Iraqi J Pharm Sci, Vol.25(1) 2016 Serum ferritin in women with breast cancer 23 Detection of Serum Ferritin in Women with Breast Cancer Eman S. Nassir *, 1 * Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad ,Baghdad, Iraq. Abstract Breast cancer is one of the most common cancers in females. In Iraq there are noticeable elevation in incidence rates and prevalence of advanced stages of breast cancer. Ferritin is intracellular iron storage protein abundant in circulation and its main application in differential diagnosis of anemia. The level of serum ferritin was found raised in various cancers including breast cancer. The aim of this study was to assess whether the serum ferritin concentration would be altered in Iraqi women with breast cancer and it could be related to progression of disease. Sixty eight females participated in this study .The mean age of these females was 53.25± 9.52 .The level of serum ferritin was measured in 24 Iraqi women of early stage of breast cancer (stage I and II) and 24 Iraqi women of advanced stage (III and IV). These levels were compared with 20 healthy females as controls. Serum ferritin was estimated by using enzyme linked immune sorbent assay method. This serum ferritin was found to be raised in all breast cancer patients (p<0.05) as compared to controls. The rise in ferritin level was significant in advanced stage (p<0.05) as compared to early stage. Thus the estimation of ferritin may aid in diagnosis, assessment of severity and monitoring of Iraqi women with breast cancer. Key words: - Ferritin, Breast cancer, Iraq. قياس مستوى الفرتييه في المصل في النساء المصابات بسرطان الثدي ايمان صادق ناصر *،1 * . فشع انعهٕو انًخخبشٚت انسشٚشٚت، كهٛت انصٛذنت، جايعت بغذاد، بغذاد انعشاق الخالصة انثذ٘ ٔاحذ يٍ اْى انسشطاَاث انشائعت نذٖ االَاد فٙ انعشاق ُْاك صٚادة ٔاضحت فٙ َسبت حذٔد انًشض ٚعخبش سشطاٌ ٍ ْٕ انبشٔحٍٛ انًسؤٔل عٍ خضٌ انحذٚذ يٕجٕد داخم انخالٚا ٔاٚضا فٙ انذٔسة انذيٕٚت. ٛٔٔصٕنّ انٗ يشاحم يخطٕسة .انفشحٛ ٍ انًٕجٕد فٙ يصم انذو ٔجذ اَّ ٚشحفع فٙ حاالث انسشطاٌ ٔيٍ ٛسخٕٖ انفشحٛاْى حطبٛقاحّ ْٕ نهخشخٛص انخًٛض٘ الَٕاع فقش انذو. ي يصم انذو ٚخغٛش فٙ انُساء انعشاقٛاث فٙضًُٓا سشطاٌ انثذ٘. انٓذف يٍ ْزِ انذساست ْٕ نخقٛٛى فًٛا ارا كاٌ يسخٕٖ انفشحٍٛٛ ٛت )يشاحم انًشض(.انًصاباث بسشطاٌ انثذ٘ ْٔم اٌ يسخٕاِ فٙ انذو نّ عالقت بخقذو انحانت انًشض ٍ فٙ ٛفٙ ْزِ انذساست حى قٛاط يسخٕٖ انفشحٛ ,25,9 ,259ثًاَٛت ٔسخٌٕ ايشاة شاسكج فٙ ْزِ انذساست ٔيعذل اعًاسْى ايشأة عشاقٛت 92( ٔاٚضا حى قٛاسّ فٙ 9ٔ 1ايشأة عشاقٛت يصابت بسشطاٌ انثذ٘ فٙ انًشاحم االبخذائٛت )يشاحم 92يصم انذو نـ ايشأة عشاقٛت 92( ٔحى يقاسَت انُخائج يع االصحاء باخز عُٛاث دو يٍ 2ٔ 2ٌ انثذ٘ فٙ انًشاحم انًخقذيت )يشاحم يصابت بسشطا نقذ ٔجذ اٌ يسخٕٖ فحص انًُاعٙ انًشحبظ باالَضٚى . حى فٛاط يسخٕٖ انفشحٍٛٛ فٙ يصم انذو باسخعًال طشٚقت غٛش يصابت بانًشض. بسشطاٌ انثذ٘ يقاسَت يٍ انُساء االصحاء ٔاٚضا ٔجذ اٌ يسخٕٖ انفشحٍٛ فٙ انُساء انًصاباث انفشحٍٛٛ يشحفع فٙ انُساء انًصاباث بسشطاٌ انثذ٘ فٙ انًشاحم انًخقذيت اعهٗ يٍ يثٛالحٓى فٙ انًشاحم االبخذائٛت يٍ ْزِ انُخائج َخٕقع اَّ يٍ انًًكٍ االسخفادة يٍ قٛاط انثذ٘ ٔيعشفت يذٖ حقذو انحانت ٔحطٕسْا. نًخابعت انُساء انًصاباث بسشطاٌ يسخٕٖ انفشحٍٛٛ. الفرتييه، سرطان الثدي، العراق. -الكلمات المفتاحية : Introduction Ferritin is the protein that stored iron (1) . This protein is synthesized in the liver, spleen, myocardium, placenta and other tissues. It is a large macromolecule (450kDa) which consists of 24 subunits that form protein shell (apoferritin) around an insoluble core of stored iron. There are 2 types of subunits, the basic L and the acidic H type (2) . Although the two subunits share approximately 55% of their amino sequences, in addition to their multi- helical three dimensional structures, they are functionally different (3) . The H subunit has ferroxidase activity and it is responsible for the oxidation of ferrous iron into ferric iron, whereas the L subunit contribute to the stable storage of iron in the ferritin core. The efficient storage of iron in ferritin requires cooperation of both (4) . Ferritin is an abundant protein in circulation which is known as serum ferritin in addition to its intracellular form. Serum ferritin was first detected in 1948 in animals experiencing hepatic cirrhosis or shock (5) .These observation was later confirmed in humans with different types of liver disease (6) . Serum ferritin is a dependable indicator of the body’s iron stores. Its level is significantly decreased in individuals suffering from iron deficiency anemia or undergoing phlebotomy. 