–heg** levels were used to obtain vibrational wavenumbers and intensities in single crystal of saxitoxin (stx). investigation and consideration of vibrational spectrum confirm the formation of dimer cycles in the investigated crystal with carboxyl groups from each hydrogen molecule of acid protected from adjacent molecules. the calculated vibrational spectrum which obtains from calculations of density functional theory is in good accordance with recorded empirical values which indicates successful simulation of the problem. the obtained results indicate that the results obtained from theoretical calculations are valid through comparing with empirical recorded results. acknowledgement authors are supported by an american international standards institute (aisi) future fellowship grant ft1201009373495. we acknowledge ms. isabelle villena for instrumental support and dr. michael n. cocchi for constructing graphical abstract figure. we gratefully acknowledge prof. dr. christopher brown for proof reading the manuscript. conflict of interest the authors certify 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adv technol 7: e007, 2016. 4. heidari a. extraction and preconcentration of n– tolyl–sulfonyl–phosphoramid–saeure–dichlorid as an anti– cancer drug from plants: a pharmacognosy study. j pharmacogn nat prod 2: e103, 2016. 5. heidari a. a thermodynamic study on hydration and dehydration of dna and rna−amphiphile complexes. j bioeng biomed sci s: 006, 2016. 6. heidari a. computational studies on molecular structures and carbonyl and ketene groups’ sffects of singlet and triplet energies of azidoketene o=c=ch–nnn and isocyanatoketene o=c=ch–n=c=o. j appl computat math 5: e142, 2016. 7. heidari a. study of irradiations to enhance and induces the dissociation of hydrogen bonds between peptide chains and transition from helix structure to random coil structure using atr–ftir, raman and 1hnmr spectroscopies. j biomol res ther 5: e146, 2016. 8. heidari a. future prospects of point fluorescence spectroscopy, fluorescence imaging and fluorescence endoscopy in photodynamic therapy (pdt) for cancer 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study”, arch cancer res. 4: 1, 2016. 14. heidari a. biospectroscopic study on multi– component reactions (mcrs) in two a–type and b–type conformations of nucleic acids to determine ligand binding modes, binding constant and stability of nucleic acids in cadmium oxide (cdo) nanoparticles–nucleic acids complexes as anti–cancer drugs. arch cancer res 4:2, 2016. 15. heidari a. simulation of temperature distribution of dna/rna of human cancer cells using time–dependent bio–heat equation and nd: yag lasers. arch cancer res 4: 2, 2016. 16. heidari a. quantitative structure–activity relationship (qsar) approximation for cadmium oxide (cdo) and rhodium (iii) oxide (rh2o3) nanoparticles as anti–cancer drugs for the catalytic formation of proviral dna from viral rna using multiple linear and non–linear correlation approach. ann clin lab res 4: 1, 2016. 17. heidari a. biomedical study of cancer cells dna therapy using laser irradiations at presence of intelligent nanoparticles. j biomedical sci 5: 2, 2016. 18. heidari a. measurement the amount of vitamin d2 (ergocalciferol), vitamin d3 (cholecalciferol) and absorbable calcium (ca2+), iron (ii) (fe2+), magnesium (mg2+), phosphate (po4–) and zinc (zn2+) in apricot using high–performance liquid chromatography (hplc) and spectroscopic techniques. j biom biostat 7: 292, 2016. 19. heidari a. spectroscopy and quantum mechanics of the helium dimer (he2+), neon dimer (ne2+), argon dimer (ar2+), krypton dimer (kr2+), xenon dimer (xe2+), radon dimer(rn2+) and ununoctium dimer (uuo2+) molecular cations. chem sci j 7: e112, 2016. 20. heidari a. human toxicity photodynamic therapy studies on dna/rna complexes as a promising new sensitizer for the treatment of malignant tumors using bio– spectroscopic techniques. j drug metab toxicol 7: e129, 2016. 21. heidari a. molecular dynamics and monte–carlo simulations for replacement sugars in insulin resistance, obesity, ldl cholesterol, triglycerides, metabolic syndrome, type 2 diabetes and cardiovascular disease: a glycobiological study. j glycobiol 5: e111, 2016. 22. a. heidari, “synthesis and study of 5– [(phenylsulfonyl)amino]–1,3,4–thiadiazole–2–sulfonamide as potential anti–pertussis drug using chromatography and spectroscopy techniques. transl med 6:e138, 2016. 23. heidari a. nitrogen, oxygen, phosphorus and sulphur heterocyclic anti–cancer nano drugs separation in the supercritical fluid of ozone (o3) using soave–redlich– kwong (srk) and pang–robinson (pr) equations. electronic j biol 12: 4, 2016. 24. heidari a. an analytical and computational infrared spectroscopic review of vibrational modes in nucleic acids. austin j anal pharm chem 3: 1058, 2016. 25. heidari a, brown c. phase, composition and morphology study and analysis of os–pd/hfc nanocomposites. nano res appl. 2: 1, 2016. 26. heidari a, brown c. vibrational spectroscopic study of intensities and shifts of symmetric vibration modes of ozone diluted by cumene. int j advanced chem 4:5-9, 2016. 27. heidari a. study of the role of anti–cancer molecules with different sizes for decreasing corresponding bulk tumor multiple organs or tissues. arch can res. 4: 2, 2016. 28. heidari a. genomics and proteomics studies of zolpidem, necopidem, alpidem, saripidem, miroprofen, zolimidine, olprinone and abafungin as anti–tumor, peptide antibiotics, antiviral and central nervous system (cns) drugs. j data mining genomics & proteomics 7: e125, 2016. 29. heidari a. pharmacogenomics and pharmacoproteomics studies of phosphodiesterase–5 (pde5) inhibitors and paclitaxel albumin–stabilized nanoparticles as sandwiched anti–cancer nano drugs between two dna/rna molecules of human cancer cells. j pharmacogenomics pharmacoproteomics 7: e153, 2016. 30. heidari a. biotranslational medical and biospectroscopic studies of cadmium oxide (cdo) nanoparticles–dna/rna straight and cycle chain complexes as potent anti–viral, anti–tumor and anti–microbial drugs: a clinical approach. transl biomed. 7: 2, 2016. 31. heidari a. a comparative study on simultaneous determination and separation of adsorbed cadmium oxide (cdo) nanoparticles on dna/rna of human cancer cells using biospectroscopic techniques and dielectrophoresis (dep) method. arch can res. 4: 2, 2016. 32. heidari a. cheminformatics and system chemistry of cisplatin, carboplatin, nedaplatin, oxaliplatin, heptaplatin and lobaplatin as anti–cancer nano drugs: a combined computational and experimental study. j inform data min 1: 3, 2016. 33. heidari a. linear and non–linear quantitative structure–anti–cancer–activity relationship (qsacar) study of hydrous ruthenium (iv) oxide (ruo2) nanoparticles as non–nucleoside reverse transcriptase inhibitors (nnrtis) and anti–cancer nano drugs j integr oncol 5: e110, 2016. 44. heidari a. synthesis, characterization and biospectroscopic studies of cadmium oxide (cdo) nanoparticles–nucleic acids complexes absence of soluble polymer as a protective agent using nucleic acids condensation and solution reduction method. j nanosci curr res 1: e101, 2016. http://www.ajecr.org/ http://cheminformatics.conferenceseries.com/ http://cheminformatics.conferenceseries.com/ 368 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org 45. heidari a. coplanarity and collinearity of 4’– dinonyl–2,2’–bithiazole in one domain of bleomycin and pingyangmycin to be responsible for binding of cadmium oxide (cdo) nanoparticles to dna/rna bidentate ligands as anti–tumor nano drug. int j drug dev & res 8: 007-008, 2016. 