Study the Effects of Vitamin D as Immune-Modulatory Agent in Type II Diabetes Mellitus Patients Afaf Th. Marzook Dept. of Chemistry/College of Education for Pure Science(Ibn Al-Haitham)/ University of Baghdad E-mail: afaf_ch52@yahoo.com Received in :12 May 2013 , Accepted in : 24 July 2013 Abstract This study was designed to show the roles of vitamin D as immune-modulatory agent in serum type II Diabetes Mellitus Patients collected from type II Diabetes Mellitus and controls. They have been classified into two groups as the following: 1) Patients of type II DM group includes (20) individuals from both sexes with age range (35–65) years. 2) Control group: includes (20) healthy individuals from both sexes, with age range (30 – 45) years and no previous disease which may interfere with the parameters analyzed in this research. All the blood samples were analyzed for vitamin D3, albumin, C- reactive protein (CRP), erythrocyte sedimentation rate (ESR), immunoglobulins (IgG, IgM, IgA), α1- antitrypsin and total protein (TP). Keywords: vitamin D, type II diabetes mellitus, inflammation, Acute - phase proteins. 261 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Introduction Diabetes Mellitus (DM) is a heterogeneous, metabolic disease which is characterized by hyperglycemia and long term complications [1]. Type II DM is caused by combination of insulin resistance (impaired sensitivity of tissues to insulin action) and relative insulin deficiency with increased hepatic glucose production [2]. Cholecalcifrol is a prohormone that is synthesized in the skin by photochemical of 7-dehydrocholestrol, it is subsequently hydroxylated to 25-hydroxy-cholecalciferol [25(OH) D3] in the liver and finally to the active metabolite, 1,25-dihydroxy – cholecalciferol [1,25(OH)2D3] in the kidney [3]. Opinions differ on how to define vitamin D deficiency. A recent statement by the institutes of medicine agreed upon level of 10 ng/ml (25nmol/L) to be clear that vitamin D deficiency is determined as the level where parathyroid hormones (PTH) start to rise [4]. The vitamin D hormone, 1,25 (OH)2 D exerts its effects mainly by activating the nuclear vitamin D receptor (VDR), a member of the nuclear transcription super family of ligand-activated transcription factors, and when bound to this vitamin D an attractive molecule to investigate in the context of diabetes treatment [5,6]. Indeed, the activated form of vitamin D, 1, 25(OH)2 D3 influence insulin secretion and is an important immune modulator [7,8]. Vitamin D deficiency is more common in type II DM than in type I DM independently of age, sex or insulin therapy [9]. The initial observations linking vitamin D to type II DM in human came from studies showing that both healthy and diabetic subjects had a seasonal variation of glycemic control currently, there is evidence supporting that vitamin D status is important to regulate some pathways related to type II DM development since the activation of inflammatory pathway interferes with normal metabolism and disrupt proper insulin signaling, it is hypothesized that vitamin D could influence glucose homeostasis by modulating inflammatory response [10]. The aim of this study is to show the roles of vitamin D include immune-modulation effect and anti-inflammatory agent in type II DM patients compared with control Experimental Sampling is classified in two groups: 1) Patients of type II Diabetes mellitus group: include (20) patients from both sexes, with age range (35–65) years. 