1 Corresponding author E-mail: emansadiq1976@yahoo.com Received: 4/11/ 2015 Accepted: 20/1/2016 Iraqi J Pharm Sci, Vol.25(1) 2016 Serum ferritin in women with breast cancer 24 In contrast serum ferritin levels are increased in patients with iron over load (hemochromatosis or hemosidriosis), infection or inflammation, malignancies and damage of liver tissues (7, 8) . Ferritin has recently been implicated in the pathogenesis of disease including cancer. A number of mechanisms such as pro-oxidant and pro-inflammatory pathways are responsible for this (9) . Ferritin has long been disreputable for its association with breast cancer either as cause or result of the disease (10) . Breast cancer is the mainly frequent cancer among women, comprising about 23% of all females’ cancer (11) . It is also the most important cause of cancer-related death worldwide, case fatality rates being highest in low resource countries (12) . In Iraq, breast cancer is the commonest type of female malignancy accounting for approximately one third of the registered female cancers according to the latest Iraqi cancer registry. This shows that the breast is the chief cancer site among the Iraqi women (13) . As projected by the world health organization, early detection and screening of the disease, especially when combined with adequate therapy, suggest the most immediate hope for decline in breast cancer mortality (14) . The tumor markers in breast cancer like CA 15-3 and CEA are generally useful in follow- up of patients with metastatic disease with other diagnostic technique like X-rays and CT scan. This disease is still in the need of more precise biomarkers which might help in early detection, assessment of severity and for prediction of therapy response (15) . Therefore this study was planned to estimate the level of serum ferritin in patients with breast cancer at different stages (early and advance). The aim was to inspect potential relations between the level of serum ferritin in Iraqi women with breast cancer and the progression of disease. Material and Methods The present study was conducted at the teaching hospital of oncology-Baghdad under specialized senior supervision for the period of November 2013- July 2014. Institutional ethics committee approval was obtained prior to initiation of the study. An informed written approval was recorded from all the study subjects previous to their enrolment in the study. Sixty eight females were enrolled for this study. Out of these, 48 were patients with breast cancer (age range 33- 69) and 20 were healthy females (age range 35-67).The patients were staged according to American Joint Committee of Cancer (AJCC) staging 2010 (TNM) (9) . The subjects were divided into three groups: Group A: 20 healthy females with mean age 51.9 ± 9.71. Group B: 24 patients with histopathologically proven breast cancer in early stage of disease with mean age 53.1± 10.25 (stage I and II, AJCC-TNM stage). Group C: 24 patients with histopathologically proven breast cancer in advanced stage of disease with mean age 55.8 ± 8.80(stage III and IV, AJCC-TNM stage). Inclusion criteria: healthy non anemic females were included in this study as control group .for patients group, subjects with untreated histopathologically proven breast cancer were chosen. Exclusion criteria: for control group, subjects with fasting plasma glucose more than 120mg /dl and blood pressure more than130 /85mmHg were excluded from this study. For patients group, subjects with history of liver or kidney damage, acute inflammatory and infectious disease, anemia (Hb<10gm%), diabetes and those on medication like iron supplement, or thyroxin or having benign tumor or mass anywhere else in the body are excluded from the study as any of these factors may influence the serum ferritin levels. 