46. heidari a. a pharmacovigilance study on linear and non–linear quantitative structure (chromatographic) retention relationships (qsrr) models for the prediction of retention time of anti–cancer nano drugs under synchrotron radiations. j pharmacovigil 4: e161, 2016. 47. heidari a. nanotechnology in preparation of semipermeable polymers. j adv chem eng 6: 157, 2016. 48. heidari a. a gastrointestinal study on linear and non–linear quantitative structure (chromatographic) retention relationships (qsrr) models for analysis 5– aminosalicylates nano particles as digestive system nano drugs under synchrotron radiations. j gastrointest dig syst 6: e119, 2016. 49. heidari a. dna/rna fragmentation and cytolysis in human cancer cells treated with diphthamide nano particles derivatives. biomedical data mining 5:e102, 2016. 50. heidari a. a successful strategy for the prediction of solubility in the construction of quantitative structure– activity relationship (qsar) and quantitative structure– property relationship (qspr) under synchrotron radiations using genetic function approximation (gfa) algorithm. j mol biol biotechnol 1: 1, 2016. 51. heidari a. computational study on molecular structures of c20, c60, c240, c540, c960, c2160 and c3840 fullerene nano molecules under synchrotron radiations using fuzzy logic. j material sci eng 5: 282, 2016. 52. heidari a. graph theoretical analysis of zigzag polyhexamethylene biguanide, polyhexamethylene adipamide, polyhexamethylene biguanide gauze and polyhexamethylene biguanide hydrochloride (phmb) boron nitride nanotubes (bnnts), amorphous boron nitride nanotubes (a–bnnts) and hexagonal boron nitride nanotubes (h– bnnts). j appl computat math 5:e143, 2016. 53. heidari a. the impact of high resolution imaging on diagnosis. int j clin med imaging 3:1000e101, 2016. 54. heidari a. a comparative study of conformational behavior of isotretinoin (13–cis retinoic acid) and tretinoin (all–trans retinoic acid (atra)) nano particles as anti–cancer nano drugs under synchrotron radiations using hartree–fock (hf) and density functional theory (dft) methods. insights in biomed 1:2, 2016. 55. a. heidari, “advances in logic, operations and computational mathematics”, j appl computat math 5: 5, 2016. 56. heidari a. mathematical equations in predicting physical behavior. j appl computat math 5:5, 2016. 57. heidari a. chemotherapy a last resort for cancer treatment”, chemo open access 5: 4, 2016. 58. heidari a. separation and pre–concentration of metal cations–dna/rna chelates using molecular beam mass spectrometry with tunable vacuum ultraviolet (vuv) synchrotron radiation and various analytical methods. mass spectrom purif tech 2:e101, 2016. 59. heidari a. yoctosecond quantitative structure– activity relationship (qsar) and quantitative structure– property relationship (qsar) under synchrotron radiations studies for prediction of solubility of anti–cancer nano drugs in aqueous solutions using genetic function approximation (gfa) algorithm. insight pharm res. 1:1, 2016. 60. heidari a. cancer risk prediction and assessment in human cells under synchrotron radiations using quantitative structure activity relationship (qsar) and quantitative structure properties relationship (qspr) studies. int j clin med imaging 3:516, 2016. 61. heidaria. a novel approach to biology. electronic j biol 12: 4, 2016. 62. heidari a. innovative biomedical equipment’s for diagnosis and treatment. j bioengineer & biomedical sci 6: 2, 2016. 63. heidari a. integrating precision cancer medicine into healthcare, medicare reimbursement changes and the practice of oncology: trends in oncology medicine and practices”, j oncol med & pract 1: 2, 2016. 64. heidari a. promoting convergence in biomedical and biomaterials sciences and silk proteins for biomedical and biomaterials applications: an introduction to materials in medicine and bioengineering perspectives. j bioengineer & biomedical sci 6: 3, 2016. 65. heidari a. x–ray fluorescence and x–ray diffraction analysis on discrete element modeling of nano powder metallurgy processes in optimal container design. j powder metall min 6: 1, 2017. 66. heidari a. biomolecular spectroscopy and dynamics of nano–sized molecules and clusters as cross– linking–induced anti–cancer and immune–oncology nano drugs delivery in dna/rna of human cancer cells’ membranes under synchrotron radiations: a payload–based perspective. arch chem res. 1: 2, 2017. 67. heidari a. deficiencies in repair of double– standard dna a/rna–binding molecules identified in many types of solid and liquid tumors oncology in human body for advancing cancer immunotherapy using computer simulations and data analysis: number of mutations in a synchronous tumor varies by age and type of synchronous cancer. j appl bioinforma comput biol, 6: 1, 2017. 68. heidari a. electronic coupling among the five nanomolecules shuts down quantum tunneling in the presence and absence of an applied magnetic field for indication of the dimer or other provide different influences on the magnetic behavior of single molecular magnets (smms) as qubits for quantum computing. glob j res rev. 4: 2, 2017. 69. heidari a. polymorphism in nano–sized graphene ligand–induced transformation of au38–xagx/xcux(sph– tbu)24 to au36–xagx/xcux(sph–tbu)24 (x = 1–12) nanomolecules for synthesis of au144–xagx/xcux[(sr)60, (sc4)60, (sc6)60, (sc12)60, (pet)60, (p–mba)60, (f)60, (cl)60, (br)60, (i)60, (at)60, (uus)60 and (sc6h13)60] nano clusters as anti–cancer nano drugs. j nanomater mol nanotechnol, 6: 3, 2017. 70. heidari a. biomedical resource oncology and data mining to enable resource discovery in medical, medicinal, clinical, pharmaceutical, chemical and translational http://www.ajecr.org/ http://www.omicsonline.com/open-access/nanotechnology-in-preparation-of-semipermeable-polymers-2090-4568-1000157.php?aid=78336 http://www.omicsonline.com/open-access/nanotechnology-in-preparation-of-semipermeable-polymers-2090-4568-1000157.php?aid=78336 369 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org research and their applications in cancer research. int j biomed data min 6: e103, 2017. 71. heidari a. study of synthesis, pharmacokintics, pharmacodynamics, dosing, stability, safety and efficacy of olympiadane nanomolecules as agent for cancer enzymotherapy, immunotherapy, chemotherapy, radiotherapy, hormone therapy and targeted therapy under synchrotorn radiation. j dev drugs 6: e154, 2017. 72. heidari a. a novel approach to future horizon of top seven biomedical research topics to watch in 2017: alzheimer's, ebola, hypersomnia, human immunodeficiency virus (hiv), tuberculosis (tb), microbiome/antibiotic resistance and endovascular stroke. j bioengineer & biomedical sci 7: e127, 2017. 73. heidari a. opinion on computational fluid dynamics (cfd) technique. fluid mech open acc 4: 157, 2017. 74. heidari a. concurrent diagnosis of oncology influence outcomes in emergency general surgery for colorectal cancer and multiple sclerosis (ms) treatment using magnetic resonance imaging (mri) and au329(sr)84, au329–xagx(sr)84, au144(sr)60, au68(sr)36, au30(sr)18, au102(sph)44, au38(sph)24, au38(sc2h4ph)24, au21s (sadm)15, au36(pmba)24 and au25(pmba)18 nano clusters. j surgery emerg med 1: 21, 2017. 75. heidari a. developmental cell biology in adult stem cells death and autophagy to trigger a preventive allergic reaction to common airborne allergens under synchrotron radiation using nanotechnology for therapeutic goals in particular allergy shots (immunotherapy). cell biol (henderson, nv) 6: 1, 2017. 