2) Control group: includes (20) healthy individual from both sexes, with age years (30–45) years and no previous disease which may interfere with the parameters analyzed in this study. Specimen collection and preparation Ten milliliters (ml) disposable plastic syringes of 21 G needles were used to draw eight ml of venous blood from each patient and control groups after 12 hours fasting. The blood samples were divided into two tubes: 1. Two ml of blood samples were transferred into plastic tubes containing Ethylene Diamine Tetra Acetic acid (EDTA) and left for 20 - 30 minutes at 37Cº. The blood was later used for the determination of (ESR). 2. The second part of blood samples were transferred into plastic plane tubes no anticoagulant: blood was left to clot for 20 - 30 minutes at 37Cº. Serum was obtained by centrifugation for 10 minutes at 3000 rpm and was divided into small epindrof tubes capacity 1.5 ml and kept at – 20Cº until time of analysis. The separated serum was later used for the determination of the levels of vitamin D3, C-reactive protein (CRP), Albumin, ∝1-antitrypsin, immunoglobulins (IgM, IgG, IgA) and total protein. 262 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Determination of vitamin D3: Vitamin D3 was measured using high performance liquid chromate graphy (HPLC) technique according to (AL- Dulaimy, W. Y- M. and Al-sarrag, N.F.Y, 2013 method) [11]. Determination of Albumin: Albumin level was determined in serum sample of all studied groups according to (Doumas, etal method) [12]. Determination of C - reactive protein (CRP): CRP was measured in serum samples of all studied groups according to (Young, D.S. method) [13]. Determination of Erythrocyte Sedimentation Rate (ESR): Erythrocyte Sedimentation Rate (ESR) was determined in whole samples of all studied groups according to (Bick, R. L method) [14]. Determination of Serum Immunoglobulins (IgG , IgM, IgA): Immunoglobulins (IgG, IgM, IgA) have been determined in serum samples of all studied groups by a ready kit purchased from (parsazmum company), Iran. The method depends on immune turbidometric test which the immunoglobulins form a complex with antibodies in solution which the absorbance is read by spectrophotometry [15]. Determination of ∝𝟏 - antitrypsin: ∝1– antitrypsin was measured in serum samples of all studied according to the method depends on immune turbidometric test [15]. Determination of total protein (TP): Total protein was determined in serum of all studied group according to Biuret method [16]. Statistical Analysis: Data presented in table (1) were the means and standard deviation student's t-test was used to compare the significance of the difference in the mean values of any two groups, P value less than 0.05 was considered statistically significant. Results and discussion This study evaluate the biochemical parameters levels of [vitamin D3, Albumin, CRP, ESR, IgG, IgM, IgA, ∝1– antitrypsin and total protein TP] in sera of type II diabetes mellitus patient group compared with control group. Data in table (1) shows a significant decrease in vitamin D3, albumin and IgM in sera patients of type II DM compared with control group (P ≤ 0.05). While table (1) shows that a significant increase in the level of c-reactive protein (CRP) , ESR, IgG, IgA and ∝1– antitrypsin in sera of type II Diabetes mellitus patient group compared with control group (P ≤ 0.05). Also table (1) shows no significant difference in total protein (TP) in sera of type II Diabetic Mellitus Patient group compared with control group (P > 0.05). The result of a significant decrease in level of vitamin D3 in sera of type II diabetic patient group compared with control group agrees with the study of (Palomer, X. et al 2008) [17], which suggested that vitamin D3 has a role in abnormal glucose metabolism as well as in type II diabetes serum albumin level has linked clinical practice to several diseases. Low albumin levels can suggest inflammation, this is the study of (Sesmilo, G. et al 2004) [18]. 