5ml fasting blood sample was withdrawn from median cubital vein of each study participant with all necessary aseptic precautions. All samples were allowed to stand for 10 min to obtain serum. After centrifugation, non hemolysed sera were kept at -20 0 C for subsequent analysis as early as possible. Ferritin was estimated by using enzyme linked immunosorbent assay method (ELISA).The Ferritin ELISA kit is based on the sandwich principle. This commercially available kit was supplied by Monobind Inc, USA (Accu-Bind). Serum sample were allowed to incubate in wells coated with specific anti-ferritin antibodies. This was further added with Horse-radish peroxidase conjugated anti ferritn antibodies. The amount of bound peroxidase is in direct proportion to the content of ferritin in the sample and the intensity of color formed is proportional to the quantity of ferritin in the samples. Later on stopping the reaction, the color intensity of final mixture was measured at 450nm wavelength with micro plate reader (10) . Statistical analysis and interpretation of data were done by using student’s unpaired “t” test (for two group comparison) and ANOVA test (for comparison of three groups of variable sample size). All the data have been expressed as mean ± SEM [standard error of mean]. The levels of significance were calculated for all Iraqi J Pharm Sci, Vol.25(1) 2016 Serum ferritin in women with breast cancer 25 three groups. Probability value “p” greater than 0.05 was considered as statistically non- significant alteration while P less than 0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS (version 17) software. Results The demographic characteristics of healthy women and patients with breast cancer in the current study were described in table (1). Table (1): Demographic characteristics of healthy women and patients with breast cancer. Age(years) less than 45 more than 45 Group A 5(25%) 15(%75) Group B 4 (16.6%) 20 (83%) Group C 3 (13%) 21 (87%) Marital status Married Single Widow/divorced Group A 18(90%) 2(10%) Group B 18 (75%) 4 (17%) 2 (8%) Group C 20 (83%) 3(13%) 1 (4%) Family history with breast cancer Positive Negative Group A 20(100%) Group B 15 (62.5% 9 (37.5% Group C 13 (54%) 11 (46%) BMI Normal Over weight Group A 7(35%) 13(65%) Group B 1(4%) 23 (96%) Group C 2(8%) 22(92%) BMI: body mass index describes relative weight for height. The healthy weight falls between BMI values of 18.5- 24.9. Overweight falls between 25-30 (16) . Table (2): Ferritin concentrations in healthy and women with breast cancer. Parameter Group A Mean ±SEM Group B Mean ±SEM Group C Mean ±SEM Ferritin serum level (ng/ml) 44.1±6.19 196.6±27.68 * 248.9±32.32 *,** * Significant difference from group A (p<0.05) ** Significant difference from group B (p<0.05) In group A, the determined mean of serum ferritin was (44.1) while in group B the determined mean of serum ferritin was (196.6), and in group C, the determined mean of serum ferritin was (248.9). The levels of ferritin were found to be significantly raised in patients with breast cancer (group B and C) as compared to control (group A) as shown in table (2).Significant difference (p<0.05) was observed when compared group B and C to group A. statistically significant difference (p<0.05) was observed between group B and C. Discussion Cancer is the fourth ranked cause of death in the Eastern Mediterranean Region (EMR), after cardiovascular diseases, infectious /parasitic diseases and injuries according to world health organization (WHO) mortality estimates (17) . The major increase in cancer incidence among the WHO regions in the EMR, where breast cancer is recorded as the commonest type of female malignancy in almost all national cancer registers (18) . In Iraq in addition to being the most important cancer there are noticeable increase in incidence rates and prevalence of advanced stages at presentation associated with more aggressive tumor