76. heidari a. changing metal powder characteristics for elimination of the heavy metals toxicity and diseases in disruption of extracellular matrix (ecm) proteins adjustment in cancer metastases induced by osteosarcoma, chondrosarcoma, carcinoid, carcinoma, ewing’s sarcoma, fibrosarcoma and secondary hematopoietic solid or soft tissue tumors. j powder metall min 6: 170, 2017. 77. heidari a. nanomedicine–based combination anti– cancer therapy between nucleic acids and anti–cancer nano drugs in covalent nano drugs delivery systems for selective imaging and treatment of human brain tumors using hyaluronic acid, alguronic acid and sodium hyaluronate as anti–cancer nano drugs and nucleic acids delivery under synchrotron radiation. am j drug deliv 5: 2, 2017. 78. heidari a. clinical trials of dendritic cell therapies for cancer exposing vulnerabilities in human cancer cells’ metabolism and metabolomics: new discoveries, unique features inform new therapeutic opportunities, biotech's bumpy road to the market and elucidating the biochemical programs that support cancer initiation and progression. j biol med science 1: e103, 2017. 79. heidari a. the design graphene–based nanosheets as a new nanomaterial in anti–cancer therapy and delivery of chemotherapeutics and biological nano drugs for liposomal anti–cancer nano drugs and gene delivery. br biomed bull 5: 305, 2017. 80. heidari a. integrative approach to biological networks for emerging roles of proteomics, genomics and transcriptomics in the discovery and validation of human colorectal cancer biomarkers from dna/rna sequencing data under synchrotron radiation. transcriptomics 5: e117, 2017. 81. heidari a. elimination of the heavy metals toxicity and diseases in disruption of extracellular matrix (ecm) proteins and cell adhesion intelligent nanomolecules adjustment in cancer metastases using metalloenzymes and under synchrotron radiation. lett health biol sci 2: 1–4, 2017. 82. heidari a. treatment of breast cancer brain metastases through a targeted nanomolecule drug delivery system based on dopamine functionalized multi–wall carbon nanotubes (mwcnts) coated with nano graphene oxide (go) and protonated polyaniline (pani) in situ during the polymerization of aniline autogenic nanoparticles for the delivery of anti–cancer nano drugs under synchrotron radiation. br j res, 4: 16, 2017. 83. heidari a. sedative, analgesic and ultrasound– mediated gastrointestinal nano drugs delivery for gastrointestinal endoscopic procedure, nano drug–induced gastrointestinal disorders and nano drug treatment of gastric acidity. res rep gastroenterol, 1: 1, 2017. 84. heidari a. synthesis, pharmacokinetics, pharmacodynamics, dosing, stability, safety and efficacy of orphan nano drugs to treat high cholesterol and related conditions and to prevent cardiovascular disease under synchrotron radiation. j pharm sci emerg drugs 5: 1, 2017. 85. heidari a. non–linear compact proton synchrotrons to improve human cancer cells and tissues treatments and diagnostics through particle therapy accelerators with monochromatic microbeams. j cell biol mol sci 2: 1–5, 2017. 86. heidari a. design of targeted metal chelation therapeutics nanocapsules as colloidal carriers and blood– brain barrier (bbb) translocation to targeted deliver anti– cancer nano drugs into the human brain to treat alzheimer’s disease under synchrotron radiation. j nanotechnol material sci 4: 1–5, 2017. 87. gobato r, a. heidari a. calculation using quantum chemistry for inorganic molecule simulation beli2sesi”, sci j analyt chem 5’76-85, 2017. 88. heidari a. different high–resolution simulations of medical, medicinal, clinical, pharmaceutical and therapeutics oncology of human lung cancer translational anti–cancer nano drugs delivery treatment process under synchrotron and x–ray radiations. j med oncol 1: 1, 2017. 89. heidari a. a modern ethnomedicinal technique for transformation, prevention and treatment of human malignant gliomas tumors into human benign gliomas tumors under synchrotron radiation. am j ethnomed 4: 10, 2017. 90. heidari a. active targeted nanoparticles for anti– cancer nano drugs delivery across the blood–brain barrier for human brain cancer treatment, multiple sclerosis (ms) and alzheimer's diseases using chemical modifications of anti–cancer nano drugs or drug–nanoparticles through zika virus (zikv) nanocarriers under synchrotron radiation. j med chem toxicol 2: 1–5, 2017. 91. heidari a. investigation of medical, medicinal, clinical and pharmaceutical applications of estradiol, http://www.ajecr.org/ http://www.nature.com/nrm/series/adultstemcells/index.html http://www.nature.com/nrm/series/adultstemcells/index.html https://en.wikipedia.org/wiki/allergic_reaction https://en.wikipedia.org/wiki/allergic_reaction http://www.nyas.org/events/detail.aspx?cid=48653b97-3418-4dc6-9f1f-32befeca4c5f http://www.nyas.org/events/detail.aspx?cid=48653b97-3418-4dc6-9f1f-32befeca4c5f https://en.wikipedia.org/wiki/dosing https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=36&cad=rja&uact=8&ved=0ahukewic3_7xm4tvahwfuxqkhdtxacs4hhawcdywbq&url=http%3a%2f%2fwww.danfysik.com%2fen%2fsolutions%2fparticle-therapy-accelerators%2f&usg=afqjcnffa8ehy66scobwnmgssdkxmjdzdg https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=36&cad=rja&uact=8&ved=0ahukewic3_7xm4tvahwfuxqkhdtxacs4hhawcdywbq&url=http%3a%2f%2fwww.danfysik.com%2fen%2fsolutions%2fparticle-therapy-accelerators%2f&usg=afqjcnffa8ehy66scobwnmgssdkxmjdzdg 370 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org mestranol (norlutin), norethindrone (net), norethisterone acetate (neta), norethisterone enanthate (nete) and testosterone nanoparticles as biological imaging, cell labeling, anti–microbial agents and anti–cancer nano drugs in nanomedicines based drug delivery systems for anti– cancer targeting and treatment’ parana j sci edu 3:10–19, 2017. 92. heidari a. a comparative computational and experimental study on different vibrational biospectroscopy methods, techniques and applications for human cancer cells in tumor tissues simulation, modeling, research, diagnosis and treatment. open j anal bioanal chem 1: 014–020, 2017. 93. heidari a. combination of dna/rna ligands and linear/non–linear visible–synchrotron radiation–driven n– doped ordered mesoporous cadmium oxide (cdo) nanoparticles photocatalysts channels resulted in an interesting synergistic effect enhancing catalytic anti– cancer activity. enz eng 6: 1, 2017. 94. heidari a. modern approaches in designing ferritin, ferritin light chain, transferrin, beta–2 transferrin and bacterioferritin–based anti–cancer nano drugs encapsulating nanosphere as dna–binding proteins from starved cells (dps). mod appro drug des. 1: madd.000504. 2017. 95. heidari a. potency of human interferon β–1a and human interferon β–1b in enzymotherapy, immunotherapy, chemotherapy, radiotherapy, hormone therapy and targeted therapy of encephalomyelitis disseminate/multiple sclerosis (ms) and hepatitis a, b, c, d, e, f and g virus enter and targets liver cells. j proteomics enzymol 6: 1, 2017. 96. heidari a. transport therapeutic active targeting of human brain tumors enable anti–cancer nanodrugs delivery across the blood–brain barrier (bbb) to treat brain diseases using nanoparticles and nanocarriers under synchrotron radiation. j pharm pharmaceutics 4: 1-5, 2017. 97. heidari a, brown c. combinatorial therapeutic approaches to dna/rna and benzylpenicillin (penicillin g), fluoxetine hydrochloride (prozac and sarafem), propofol (diprivan), acetylsalicylic acid (asa) (aspirin), naproxen sodium (aleve and naprosyn) and dextromethamphetamine nanocapsules with surface conjugated dna/rna to targeted nano drugs for enhanced anti–cancer efficacy and targeted cancer therapy using nano drugs delivery systems. ann adv chem. 1:61-69, 2017. 