263 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 This study is compatible with our result in table (1) showing a significant decrease in albumin level in sera of patients of type II DM compared with control group (P ≤ 0.05). A significant increase in the level of C- reactive protein (CRP) in sera of type II DM patient group compared with control group (P ≤ 0.05). Our result agrees with the study of (Badawi, A. et al 2010) [19]. That suggests C-reactive protein is an acute-phase reactant inflammation biomarker and that elevated synthesis of Pro-inflammatory cytokines and acute-phase proteins characterize the pre-clinical stages of type II DM and exhibit a graded increase with disease progression. Also a recent study estimated that one-third of type II DM cases can be associated with elevated serum CRP [20]. It is known that adipose tissue can synthesis and release the main pro-inflammatory cytokine alfa-tumor necrosis factor (TNF-alpha), interleukin-1 (IL–1) and interleukin-6 (IL–6) and that the pro-inflammatory cytokines and acute phase reactants are involved in multiple metabolic pathways which are relevant to insulin resistance [21]. A significant increase in the level of ESR in sera of patients with type II DM compared with control (P ≤ 0.05). Our result agrees with study of (Donth, et al 2003) [22]. Donth study suggests that inflammations play a role in the pathogensis of type II DM most probably by determinal effects of inflammation on beta cells function. Also in diabetes mellitus disease, the levels of variety of plasma proteins increase as a result of increased red cell agglomeration leading to accelerated sedimentation [14]. Table (1) shows that both immunoglobulins IgG and IgA were elevated while IgM was decreased in sera of type II patient group compared with control group. These findings are compatible with the results by (Michael, L. Bishop et al., 2005) [23] suggested that infections and inflammation diseases indicating in flammatory condition seen when there is an increase in both IgG, IgA immuogobulins, ∝- antitrypsin , CRP levels while there is a decrease in IgM and albumin level that stimulate the immune system . Table (1) shows a significant increase in the ∝- antitrypsin level in sera of type II DM patient group compared with control group. This result agrees with the previous study of (Michael, L. Bishop et al., 2005) [23]. While table (1) shows no significant difference in total protein (TP) level in sera of type II patient group compared with control group. Vitamin D has long been known to play important role in immune function. All cells of the immune system express the vitamin D receptor (VDR) including monocytes, macrophages, dendritic cell, neutrophils, T-Lymphocytes as well as B-Lymphocytes (Veldman et al 2000) [24]. Vitamin D modulates the adapative immune system as well, through direct effects on T-cell activation and on the phenotype and function of antigen-presenting (Kamen, DL et al 2010) [25] and (Von Essen, Mr. et al 2010) [26]. Type II diabetes development involves impaired pancreatic B cell function, insulin resistance and inflammation. It has been suggested that both environmental and genetic factors seem to be involved in type II diabetes development [27]; also, human and experimental data support the role of vitamin D on these pathways. Due to the presence of both 1-∝- hydroxylase and VDR in pancreatic Beta cells, vitamin D is important for insulin synthesis and release [28]. This study demonstrated that type II DM an inflammatory diseases through the determination of biochemical parameters (Albumin, CRP, ESR, IgG, IgM, IgA, ∝ - antitrypsin and total protein (TP) and the deficiency in vitamin D play a crucial role in progression of type II diabetes mellitus. 