behaviors, resulting in larger fatality rates (14) . In this study, breast cancer was diagnosed in 48 females. The percentage of patients with breast cancer increased with increasing age over 45. In table (1) we observed that 83% of the patients in group B are in age over 45 and 87% of the patients in group C are in age over 45. Approximately half of cancer cases in EMR occur before the age of 55 and the age standardized incidence rates of all cancer in this region is expected to double as risk factor exposure increases as revealed by WHO estimates (17) . In table (1) high numbers of patients with positive family history observed in this study, 63% of the patients in group B were with Iraqi J Pharm Sci, Vol.25(1) 2016 Serum ferritin in women with breast cancer 26 positive family history and 54% of the patients in group C were with positive family history. The customary consanguineous marriages which are known to be common throughout this region could be the main cause (14) . Weight gain and being overweight are risk factor for breast cancer in women who have gone through the menopause (19) . In this study as we observed in table (1) 96% of the patients in group B were overweight and 92% of the patients in group C were overweight. It is believed that obesity is proinflammatory state as it results in release of inflammatory mediators that promote tumor growth (20) . One of the most vital nutritional elements required in physiological activities in the body is iron. Many processes like transport of oxygen, generation of energy and synthesis of DNA are dependent on Iron (21) . Despite the fact that iron is essential for growth and development of cells, it has been proven to be harmful when present in excess amount in the body. In recent times, several lines of evidence have established that the iron storage protein which is termed ferritin is a multi-functional protein that have possible role in proliferation, angiogenesis, immunosuppression and delivery of iron. In the context of cancer ferritin is detected at higher levels in the sera of many cancer patients (7) . In the current study as we observed in table (2) the levels of ferritin were found to be significantly raised in breast cancer patients (in both groups B and C) as compared to control (group A). This increase may either due to increase expression of a tumor derived protein which interferes with iron metabolism or due to nonspecific effect of malignancy on reticule-endothelial iron metabolism (22) . The rise in ferritin levels is reported to be linked with more advanced stage breast cancer (22, 23) . In our study, also the levels in group C were significantly higher as compared to group B (p<0.05). Elevation of serum ferritin levels may be attributed to increased iron requirement by malignant cells for growth and for modulation of transferrin receptor. Transferrin is considered potential markers for identifying cells undergoing divisional activity and requiring the incorporation of additional iron (24) . In addition to increased synthesis by malignant cells, other causes of raised ferritin levels include presence of inflammation, hepatic necrosis due to metastasis and reduced hepatic clearance of ferritin (25) . Hypoxia often associated with solid tumors and it considers one of the factors that encourage production of ferritin (26) .Ferritin receptor expression is post transcriptionally regulated by conserved mRNA sequence termed iron responsive element (IRE), to which a transacting protein called iron regulatory protein (IRP) is bound. Early studies demonstrated that hypoxia induces the specific and reversible expression of ferritin. This induction occurs primarily on post transcriptional level due to decreased mRNA binding capacity of IRP (27) . The tumor markers being presently used for breast cancer like CA15 -3 and CEA are not specific and are found raised in other disease too. The utility of these markers is more reliable if analyzed in conjunction with other markers (9) . 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