157 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 Interleukin-35 in Iraqi's Menopausal Women with Osteoporosis Husam S. Jasim Dept. of Chemistry / College of Education For Pure Sciences( Ibn Al- Haitham)/ University of Baghdad Received in: 19 May 2014, Accepted in:5 January 2014 Abstract Osteoporosis is related to the loss of bone mass that occurs as part of the natural process of aging and in menopausal women. The aim of this study is to evaluate the levels of interleukin-35 in menopausal women with osteoporosis, that may be used as an early predicted marker of osteoblastic and osteoclastic activity in osteoporosis patients in relation with other diagnostic parameters. Fasting blood samples were obtained from forty Iraqi menopausal women with mean age of (61.06±6.59)years who were newly diagnosed of osteoporosis by physicians at Baghdad Teaching Hospital as patients group(G1). In addition to , twenty two healthy women were included in the study as a control group(G2) with mean age(60.75±6.35). Serum samples were used for determination of Rheumatoid Factor (RF), Alkaline Phosphatase(ALP), Calcium(Ca+2), Phosphate(PO4-3) and Interleukin-35(IL-35),while blood sample was used for determination of Erythrocyte Sedimentation Rate (ESR). The results revealed a highly significant increase in IL-35 level in patients group(G1) compared to the control group(G2) while there are no significant differences in ALP, Ca+2 and PO4-3 levels between G1 and G2. The correlation between serum levels of IL-35 and ALP ,Ca+2 and PO4-3 in menopausal osteoporosis patients was calculated; A significant positive correlation between IL-35 and ALP was found (r=0.277,P<0.05), while there was no significant correlation between IL-35 and each of Ca+2 and PO4-3 levels (r=0.191,P>0.05),(r=0.124,P>0.05) respectively. The conclusion obtained from this study that IL-35 may be used as an additional biomarker in predicting osteoporosis in postmenopausal women. Key Words: Osteoporosis, Interleukin-35 158 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 Introduction Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk[1].Although osteoporosis can occur in men, it is most common in women who have gone through menopause[2]. The interleukin-12 (IL-12) family, is composed of IL-12, IL-23, IL-27, and the newly identified IL-35[3]. The IL-12 cytokine family share binding partners and structural features, there are notable differences in their pattern of expression and secretion. IL-12, IL-23, and IL- 27 are all primarily produced by antigen-presenting cells, following their activation by recognition of pathogen-specific patterns. In contrast, secretion of bioactive IL-35 has been described only in regulatory T cells (Tregs)[4-6]. Interleukin-12,IL-23,and IL-27 share the common feature of inducing interferon- production and promoting T-helper 1(Th1) cell differentiation and proliferation, they act differentially on subsets of T cells and with different kinetics. In contrast, IL-35 appears to function solely in an anti-inflammatory fashion by inhibiting T-cell proliferation and perhaps other parameters[3]. The biological effects of IL-12,IL-23,IL-27, and presumably IL-35 are mediated through their interaction with their receptor chains which bind to and induce phosphorylation of the Janus kinase (Jak) family proteins[7]. Jak proteins then phosphorylate tyrosine residues on the intracellular domains of the receptor chains, which act as docking sites for various members of the signal transducer and activator of transcription(STAT) family. Phosphorylated STAT proteins form intramolecular complexes that translocate into the nucleus and bind DNA to initiate the gene expression profile and cellular response that typifies the given cytokine[8,9]. The aim of this study is to evaluate the levels of IL-35 in postmenopausal women with osteoporosis, that may be used as an early predicted marker of osteoblastic and osteoclastic activity in osteoporosis patients in relation with other diagnostic parameters. Materials and Methods Fasting blood samples were obtained from forty