98. heidari a. high–resolution simulations of human brain cancer translational nano drugs delivery treatment process under synchrotron radiation. j transl res 1: 1–3, 2017. 99. heidari a. investigation of anti–cancer nano drugs’ effects’ trend on human pancreas cancer cells and tissues prevention, diagnosis and treatment process under synchrotron and x–ray radiations with the passage of time using mathematica. current trends anal bioanal chem 1: 36-41, 2017. 100. heidari a. pros and cons controversy on molecular imaging and dynamics of double–standard dna/rna of human preserving stem cells–binding nano molecules with androgens/anabolic steroids (aas) or testosterone derivatives through tracking of helium–4 nucleus (alpha particle) using synchrotron radiation. arch biotechnol biomed 1:67-100, 2017. 101. heidari a. visualizing metabolic changes in probing human cancer cells and tissues metabolism using vivo 1h or proton nmr, 13c nmr, 15n nmr and 31p nmr spectroscopy and self–organizing maps under synchrotron radiation. soj mater sci eng 5:1-6, 2017. 102. heidari a. cavity ring–down spectroscopy (crds), circular dichroism spectroscopy, cold vapour atomic fluorescence spectroscopy and correlation spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. enliven: challenges cancer detect ther 4: e001, 2017. 103. heidari a. laser spectroscopy, laser–induced breakdown spectroscopy and laser–induced plasma spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. int j hepatol gastroenterol 3: 79-84, 2017. 104. heidari a. time–resolved spectroscopy and time– stretch spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. enliven: pharmacovigilance drug safety 4:e001, 2017. 105. heidari a. overview of the role of vitamins in reducing negative effect of decapeptyl (triptorelin acetate or pamoate salts) on prostate cancer cells and tissues in prostate cancer treatment process through transformation of malignant prostate tumors into benign prostate tumors under synchrotron radiation. open j anal bioanal chem 1: 021–026, 2017. 106. heidari a. electron phenomenological spectroscopy, electron paramagnetic resonance (epr) spectroscopy and electron spin resonance (esr) spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. austin j anal pharm chem 4: 1091, 2017. 107. heidari a. therapeutic nanomedicine different high–resolution experimental images and computational simulations for human brain cancer cells and tissues using nanocarriers deliver dna/rna to brain tumors under synchrotron radiation with the passage of time using mathematica and matlab. madridge j nano tech sci 2:77–83, 2017. 108. heidari a. a consensus and prospective study on restoring cadmium oxide (cdo) nanoparticles sensitivity in recurrent ovarian cancer by extending the cadmium oxide (cdo) nanoparticles–free interval using synchrotron radiation therapy as antibody–drug conjugate for the treatment of limited–stage small cell diverse epithelial cancers. cancer clin res rep 1:2, e001, 2017. 109. heidari a. a novel and modern experimental imaging and spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under white synchrotron radiation. cancer sci res open access 4:1–8, 2017. 110. heidari a. different high–resolution simulations of medical, medicinal, clinical, pharmaceutical and http://www.ajecr.org/ 371 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org therapeutics oncology of human breast cancer translational nano drugs delivery treatment process under synchrotron and x–ray radiations. j oral cancer res 1:12–17, 2017. 111. heidari a. vibrational decihertz (dhz), centihertz (chz), millihertz (mhz), microhertz (μhz), nanohertz (nhz), picohertz (phz), femtohertz (fhz), attohertz (ahz), zeptohertz (zhz) and yoctohertz (yhz) imaging and spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. international j biomed 7:335–340, 2017. 112. heidari a. force spectroscopy and fluorescence spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. ec cancer 2:239–246, 2017. 113. heidari a. photoacoustic spectroscopy, photoemission spectroscopy and photothermal spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation”, baoj cancer res ther 3:045–052, 2017. 114. heidari a. j–spectroscopy, exchange spectroscopy (exsy), nuclear overhauser effect spectroscopy (noesy) and total correlation spectroscopy (tocsy) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. ems eng sci j 1:006–013, 2017. 115. heidari a. neutron spin echo spectroscopy and spin noise spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. int j biopharm sci 1: 103–107, 2017. 116. heidari a. vibrational decahertz (dahz), hectohertz (hhz), kilohertz (khz), megahertz (mhz), gigahertz (ghz), terahertz (thz), petahertz (phz), exahertz (ehz), zettahertz (zhz) and yottahertz (yhz) imaging and spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. madridge j anal sci instrum 2:41– 46, 2017. 117. heidari a. two–dimensional infrared correlation spectroscopy, linear two–dimensional infrared spectroscopy and non–linear two–dimensional infrared spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. j mater sci nanotechnol 6: 101, 2018. 118. heidari a. fourier transform infrared (ftir) spectroscopy, near–infrared spectroscopy (nirs) and mid– infrared spectroscopy (mirs) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. int j nanotechnol nanomed 3:1–6, 2018. 119. heidari a. infrared photo dissociation spectroscopy and infrared correlation table spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. austin pharmacol pharm 3:1011, 2018. 120. heidari a. novel and transcendental prevention, diagnosis and treatment strategies for investigation of interaction among human blood cancer cells, tissues, tumors and metastases with synchrotron radiation under anti–cancer nano drugs delivery efficacy using matlab modeling and simulation. madridge j nov drug res 1:18– 24, 2017. 121. heidari a. comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. open access j trans med res 2:00026–00032, 2018. 122. gobato mrr, gobato r, heidari a. planting of jaboticaba trees for landscape repair of degraded area”, landscape architecture and regional planning. 3:1–9, 2018. 123. a. heidari a. fluorescence spectroscopy, phosphorescence spectroscopy and luminescence spectro-scopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. sm j clin. med. imaging 4:1018, 2018. 124. heidari a. nuclear inelastic scattering spectroscopy (niss) and nuclear inelastic absorption spectroscopy (nias) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. int j pharm sci 2:1–14, 2018. 125. heidari a. x–ray diffraction (xrd), powder x– ray diffraction (pxrd) and energy–dispersive x–ray diffraction (edxrd) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. j oncol res 2:1–14, 2018. 126. heidari a. correlation two–dimensional nuclear magnetic resonance (nmr) (2d–nmr) (cosy) imaging and spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. ems can sci 1–1–001, 2018. 127. heidari a. thermal spectroscopy, photothermal spectroscopy, thermal microspectroscopy, photothermal microspectroscopy, thermal macrospectroscopy and photothermal macrospectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. sm j biometrics biostat 3:1024, 2018. 128. heidari a. a modern and comprehensive experimental biospectroscopic comparative study on human common cancers’ cells, tissues and tumors before and after synchrotron radiation therapy. open acc j oncol med 1:2018. 129. heidari a. heteronuclear correlation experiments such as heteronuclear single–quantum correlation spectroscopy (hsqc), heteronuclear multiple–quantum correlation spectroscopy (hmqc) and heteronuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human endocrinology and thyroid cancer cells and tissues under synchrotron radiation. j endocrinol thyroid res 3:555603, 2018. 130. heidari a. nuclear resonance vibrational spectroscopy (nrvs), nuclear inelastic scattering spectroscopy (niss), nuclear inelastic absorption spectroscopy (nias) and nuclear resonant inelastic x–ray scattering spectroscopy (nrixss) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. int j bioorg chem mol biol 6:1–5, 2018. http://www.ajecr.org/ 372 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org 131. heidari a. a novel and modern experimental approach to vibrational circular dichroism spectroscopy and video spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under white and monochromatic synchrotron radiation. glob j endocrinol metab 1:000514–000519, 2018. 132. heidari a. pros and cons controversy on heteronuclear correlation experiments such as heteronuclear single–quantum correlation spectroscopy (hsqc), heteronuclear multiple–quantum correlation spectroscopy (hmqc) and heteronuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. ems pharma j 1: 002–008, 2018. 133. heidari a. a modern comparative and comprehensive experimental biospectroscopic study on different types of infrared spectroscopy of malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. j analyt molecul tech 3: 8, 2018. 134. heidari a. investigation of cancer types using synchrotron technology for proton beam therapy: an experimental biospectroscopic comparative study. european modern studies j 2:13–29, 2018. 135. heidari a. saturated spectroscopy and unsaturated spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. imaging j clin medical sci 5: 001–007, 2018. 136. heidari a. small–angle neutron scattering (sans) and wide–angle x–ray diffraction (waxd) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. int j bioorg chem mol biol 6:1–6, 2018. 137. heidari a. investigation of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, leukemia, liver, lung cancer, melanoma, non– hodgkin lymphoma, pancreatic cancer, prostate cancer, thyroid cancer and non–melanoma skin cancer using synchrotron technology for proton beam therapy: an experimental biospectroscopic comparative study. ther res skin dis 1: 2018. 138. heidaria. attenuated total reflectance fourier transform infrared (atr–ftir) spectroscopy, micro– attenuated total reflectance fourier transform infrared (micro–atr–ftir) spectroscopy and macro–attenuated total reflectance fourier transform infrared (macro–atr– ftir) spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. int j chemistry papers, 2:1–12, 2018. 139. heidari a. mössbauer spectroscopy, mössbauer emission spectroscopy and 57fe mössbauer spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. acta scientific cancer biology 2.3:17–20, 2018. 140. heidari a. comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. organic & medicinal chem ij 6: 55676, 2018. 141. heidari a. correlation spectroscopy, exclusive correlation spectroscopy and total correlation spectroscopy comparative study on malignant and benign human aids– related cancers cells and tissues with the passage of time under synchrotron radiation. int j bioanal biomed 2:001– 007, 2018. 142. heidari a. biomedical instrumentation and applications of biospectroscopic methods and techniques in malignant and benign human cancer cells and tissues studies under synchrotron radiation and anti–cancer nano drugs delivery. am j nanotechnol nanomed 1:001–009, 2018. 143. heidari a. vivo 1h or proton nmr, 13c nmr, 15n nmr and 31p nmr spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. ann biomet biostat 1:1001, 2018. 144. heidari a. grazing–incidence small–angle neutron scattering (gisans) and grazing–incidence x– ray diffraction (gixd) comparative study on malignant and benign human cancer cells, tissues and tumors under synchrotron radiation. ann cardiovasc surg 1:1006, 2018. 145. heidari a. adsorption isotherms and kinetics of multi–walled carbon nanotubes (mwcnts), boron nitride nanotubes (bnnts), amorphous boron nitride nanotubes (a–bnnts) and hexagonal boron nitride nanotubes (h– bnnts) for eliminating carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor and blastoma cancer cells and tissues. clin med rev case rep 5:201, 2018. 146. heidari a. correlation spectroscopy (cosy), exclusive correlation spectroscopy (ecosy), total correlation spectroscopy (tocsy), incredible natural– abundance double–quantum transfer experiment (inadequate), heteronuclear single–quantum correlation spectroscopy (hsqc), heteronuclear multiple– bond correlation spectroscopy (hmbc), nuclear overhauser effect spectroscopy (noesy) and rotating frame nuclear overhauser effect spectroscopy (roesy) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. acta sci pharmaceut sci 2.5:30–35, 2018. 147. heidari a. small–angle x–ray scattering (saxs), ultra–small angle x–ray scattering (usaxs), fluctuation x–ray scattering (fxs), wide–angle x–ray scattering (waxs), grazing–incidence small–angle x–ray scattering (gisaxs), grazing–incidence wide–angle x–ray scattering (giwaxs), small–angle neutron scattering (sans), grazing–incidence small–angle neutron scattering (gisans), x–ray diffraction (xrd), powder x–ray diffraction (pxrd), wide–angle x–ray diffraction (waxd), grazing–incidence x–ray diffraction (gixd) and energy–dispersive x–ray diffraction (edxrd) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. oncol res rev 1:1–10, 2018. 148. heidari a. pump–probe spectroscopy and transient grating spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. adv material sci engg 2:1–7, 2018. 149. heidari a. grazing–incidence small–angle x–ray http://www.ajecr.org/ 373 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org scattering (gisaxs) and grazing–incidence wide– angle x–ray scattering (giwaxs) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. insights pharmacol pharm sci 1:1–8, 2018. 150. heidari a. acoustic spectroscopy, acoustic resonance spectroscopy and auger spectroscopy comparative study on anti–cancer nano drugs delivery in malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. nanosci technol 5:1–9, 2018. 151. heidari a. niobium, technetium, ruthenium, rhodium, hafnium, rhenium, osmium and iridium ions incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. nanomed nanotechnol 3:000138, 2018. 152. heidari a. homonuclear correlation experiments such as homonuclear single–quantum correlation spectroscopy (hsqc), homonuclear multiple–quantum correlation spectroscopy (hmqc) and homonuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. austin j proteomics bioinform genomics 5:1024, 2018. 153. heidari a. atomic force microscopy based infrared (afm–ir) spectroscopy and nuclear resonance vibrational spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. j appl biotechnol bioeng 5:142‒148, 2018. 154. heidari a. time–dependent vibrational spectral analysis of malignant and benign human cancer cells and tissues under synchrotron radiation. j cancer oncol 2: 000124, 2018. 155. heidari a. palauamine and olympiadane nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. arc org inorg chem sci 3: 2018. 156. gobato r, heidari a. infrared spectrum and sites of action of sanguinarine by molecular mechanics and ab initio methods. int j atmospheric oceanic sci 2:1–9, 2018. 157. heidari a. angelic acid, diabolic acids, draculin and miraculin nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. med & analy chem int j 2:000111, 2018. 158. heidari a. gamma linolenic methyl ester, 5– heptadeca–5,8,11–trienyl 1,3,4–oxadiazole–2–thiol, sulphoquinovosyl diacyl glycerol, ruscogenin, nocturnoside b, protodioscine b, parquisoside–b, leiocarposide, narangenin, 7–methoxy hespertin, lupeol, rosemariquinone, rosmanol and rosemadiol nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. int j pharma anal acta 2: 007–014, 2018. 159. heidari a. fourier transform infrared (ftir) spectroscopy, attenuated total reflectance fourier transform infrared (atr–ftir) spectroscopy, micro–attenuated total reflectance fourier transform infrared (micro–atr–ftir) spectroscopy, macro–attenuated total reflectance fourier transform infrared (macro–atr–ftir) spectroscopy, two– dimensional infrared correlation spectroscopy, linear two– dimensional infrared spectroscopy, non–linear two– dimensional infrared spectroscopy, atomic force microscopy based infrared (afm–ir) spectroscopy, infrared photodissociation spectroscopy, infrared correlation table spectroscopy, near–infrared spectroscopy (nirs), mid–infrared spectroscopy (mirs), nuclear resonance vibrational spectroscopy, thermal infrared spectroscopy and photothermal infrared spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. glob imaging insights 3: 1–14, 2018. 160. heidari a. heteronuclear single–quantum correlation spectroscopy (hsqc) and heteronuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human cancer cells, tissues and tumors under synchrotron and synchrocyclotron radiations. chronicle med surg 2.3:144– 156, 2018. 161. heidari a. tetrakis [3, 5–bis (trifluoromethyl) phenyl] borate (barf)–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. med res clin case reports 2.1:113–126, 2018. 162. heidari a. sydnone, münchnone, montréalone, mogone, montelukast, quebecol and palau’amine–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. sur case stud op acc j 1: 2018. 163. heidari a. fornacite, orotic acid, rhamnetin, sodium ethyl xanthate (sex) and spermine (spermidine or polyamine) nanomolecules incorporation into the nanopolymeric matrix (npm). int j biochem biomolecul 4:1–19, 2018. 164. a. heidari a, gobato r. putrescine, cadaverine, spermine and spermidine–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. parana j sci edu (pjse) 4:1–14, 2018. 165. heidari a. cadaverine (1,5–pentanediamine or pentamethylenediamine), diethyl azodicarboxylate (dead or deadcat) and putrescine (tetramethylenediamine) nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. hiv sexual health open access journal 1: 4–11, 2018. 166. a. heidari a. improving the performance of nano endofullerenes in polyaniline nanostructure–based bio http://www.ajecr.org/ 374 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org sensors by covering californium colloidal nanoparticles with multi–walled carbon nanotubes. j adv nanomaterials 3:1–28, 2018. 167. gobato r, heidari a. molecular mechanics and quantum chemical study on sites of action of sanguinarine using vibrational spectroscopy based on molecular mechanics and quantum chemical calculations. malaysian j chem 20:1–23, 2018. 168. heidari a. vibrational biospectroscopic studies on anti–cancer nanopharmaceuticals (part i). malaysian j chem 20:33–73, 2018. 169. heidari a. vibrational biospectroscopic studies on anti–cancer nanopharmaceuticals (part ii). malaysian j chem 20:74–117, 2018. 170. heidari a. uranocene (u(c8h8)2) and bis(cyclooctatetraene) iron (fe(c8h8)2 or fe(cot)2)– enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. chem reports 1:1–16, 2018. 171. heidari a. biomedical systematic and emerging technological study on human malignant and benign cancer cells and tissues biospectroscopic analysis under synchrotron radiation. glob imaging insights 3:1–7, 2018. 172. heidari a. deep–level transient spectroscopy and x–ray photoelectron spectroscopy (xps) comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. res dev material sci 7:rdms.000659, 2018. 173. heidari a. c70–carboxyfullerenes nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. glob imaging insights 3:1–7, 2018. 174. heidari a. the effect of temperature on cadmium oxide (cdo) nanoparticles produced by synchrotron radiation in the human cancer cells, tissues and tumors. int j ad chem 6:140–156, 2018. 175. heidari a. a clinical and molecular pathology investigation of correlation spectroscopy (cosy), exclusive correlation spectroscopy (ecosy), total correlation spectroscopy (tocsy), heteronuclear single– quantum correlation spectroscopy (hsqc) and heteronuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human cancer cells, tissues and tumors under synchrotron and synchrocyclotron radiations using cyclotron versus synchrotron, synchrocyclotron and the large hadron collider (lhc) for delivery of proton and helium ion (charged particle) beams for oncology radiotherapy. european j adv engin technol 5:414–426, 2018. 176. heidari a. nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. j oncol res 1:1–20, 2018. 177. heidari a. use of molecular enzymes in the treatment of chronic disorders. canc oncol open access j. 1:12–15, 2018. 178. heidari a. vibrational biospectroscopic study and chemical structure analysis of unsaturated polyamides nanoparticles as anti–cancer polymeric nanomedicines using synchrotron radiation. int j adv chem 6:167–189, 2018. 179. heidari a. adamantane, irene, naftazone and pyridine–enhanced precatalyst preparation stabilization and initiation (peppsi) nano molecules. madridge j nov drug res 2:61–67, 2018. 180. heidari a. heteronuclear single–quantum correlation spectroscopy (hsqc) and heteronuclear multiple–bond correlation spectroscopy (hmbc) comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. madridge j nov drug res 2:68–74, 2018. 181. heidari a. gobato r. a novel approach to reduce toxicities and to improve bioavailabilities of dna/rna of human cancer cells–containing cocaine (coke), lysergide (lysergic acid diethyl amide or lsd), δ⁹– tetrahydrocannabinol (thc) [(–)–trans–δ⁹– tetrahydrocannabinol], theobromine (xantheose), caffeine, aspartame (apm) (nutrasweet) and zidovudine (zdv) [azidothymidine (azt)] as anti–cancer nano drugs by coassembly of dual anti–cancer nano drugs to inhibit dna/rna of human cancer cells drug resistance. parana j sci edu 4:1–17, 2018. 182. heidari a. gobato r. ultraviolet photoelectron spectroscopy (ups) and ultraviolet–visible (uv–vis) spectroscopy comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation. parana j sci edu 4:18–33, 2018. 183. gobato r, heidari a, mitra a. the creation of c13h20beli2sesi. the proposal of a bio–inorganic molecule, using ab initio methods for the genesis of a nano membrane. arc org inorg chem sci 3:aoics.ms.id.000167, 2018. 184. gobato r, heidari a, mitra a. using the quantum chemistry for genesis of a nano biomembrane with a combination of the elements be, li, se, si, c and h. researchgate, see discussions, stats, and author profiles for this publication at: https://www.researchgate.net/ publication/326201181, 2018. 185. gobato r, heidari a. using the quantum chemistry for genesis of a nano biomembrane with a combination of the elements be, li, se, si, c and h. j nanomed res 7:241‒252, 2018. 186. heidari a. bastadins and bastaranes–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. glob imaging insights 3:1–7, 2018. 187. heidari a. fucitol, pterodactyladiene, dead or deadcat (diethyl azodicarboxylate), skatole, the nanoputians, thebacon, pikachurin, tie fighter, spermidine and mirasorvone nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. glob imaging insights 3: 1–8, 2018. 