264 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 References 1. Ionecu, T.C. (1998). "Proposed for a new classification of diabetes mellitus". Rom. J. inter. Med, 36 (1-2): P121-34. 2. American Diabectes Association, Inc: (2007). "Diabetes magnitude and mechanism", clinical Journal 25: 25-28. 3. Holick, M. (2002). "Vitamin D: the underappereciated D-lightful hormone that is important for skeletal and cellular health "CurropinEndocrinol Diabetes; 9:87-98. 4. Ross, A. C; Munson, J.E; Abrams, S.A; Aloia, J.F.; Brannon, P.M; Clinton, S.K.; Durazo- Arvizu, R.A; Gallagher, J.C; Gallo, R.I; Jones, G; Kovacs, C. S; and Mayne S. T. "The 2011 report on dietary reference intakes for calcium and vitamin D from the institute of medicine what clinicians need to know" J. Clin. Pract 64:1084-1089. 5. Christakoss, S; Barletta F; Huening, M; Dhawan P; Liu, Y; and Porta, A. et al (2003). "Vitamin D target proteins: function and regulation". J. Cell Biochem 88 :238–44. 6. Vidal, M; Ramana C.V; and Dusso, A.S. (2000). "Stat 1-vitamin D receptor interactions antagonize 1, 25-dihydroxy vitamin D transcriptional activity and enhance stat 1 mediated transcription", Mol. Cell. Biol. 22:2777–87. 7. Mathieu, C; and Adorinol (2002). "The coming of age of 1, 25-dihydroxy vitamin D3 analogs as immunomodulatory agents", Trends Mol. Med. 8:174–9. 8. Matheiu, C; Gysemans, C; Giulietti, A; and Bouillon, R. (2005). "Vitamin D and diabetes", Diabetologia, 48:1247–57. 9. Dicesa, R.D.j; ploutz-synder, R; Weinstock, R.S; and Moses, A.M. (2006). "Vitamin D deficiency is more common in type II DM than in type I DM". Diabetes care, 29 :174. 10. Chagas, C.E.A; Borges, M.C; Martini, L.A; and Rogero, M.M. (2012). "Focuson Vitamin D, inflammation and type IIDiabetes", Nutrients, 4:52–67. 11. Al-Dulaimy, W.Y.M; and Al-Sarrag, N.F.Y. (2013). "study of some non-traditional roles a scribed to Vitamin D include immunemodulating effects and anti – inflammatory insera of Iraqi myocardial infarction patients" ,word Family medicine Journal. 11::issue 4. 12. Doumas, B. T; Watson, W. A; and Biffs, H.G. (1977). Clin. Acta (31):87–96. 13. Young, D. S. (1995), "Effect of drugs on clinical laboratory tests". 4thed. AACC press. 14. Bick, R.L. (1993). "Heamatology clinical Laboratory practice" 1sted, P: 253, Baltimore publishing company. 15. Shivanada, Nayak B. (2008). Manpal Manual of clinical Biochemistry, (For laboratory and MSC. students): (2008) 3rd. ed, p: 104-106 , medical publishers (p) Ltd , New Delhi , india 16. Tietz, N.W. (1987). "Fundamentals of clinical chemistry" p: 940, W.B. Sannders Co. Philadelphia, P. A. 17. Palomer, X; Gonzalz-Clemente, J.M; Blanco Vaca, F; and Mauricio, D. (2008). "Role of vitamin D in the pathogensis of type II diabetes mellitus". Diabetes Obes. Metab. 10: 185– 197. 18. Sesmilo, G. et al. (2004). Effect of vitamin D on glucose metabolism. Ann Intern Med, 133(2):111–122. 19. Badawi, A.; Klip, A; Haddad, P. Cole; E.C; David; Bailo, B.G; El-Sohemy, A; and Karmali, M. (2010). "Type II diabetes mellitus and inflammation": prospects for biomarkers of risk and nutritional intervention. Diabetes Metab. Synd Obes. 3:173–186. 20. Dehghan, A; Kardys, I; de Maat, M.P., et al (2007). "Genetic variation, C-reactive protein levels, and incidence of diabetes". Diabetes, 56:872–878. 21. Mahajan, V.V.; APTE, I.C; and shende, S.S. (2011). "Acute phase Reactants in type II Diabetes Mellitus and Their Correlation with the Duration of Diabetes Mellitus" J. Of clinical and Research No.6 (5):1165–1168. 