Iraqi postmenopausal women with mean age of (61.06±6.59) years who were newly diagnosed of osteoporosis by physicians at Baghdad Teaching Hospital as patients group(G1) . In addition to, twenty two healthy women were included in the study as a control group(G2) with mean age of (60.75±6.35). Serum samples were used for determination of RF, ALP, Calcium and Phosphate While Blood sample was used for determination of ESR, using standard procedures of the biochemistry laboratory of the hospital. Serum IL-35 levels were measured using specific enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO) human IL-35 for in vitro quantitative measurement, according to the manufactures protocol[10]. The data were expressed as mean±SD. The comparison between patients and control group were analyzed using student t-test. Pearson's correlation coefficient was used to find the correlation between IL-35 with ALP, Ca+2 and PO4-3 in patients group. P-value of < 0.001 and <0.05 were considered highly significant and significant respectively. Results and Discussion Descriptive and diagnostic parameters for the two studied groups(G1,G2) are shown in table(1). The results revealed highly significant increase in IL-35, ESR and RF levels in patients group(G1) compared to control group(G2) while there are no significant differences in ALP, Ca+2 and PO4-3 levels between G1 and G2. 159 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 Calcium is an essential element in the human body and is necessary to many cell functions. Calcium and vitamin D have long been recognized as important and required nutrients for bone health and maintenance. Calcium is a vital component of bone architecture and is required for deposition of bone mineral throughout life. It is the levels of plasma calcium that dictate calcium balance. Adequate intake of calcium is necessary to maintain this balance[11]. The amount of phosphate in the blood affects the level of calcium there. Calcium and phosphate in the body react in opposite ways: As blood calcium levels rise, phosphate levels fall[12,13]. Alkaline phosphatase (ALP)[EC 3.1.3.1] are a group of enzymes that are present in many different tissues, it is made mostly in the liver and in bone with some made in the intestines and kidneys[14]. It is also made by the placenta of a pregnant woman. The liver makes more ALP than the other organs or the bones. Some conditions cause large amounts of ALP in the blood. These conditions include rapid bone growth (during puberty), bone disease (osteomalacia or Paget's disease), or a disease that affects how much calcium is in the blood (hyperparathyroidism),vitamin D deficiency, or damaged liver cells[15]. The results of the present study are in agreement with previous study that suggested elevated concentration of alkaline phosphatase even levels within the normal range of the enzyme may indicate an increased level of bone cell activity and, therefore, potential bone loss[16]. Bone cell activity is a normal occurrence marked by the breaking down and rebuilding of bone at remodeling sites. When estrogen levels decrease after menopause, the remodeling rate increases, but the process of rebuilding the bone is not as efficient. Estrogen helps to keep calcium in the bones, postmenopausal women are at increased risk for bone loss and osteoporosis[17]. The results in table(2) showed the correlation relation between serum levels of IL-35 with ALP ,Ca+2 and PO4-3 in menopausal osteoporosis patients, A significant positive correlation between IL-35 and ALP was noted (r=0.277,P<0.05), while there are no significant correlation between IL-35 levels and Ca+2 , PO4-3 levels were found (r=0.191,P>0.05),(r=0.124,P>0.05) respectively. Bone resorption is tightly and dynamically regulated by multiple mediators, including cytokines that act directly on osteoclasts and their precursors, or indirectly by modulating osteoblast lineage cells that in turn regulate osteoclast differentiation. It has been extensively documented that various inflammatory factors produced by activated immune cells act as antiosteoclastogenic