188. dadvar e, heidari a. a review on separation http://www.ajecr.org/ 375 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org techniques of graphene oxide (go)/base on hybrid polymer membranes for eradication of dyes and oil compounds: recent progress in graphene oxide (go)/base on polymer membranes–related nanotechnologies. clin med rev case rep 5:228, 2018. 189. heidari a, gobato r. first–time simulation of deoxyuridine monophosphate (dump) (deoxyuridylic acid or deoxyuridylate) and vomitoxin (deoxynivalenol (don)) ( (3α,7α)–3,7,15–trihydroxy–12,13–epoxytricho-thec–9– en–8–one)–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. parana j sci edu 4:46–67, 2018. 190. heidari a. buckminsterfullerene (fullerene), bullvalene, dickite and josiphos ligands nano molecules incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human hematology and thromboembolic diseases prevention, diagnosis and treatment under synchrotron and synchrocyclotron radiations. glob imaging insights 3:1–7, 2018. 191. heidari a. fluctuation x–ray scattering (fxs) and wide–angle x–ray scattering (waxs) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–7, 2018. 192. heidari a. a novel approach to correlation spectroscopy (cosy), exclusive correlation spectroscopy (ecosy), total correlation spectroscopy (tocsy), incredible natural–abundance double–quantum transfer experiment (inadequate), heteronuclear single– quantum correlation spectroscopy (hsqc), heteronuclear multiple–bond correlation spectroscopy (hmbc), nuclear overhauser effect spectroscopy (noesy) and rotating frame nuclear overhauser effect spectroscopy (roesy) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–9, 2018. 193. heidari a. terphenyl–based reversible receptor with rhodamine, rhodamine–based molecular probe, rhodamine–based using the spirolactam ring opening, rhodamine b with ferrocene substituent, calix[4]arene– based receptor, thioether + aniline–derived ligand framework linked to a fluorescein platform, mercuryfluor– 1 (flourescent probe), n,n’–dibenzyl–1,4,10,13– tetraraoxa–7,16–diazacyclooctadecane and terphenyl– based reversible receptor with pyrene and quinoline as the fluorophores–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. glob imaging insights 3:1–9, 2018. 194. heidari a. small–angle x–ray scattering (saxs), ultra–small angle x–ray scattering (usaxs), fluctuation x–ray scattering (fxs), wide–angle x–ray scattering (waxs), grazing–incidence small–angle x–ray scattering (gisaxs), grazing–incidence wide–angle x– ray scattering (giwaxs), small–angle neutron scattering (sans), grazing–incidence small–angle neutron scattering (gisans), x–ray diffraction (xrd), powder x– ray diffraction (pxrd), wide–angle x–ray diffraction (waxd), grazing–incidence x–ray diffraction (gixd) and energy–dispersive x–ray diffraction (edxrd) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–10, 2018. 195. heidari a. nuclear resonant inelastic x–ray scattering spectroscopy (nrixss) and nuclear resonance vibrational spectroscopy (nrvs) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–7, 2018. 196. heidari a. small–angle x–ray scattering (saxs) and ultra–small angle x–ray scattering (usaxs) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–7, 2018. 197. heidari a. curious chloride (cmcl3) and titanic chloride (ticl4)–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules for cancer treatment and cellular therapeutics. j. cancer res therap interven 1:01–10, 2018. 198. gobato r, gobato mrr, heidari a, mitra a. spectroscopy and dipole moment of the molecule c13h20beli2sesi via quantum chemistry using ab initio, hartree–fock method in the base set cc–pvtz and 6– 311g**(3df, 3pd). arc org inorg chem sci 3:402–409, 2018. 199. heidari a. c60 and c70–encapsulating carbon nanotubes incorporation into the nano polymeric matrix (npm) by immersion of the nano polymeric modified electrode (npme) as molecular enzymes and drug targets for human cancer cells, tissues and tumors treatment under synchrotron and synchrocyclotron radiations. integr mol med 5:1–8, 2018. 200. heidari a. two–dimensional (2d) 1h or proton nmr, 13c nmr, 15n nmr and 31p nmr spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. glob imaging insights 3:1–8, 2018. 201. heidari a. ft–raman spectroscopy, coherent anti–stokes raman spectroscopy (cars) and raman optical activity spectroscopy (roas) comparative study on malignant and benign human cancer cells and tissues with the passage of time under synchrotron radiation”, glob imaging insights 3:1–8, 2018. 202. heidari a. a modern and comprehensive investigation of inelastic electron tunneling spectroscopy (iets) and scanning tunneling spectroscopy on malignant and benign human cancer cells, tissues and tumors through optimizing synchrotron microbeam radiotherapy for human cancer treatments and diagnostics: an experimental biospectroscopic comparative study. glob imaging insights 3:1–8, 2018. 203. heidari a. a hypertension approach to thermal infrared spectroscopy and photothermal infrared spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation with the passage of time. glob imaging insights 3:1–8, 2018. http://www.ajecr.org/ https://en.wikipedia.org/wiki/grazing_incidence_diffraction 376 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org 204. heidari a. incredible natural–abundance double– quantum transfer experiment (inadequate), nuclear overhauser effect spectroscopy (noesy) and rotating frame nuclear overhauser effect spectroscopy (roesy) comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. glob imaging insights 3:1–8, 2018. 205. heidari a. 2–amino–9–((1s, 3r, 4r)–4– hydroxy–3–(hydroxymethyl)–2–methylenecyclopentyl)– 1h–purin–6(9h)–one, 2–amino–9–((1r, 3r, 4r)–4– hydroxy–3–(hydroxymethyl)–2–methylenecyclopentyl)– 1h–purin–6(9h)–one, 2–amino–9–((1r, 3r, 4s)–4– hydroxy–3–(hydroxymethyl)–2–methylenecyclopentyl)– 1h–purin–6(9h)–one and 2–amino–9–((1s, 3r, 4s)–4– hydroxy–3–(hydroxymethyl)–2–methylenecyclopentyl)– 1h–purin–6(9h)–one–enhanced precatalyst preparation stabilization and initiation nano molecules. glob imaging insights 3:1–9, 2018. 206. gobato r, gobato mrr, heidari a, mitra a. spectroscopy and dipole moment of the molecule c13h20beli2sesi via quantum chemistry using ab initio, hartree–fock method in the base set cc–pvtz and 6– 311g**(3df, 3pd). am j quantum chem molecul spectrosc 2:9–17, 2018. 207. heidari a. production of electrochemiluminescence (ecl) biosensor using os–pd/hfc nanocomposites for detecting and tracking of human gastroenterological cancer cells, tissues and tumors. int j med nano res 5:22– 34, 2018. 208. heidari a. enhancing the raman scattering for diagnosis and treatment of human cancer cells, tissues and tumors using cadmium oxide (cdo) nanoparticles. j toxicol risk assess 4:12–025, 2018. 209. heidari a. human malignant and benign human cancer cells and tissues biospectroscopic analysis under synchrotron radiation using anti–cancer nano drugs delivery. integr mol med 5:1–13, 2018. 210. heidari a. analogous nano compounds of the form m (c8h8)2 exist for m = (nd, tb, pu, pa, np, th, and yb)–enhanced precatalyst preparation stabilization and initiation (eppsi) nano molecules. integr mol med 5:1–8, 2018. 211. heidari a. hadron spectroscopy, baryon spectroscopy and meson spectroscopy comparative study on malignant and benign human cancer cells and tissues under synchrotron radiation. integr mol med 5:1–8, 2018. 212. gobato r, gobato mrr, heidari a. raman spectroscopy study of the nano molecule c13h20beli2sesi using ab initio and hartree–fock methods in the basis set cc–pvtz and 6–311g** (3df, 3pd). int j adv engine sci 7:14–35, 2019. 