265 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 22. Donah, M.Y; Starling, J; Maedier, K; and Manruppoulsem, T. (2003). "Inflammatory mediators and islet beta cell failure": a link between type I and type II diabetes" J. Mol. Med. 81: 455–470. 23. Michael, L. Bishop; Edwared, P; Fody, and Larry Schoeff. (2005). "Clinical chemistry principle, procedures, correlation." 5th ed. Lippincott Williams and Wilkins. P :(194–195), P:(286–270) . 24. Veldman, C. M; and Cantorna, M.I.H.F. (2000). "Expression of 1, 25-dihydroxy vitamin D3 receptor in the immune system". Arch. Biochem. Biophys 374:334–338 . 25. Kamen, D.L; and Tangpricha, V. (2010). "Vitamin D and Molecular actions on immunesystem" : modulation of innate and autoimmunity ; J. Mol. Med, 88 : 441 – 450 . 26. Von Essen, M.R; Kongsbak, M; Schjerling. P; Olgaard k; Odum. N; and Geisler. C. (2010). "Vitamin D controls T cell antigen receptor signaling and activation of human T cells"; Nat Immunol, 11:344–349. 27. Takiishi, T.; Gysemans, C.; Bouillon R.and Mathieu, C. (2010). Endocrinol. Metab. Clin. N. Am., 39:419–446. 28. Pittas, A. and Dawson-Hughes, B. (2010). "Vitamin D and diabetes". J. Sterol. Biochem. Mol. Biol. 121:425–424. Table (1): Vitamin 𝐷3,Albumin, CRP, ESR, IgG, IgM, IgA, ∝1-antitypic and total protein (TP) levels in Sera of type II diabetes mellitus patients group and control Significance t- test Patient group N=20 Mean ±SD Control N=20 Mean±SD Biochemical parameters Significant 0.00001 33.07±11.2 46.57±9.2 Vitamin 𝐷3(ng/ml) Significant 0.00002 1.37±2.97 1.93±0.004 Albumin (g/dl) Significant 0.0004 3.6±0.12 ---- CRP (m mol/dl) Significant 0.003 12.96±3.47 4.16±1.52 ESR (mmole/hr) Significant 0.003 1214.64±101.22 797.3±184 Ig G (mg/dl) Significant 0.001 15.4±28.92 34.68±1.98 Ig M (mg/dl) Significant 0.0005 347.28±57.38 151.14±39.31 Ig A (mg/dl) Significant 0.004 77.8±1.84 52.44±18.12 ∝1-antitrypsin (mg/dl) Not-significant 0.2 2.79±0.28 2.75±0.31 TP (g/l) 266 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 مناعي في مرضى داء السكري النوع وسیط Dدراسة عن تأثیرات فیتامین الثاني عفاف ذیاب مرزوك / جامعة بغداد )ابن الھیثم(قسم الكیمیاء / كلیة التربیة للعلوم الصرفة 2013تموز 24، قبل البحث في : 2013آیار 12استلم البحث في : الخالصة وسیط مناعي في مصل دم عشرین مریضا عراقیا یعانون من داء Dصممت ھذه الدراسة لبیان تأثیرات فیتامین السكر النوع الثاني، وعشرین فردا من األصحاء مجموعة سیطرة . وقد تم تصنیفھا إلى مجموعتین: ) سنة. 65– 35المجموعة األولى تتضمن عشرین مریضا بداء السكر النوع الثاني من كال الجنسین وبمعدل عمر من ( ) سنة ولیس 45–30والمجموعة الثانیة تتضمن عشرین فردا من االصحاء مجموعة سیطرة ومن كال الجنسین بمعدل عمر ( تم قیاسھا في ھذا البحث. حللت جمیع نماذج الدم لقیاس المتغیرات لدیھم أي أمراض سابقة تؤثر في المتغیرات الحیاتیة التي التفاعلي و معدل ترسب كریات الدم الحمراء و الكلوبیولینات المناعیة Cو األلبومین و بروتین 3Dالحیاتیة مثل فیتامین أنتي تربسین والبروتین الكلي . -1 –، بروتین الفا ,IgG, IgM, IgA)وھي( بینما أظھرت IgM، االلبومین، الكلوبیولین المناعي D3ئج انخفاضاً معنویا في كل من مستوى فیتامین اذ أظھرت النتا التفاعلي، معدل ترسب كریات الدم الحمراء والكلوبیولینات المناعیة Cالنتائج ارتفاعا معنویا في مستوى كل من بروتین IgA , IgG صحاء مجموعة سیطرة بینما أظھرت النتائج عدم وجود فرق انتي تربسین مقارنة مع األ -1 –وبروتین الفا معنوي في مستوى البروتین الكلي لمرضى داء السكر النوع الثاني مقارنة مع األصحاء مجموعة سیطرة، ومن خالل ھذه اء السكر لھ تأثیر في تطور الحالة االلتھابیة عند مرضى د Dالنتائج توصلنا إلى أن النقصان الحاصل في مستوى فیتامین النوع الثاني. ، مرض السكر النوع الثاني، االلتھاب، بروتینات الطور الحاد.3Dفیتامین : الكلمات المفتاحیة 267 | Chemistry @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013