factors by different mechanisms. Suppression of osteoclastogenesis by inflammatory factors and cytokines functions as a feedback inhibition system that limits bone resorption and tissue damage associated with infection or inflammation[18]. The role of interleukins in bone metabolism has been investigated, which may help to expose new targets for gene therapy[19]. IL-12 superfamily of cytokines has pleiotropic immune functions with either activating or suppressive roles in various infectious, inflammatory models and potently inhibits receptor activator of nuclear factor kappa-B ligand (RANKL)-induced human osteoclastogenesis and osteoclastic[20,21]. IL-12 plays an inhibitory role in osteoclastogenesis by directly inhibits osteoclast precursors or by targets other cell types such as stromal/osteoblastic cells or T cells to indirectly suppress osteoclastogenesis [22]. Apoptosis induced by interactions between IL-12- induced Fas ligand (FasL) and Tumor necrosis factor (TNF-α)-induced Fas contributes to the inhibitory mechanisms of IL-12 in TNF-α-induced osteoclastogenesis[23]. The conclusion could be drown from this study that IL-35 could be used as an additional biomarker in predicting osteoporosis in menopausal women. 160 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 References 1. Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis. Report of a WHO Study Group. Geneva: WHO;( 1994). 2. Pagana, K.D.and Pagana, T.J. (2010) Mosby’s Manual of Diagnostic and Laboratory Tests, 4th ed., St. Louis, Mosby Elsevier. 3. Collision, L.W.; Workman, C. J.; Kuo, T.T.; Boyd, K.; Wang, Y.; Vignali, K.M.; Cross, R.; Sehy, D.; Blumberg, R. S. and Vignali, D. A. A.(2007) the inhibitory cytokine IL-35 contributes to regulatory T-cell function, Nature, 450,566-569. 4. Pflanz, S.; Timans, J. C. ; Cheung, J. ; Rosales, R. ; Kanzler, H. ; Gilbert, J. ; Hibbert, L. ; Churakova,T. ;Travis, M. ;Vaisberg, E.; Blumenschein, W. M. ; Mattson, J. D. ; Wagner, J. L.; To,W. ; Zurawski, S.; McClanahan, T.K. ; Gorman, D. M.; Bazan, J.F. ; Malefyt, R. W. ; Rennick, D.and Kastelein, R. A.(2002) IL-27 a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naiva CD4(+)T cells. Immunity ,16,779-790. 5. Hibbert, L.; Pflanz, S.; De Waal Malefyt, R.and Kastelein, R.A.(2003) IL-27 and IFN- alpha signal via Stat 1 and Stat 3 and induce T-Bet and IL-12 Rbeta2 in naïve T cells, J Interferon Cytokine Res.,23,513-522. 6. Ma, X.; Chow, J.M.; Gri , G.; Carra, G.; Gerosa, F.; Wolf, S.F.; Dzialo, R. and Trinchieri, G.(1996)The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells, J Exp Med., 183,147-157. 7. Nagai,T.; Devergne, O.; van Seventer, G.A. and van Seventer, J.M.(2007) Interferon- beta mediates opposing effects on interferon-gamma-dependent Interleukin-12 p70 secretion by human monocyte-derived dendritic cells, Scand J Immunol,65,107-117. 8. Stumhofer, J.S. and Hunter, C.A.(2008)Advances in understanding the anti- inflammatory properties of IL-27, Immunol Lett.,117,123-130. 9. Krumbiegel, D.; Anthogalidis-Voss, C.; Markus, H.; Zepp, F.and Meyer, C.U.(2008) Enhanced expression of IL-27 mRNA in human newborns, Pediatr allergy Immunol, 19(6), 513-516 10. Shiuanada, N.B.(2008) "Manipal Manual of Clinical Biochemistry",3rd ed., Jaypee brothers medical publishers(P) Ltd New Delhi. 11. Bringhurst, F.R.and Demay, M.B.( 2005) Bone and mineral metabolism in health and disease. In: Harrison’s Principles of Internal Medicine, 16th ed, II. New York,McGraw Medical Publishing Division, 2246–9. 12. Fischbach, F.T. and Dunning, M.B. III(2009) Manual of Laboratory and Diagnostic Tests, 8th ed., Philadelphia, Lippincott Williams and Wilkins. 13. Strauss, Saltman,P.D.(1994) Spinal bone loss in postmenopausal women supplemented with calcium and trace mineral, Jour Nutr.,124, 1064-1064. 14. Francis,H.;glorieu john, M.; pettifor and Harold Juppner(2012)"pediatric bone"2ed Ed.,Elsevier Inc,USA. 15. Chernecky, C.C. and Berger, B.J. (2008) Laboratory Tests and Diagnostic Procedures, 5th ed., St. Louis, Saunders. 16. Jane, L.; Lukacs Nancy, E. and Reame.