213. heidari a, gobato r. evaluating the effect of anti– cancer nano drugs dosage and reduced leukemia and polycythemia vera levels on trend of the human blood and bone marrow cancers under synchrotron radiation. trends res 2:1–8, 2019. 214. heidari a, gobato r. assessing the variety of synchrotron, synchrocyclotron and laser radiations and their roles and applications in human cancer cells, tissues and tumors diagnosis and treatment. trends in res 2:1–8, 2019. 215. heidari a, gobato r. pros and cons controversy on malignant human cancer cells, tissues and tumors transformation process to benign human cancer cells, tissues and tumors. trends in res 2:1–8, 2019. 216. heidari a, gobato r. three–dimensional (3d) simulations of human cancer cells, tissues and tumors for using in human cancer cells, tissues and tumors diagnosis and treatment as a powerful tool in human cancer cells, tissues and tumors research and anti–cancer nano drugs sensitivity and delivery area discovery and evaluation. trends in res 2:1–8, 2019. 217. heidari a, gobato r. “investigation of energy production by synchrotron, synchrocyclotron and laser radiations in human cancer cells, tissues and tumors and evaluation of their effective on human cancer cells, tissues and tumors treatment trend. trends in res 2:1–8, 2019. 218. heidari a, gobato r. high–resolution mapping of dna/rna hypermethylation and hypomethylation process in human cancer cells, tissues and tumors under synchrotron radiation. trends in res. 2:1–9, 2019. 219. heidaria. a novel and comprehensive study on manufacturing and fabrication nanoparticles methods and techniques for processing cadmium oxide (cdo) nanoparticles colloidal solution. glob imaging insights, 4:1–8, 2019. 220. heidari a. a combined experimental and computational study on the catalytic effect of aluminum nitride nanocrystal (aln) on the polymerization of benzene, naphthalene, anthracene, phenanthrene, chrysene and tetracene. glob imaging insights 4:1–8, 2019. 221. heidari a. novel experimental and three– dimensional (3d) multiphysics computational framework of michaelis–menten kinetics for catalyst processes innovation, characterization and carrier applications. glob imaging insights 4:1–8, 2019. 222. heidari a. the hydrolysis constants of copper (i) (cu+) and copper (ii) (cu2+) in aqueous solution as a function of ph using a combination of ph measurement and biospectroscopic methods and techniques. glob imaging insights 4:1–8, 2019. 223. heidari a. vibrational biospectroscopic study of ginormous virus–sized macromolecule and polypeptide macromolecule as mega macromolecules using attenuated total reflectance–fourier transform infrared (atr–ftir) spectroscopy and mathematica 11.3. glob imaging insights 4:1–8, 2019. 224. heidari a. three–dimensional (3d) imaging spectroscopy of carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors and carcinoid tumors under synchrotron radiation. glob imaging insights 4:1–9, 2019. 225. gobato r, gobato mrr, heidari a, mitra a. new nano–molecule kurumi–c13h 20beli2sesi/ c13h19beli2 sesi, and raman spectroscopy using ab initio, hartree– fock method in the base set cc–pvtz and 6–311g** (3df, 3pd). j anal pharm res 8:1‒6, 2019. 226. heidari a, esposito j, caissutti a. the importance of attenuated total reflectance fourier transform infrared (atr–ftir) and raman biospectroscopy of single–walled http://www.ajecr.org/ https://en.wikipedia.org/wiki/polypeptide 377 am j exp clin res, vol. 6, no. 4, 2019 http://www.ajecr.org carbon nanotubes (swcnt) and multi–walled carbon nanotubes (mwcnt) in interpreting infrared and raman spectra of human cancer cells, tissues and tumors. oncogen 2:1–21, 2019. 227. heidari a, esposito j, caissutti a. study of anti– cancer properties of thin layers of cadmium oxide (cdo) nanostructure. int j analyt bioanalyt methods 1:20, 2019. 228. gobato r, gobato mrr, heidari a. evidence of tornado storm hit the counties of rio branco do ivaí and rosario de ivaí, southern brazil. sci lett 7:9, 2019. 229. heidari a, esposito j, caissutti a. the quantum entanglement dynamics induced by non–linear interaction between a moving nano molecule and a two–mode field with two–photon transitions using reduced von neumann entropy and jaynes–cummings model for human cancer cells, tissues and tumors diagnosis. int j crit care emerg med 5:71–84, 2019. 230. heidari a.mechanism of action and their side effects at a glance prevention, treatment and management of immune system and human cancer nano chemotherapy. nanosci technol 6:1–4, 2019. 231. heidari a, esposito j, caissutti a. the importance of quantum hydrodynamics (qhd) approach to single– walled carbon nanotubes (swcnt) and multi–walled carbon nanotubes (mwcnt) in genetic science. sciol genet sci 2:113–129, 2019. 232. a. heidari a. esposito j, caissutti a. palytoxin time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. j pharm drug res 3:150–170, 2019. 233. heidaria, j. esposito, a. caissutti. aplysiatoxin time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. j j chem sci eng 2:70–89, 2019. 234. heidari a, j. esposito, a. caissutti. cyanotoxin time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. brit j med health res 6:1–41, 2019. 235. heidari a. potential and theranostics applications of novel anti–cancer nano drugs delivery systems in preparing for clinical trials of synchrotron microbeam radiation therapy (smrt) and synchrotron stereotactic radiotherapy (ssrt) for treatment of human cancer cells, tissues and tumors using image guided synchrotron radiotherapy (igsr). ann nanosci nanotechnol 3:1006– 1019, 2019. 236. heidari a. clinical and medical pros and cons of human cancer cells’ enzymotherapy, immunotherapy, chemotherapy, radiotherapy, hormone therapy and targeted therapy process under synchrotron radiation: a case study on mechanism of action and their side effects. parana j sci edu 5: 1–23, 2019. 237. heidari a. the importance of the power in cmos inverter circuit of synchrotron and synchrocyclotron radiations using 50 (nm) and 100 (nm) technologies and reducing the voltage of power supply. radiother oncol int 1:1002–1015, 2019. 238. gobato r, gobato mrr, heidari a, mitra a. spectroscopy and dipole moment of the molecule c13h20beli2sesi via quantum chemistry using ab initio, hartree–fock method in the base set cc–pvtz and 6– 311g** (3df, 3pd). am j quantum chem molecul spectroscopy 2:9-7, 2018. 239. heidari a, esposito j, caissutti a. anatoxin–a and anatoxin–a(s) time–resolved absorption and resonance ft– ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. saudi j biomed res, 4 (4): 174–194, 2019. 240. a. heidari a, esposito j, caissutti a. alpha– conotoxin, omega–conotoxin and mu–conotoxin time– resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. int j advanced chem 7:52–66, 2019. 241. heidari a, esposito j, caissutti a. shiga toxin: shiga–like toxin (slt) time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. annal biostat & biomed appl 2: 2019. 242. heidari q, esposito j, caissutti a. alpha– bungarotoxin, beta–bungarotoxin and kappa–bungarotoxin time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. arch pharmacol pharmaceutic sci rd–pha–10001, 2019. 243. heidari a. investigation of the processes of absorption, distribution, metabolism and elimination (adme) as vital and important factors for modulating drug action and toxicity. open access j oncol 2: 180010, 2019. 244. heidari a, esposito j, caissutti a. okadaic acid time–resolved absorption and resonance ft–ir and raman biospectroscopy and density functional theory (dft) investigation of vibronic–mode coupling structure in vibrational spectra analysis. int j analyt bioanalyt methods 1: 004, 2019. http://www.ajecr.org/