(2000) Concentrations of Follicle- Stimulating Hormone Correlate with Alkaline Phosphatase and a Marker for Vitamin K Status in the Perimenopause, Journal of Women's Health & Gender-Based Medicine,9,7,731-739. 17. Daniel, P.; Cardinali1,Marta, G.; Ladizesky2,Verónica Boggio1 and Rodolfo, A.(2003)Cutrera1and Carlos Mautalen2 Article first published online: 31 JAN 2, Journal of Pineal Research, 34,2, 81–87. 18. Braun,T. and Zwerina, J.(2011) Positive regulators of osteoclastogenesis and bone resorption in rheumatoid arthritis, Arthritis Res. Ther., 13(4),235. 161 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 19. Costa, R.P.; Han, S.W.; Pochini, A.C. and Reginato, R.D.(2011) Gene Therapy for osteoporosis, Acta Ortop Bras,19,1,52-7. 20. Kalliolias,G.D.; Zhao, B.; Triantafyllopoulou, A.; Park-Min,K.H. and Ivashkiv, L.B.(2010) Interleukin-27 inhibits human osteoclastogenesis by abrogating RANKLmediated induction of nuclear factor of activated T cells c1 and suppressing proximal RANK signaling, Arthritis Rheum, 62,402-413. 21. Furukawa,M.; Takaishi, H.; Takito, J.; Yoda, M.; Sakai, S.; Hikata,T.; Hakozaki, A.; Uchikawa, S.; Matsumoto, M.; Chiba, K.; Kimura, T.; Okada, Y.; Matsuo, K.; Yoshida, H. and Toyama,Y.(2009) IL-27 abrogates receptor activator of NF-kappa B ligandmediated osteoclastogenesis of human granulocyte-macrophage colonyforming unit cells through STAT1-dependent inhibition of c-Fos, J Immunol, 183,2397-2406. 22. Horwood, N.J.; Elliott, J.; Martin, T.J. and Gillespie, M.T.(2001) IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro. J Immunol, 166,4915-4921. 23. Kitaura, H.; Nagata, N.; Fujimura, Y.; Hotokezaka, H.; Yoshida, N. and Nakayama, K.(2002) Eff ect of IL-12 on TNF-alpha-mediated osteoclast formation in bone marrow cells: apoptosis mediated by Fas/Fas ligand interaction, J Immunol,169,4732- 4738. Table No.(1): Descriptive and diagnostic parameters for the patients and controls groups. Parameters Groups Age (year) BMI ESR mm/hr RF IU/ml IL-35 Pg/ml ALP (U/L) Ca+2 (mmol/L) PO4-3 (mmol/L) Patients Group (G1) N=40 61.06±6.59 30.52 ± 4.37 32.1 ± 3.14 52.10 ± 4.31 35.13 ± 5.01 78.75 ± 13.63 2.35±0.17 1.12±0.26 Control Group (G2) N=22 60.75±6.35 30.10 ± 4.08 10.91 ± 1.31 20.99 ± 6.21 22.13 ± 1.99 73.05 ± 10.75 2.21±0.14 1.22±0.20 P-Value NS NS HS HS HS NS NS NS NS(No Significant) P≥0.05, HS(Highly Significant) P<0.001 Table No.(2): Correlation between serum IL-35 with ALP, Ca and PO4 Parameters IL-35 pg/ml ALP (U/L) r 0.277 P S Ca+2 (mmol/L) r 0.191 P NS PO4-3 (mmol/L) r 0.124 P NS S (Significant) P<0.05 , NS (No Significant) P≥0.05 162 | Chemistry 2015) عام 1(العدد 28المجلد مجلة إبن الھيثم للعلوم الصرفة و التطبيقية Ibn Al-Haitham J. for Pure & Appl. Sci. Vol. 28 (1) 2015 عند النساء العراقيات المصابات بھشاشة العظام بعد انقطاع 35-انترلوكين الطمث حسام سلمان جاسم جامعة بغداد )/ابن الھيثم (كلية التربية للعلوم الصرفة /قسم الكيمياء 2015 كانون الثاني 5قبل البحث في:,2014ايار 19 البحث:استلم الخالصة سن اليأس في تعود ھشاشة العظام الى فقدان كتلة العظم وھو ما يحدث كجزء طبيعي بتقدم العمر وانقطاع الطمث عند النساء. تم الحصول على نماذج الدم من اربعين امرأة عراقية مصابة بانقطاع الطمث عند سن اليأس مع ھشاشة العظام معدل ) تم تشخيصھن حديثاً بواسطة االطباء في مستشفى بغداد التعليمي وعدون مجموعة المرضى 6.59±61.06اعمارھن ( )G1) ة منت الدراس ذلك تض ة22). ك ليمة كمجموع رأة س يطرة ( ) ام ارھن (G2س دل اعم تعمل 6.35±60.75) مع ). اس المصل في تقدير كل من: ) 3-4), phosphate (PO+2Rheumatoid Factor (RF), Alkaline phosphatase (ALP), Calcium (Ca and Interleukin-35 (IL-35). Erythrocyte Sedimentation Rate (ESR) في حين تم استعمال الدم في تقدير عند مجموعة المرضى مقارنةً بمجموعة السيطرة في حين ال IL-35اشارت النتائج الى ارتفاع معنوي في مستوى بين مجموعة المرضى ومجموعة السيطرة. كذلك بينت النتائج PO +2ALP , Ca ,4-3 يوجد اختالف معنوي في مستويات وكل IL-35في حين ال توجد عالقة معنوية بين ALP (r=0.277,P<0.05)و IL-35وجود عالقة موجبة معنوية بين على التوالي. 4PO (r=0.191,P>0.05) ,(r=0.124,P>0.05)-3و 2Ca+ من يمكن ان يعتمد كدالة مساعدة في التكھن باالصابة بھشاشة العظام عند النساء IL-35واستنتج من ھذه الدراسة ان المصابات بانقطاع الطمث في سن اليأس. 35-انترلوكين ,ھشاشة العظام الكلمات المفتاحية: