Volume: I: Issue-2: Aug-Oct ISSN

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1 Volume: I: Issue-2: Aug-Oct ISSN EVALUATION OF INSULIN RESISTANCE AND OXIDATIVE STRESS IN OBESE PATIENTS WITH POLYCYSTIC OVARY SYNDROME S. Kandasamy*, R. Inmozhi Sivagamasundai, A. Bupathy, S. Sethubathy, and V. Gobal ** *Depatment of Biochemisty, Jawahalal Institute of Postgaduate Medical Education and Reseach, Puduchey , INDIA. Division of Biochemisty, Rajamuthiah Medical College and Hospital, Annamalai Univesity, Tamil Nadu , INDIA. Depatment of Obstetics and Gynaecology, Si Manakula Vinayaga Medical College and Hospital, Puduchey , INDIA. ** College of Phamacy, Mothe Theesa Postgaduate and Reseach Institute of Health Science, Puduchey , India. BACKGROUND : Patient with Polycystic ovay syndome (PCOS) ae known to have high incidence of Insulin Resistance (IR) and oxidative stess and most of the patients ae obese. The aim of this study was to evaluate the insulin esistance and oxidative stess in obese PCOS patients. METHODS : 104 obese patients with PCOS [BMI 23 Kg/m 2 ] and 95 healthy contol subjects wee included in this study. Plasma sex steoid homones, fasting insulin, fasting glucose, wee measued in both the goups. Oal glucose toleance test was also pefomed. Total antioxidant status (TAS), malonyldialdehyde (MDH), potein cabonylation (PC), eythocyte educed gluthathione (GSH) and antioxidant enzymes like eythocyte catalase and eythocyte glutathione peoxidase (GPx) activity wee measued. Insulin esistance was evaluated by using homeostasis model assessment fo insulin esistance (HOMA-IR), quantitative insulin sensitivity check index (QUICKI) and fasting glucose / fasting insulin atio (fasting G:I atio). RESULTS : Compaed with healthy women, those with obese PCOS had significantly elevated HOMA-IR, plasma MDA, potein cabonylation, eythocyte glutathione peoxidase activity and significantly deceased QUICKI, fasting G:I atio, plasma TAS, GSH, catalase activity. CONCLUSION: The esult of the pesent study indicates, high level of insulin esistance and significantly inceased oxidative stess wee obseved in obese PCOS patients when compaed with contols. Key wods : Insulin esistance / Oxidative stess / PCOS Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 391

2 INTRODUCTION Polycystic ovay syndome (PCOS) is one of the most common and heteogeneous endocine disodes among women of epoductive age. The pevalence is estimated at 5-10% (knochenhaue et al 1998, Azziz et al 2004, Goldenbey N et al 2008). It is chaacteized by mensual iegulaities, clinical and/o biochemical hypeandogenism and hypeinsulinaemia seconday to educed insulin sensitivity (Hombug, 2003). Thee is no consensus on the diagnostic citeia and definition of PCOS. Accoding to ESHRE/ASRM (Euopean Society fo Human Repoduction and Embology / Ameican Society fo Repoductive Medicine) consensus confeence on PCOS held in othedam in 2003, PCOS was defined as at least 2 out off thee following abnomalities (a) oligo and/o anovulation, (b) clinical and/o biochemical signs of hypeandogenism (hisutism and/o acne o inceased andogens levels) (c) Detection of polycystic ovaies by ultasound and pesence of 10 o moe cyst of 2-10mm in diamete in each ovay and absence of othe endocine conditions such as thyoid disode, cushing s syndome, congenital viilizing adenal hypeplasia o hypepolactinemia. The etiology of PCOS is still unknown. Howeve insulin esistance and associated hypeinsulinemia play impotant oles in the pathogenesis of PCOS (Dunaif A, 1997). IR and hypeinsulinemia appeas to intefee with ovaian steiodogenetic defects as well as anovalatoy mechanism (Poetsky L P-999). These two factos poduce the hypeandogenism intefeing with pituitay ovaian axis, leading to inceased LH levels, anovulation, amenohea and infetility. PCOS is associated with an inceased isk of metabolic complications including impaied glucose toleance, type 2 diabetes, hypetension, dyslipidemia. Seveal studies have documented that atleast 30-40% of PCOS patients wee obese o ove weight and obesity has an aggavating ole in PCOS. (Diamanti Kandaakis et al 1999, Azziz et al 2004) Oxidative stess means an imbalance between the poduction of eactive oxygen species and the antioxidant defence system which poduces the oxidative damage. Oxidative stess has been implicated in a numbe of diseases such as cadiovascula disease, neuologic disease, malignancies, enal disease, diabetes, inflammatoy poblems, skin diseases, aging, espiatoy diseases, live diseases and diffeent types of vial infections (Kainsky N.J. 1992, Betheidge et al, 2000). Oxidative stess also impais insulin action, which has been demonstated in type 2 diabetes and this impaiment might be due seveal factos such as membane fluidity alteations, deceased availability of nitic oxide and inceased intacellula calcium content (Caimi et al 2003, Elizabeth H et al 2008). TAS is sensitive to the changes in plasma antioxidant levels and degee of oxidative stess (Gaibaldi et al 2001). MDA is a make of lipid peoxidation and inceases in oxidative stess states (Knight JA et al 1987, Batteidge 2000). Recent studies have documented inceased oxidative stess in patients with PCOS (Sabuncu T et al 2001, Fenkei et al 2003, Zhang D et al 2008) which may incease the isk of cadiovascula disease in such patients. Till date thee ae no studies elated to the same in Indian studies. Theefoe the aim of this study was to evaluate the insulin esistance and oxidative stess in obese PCOS patients. MATERIALS AND METHODS The study was appoved by Institute eseach council boad and followed by human ethical committee, Jawahalal Institute of Postgaduate Medical Education and Reseach (JIPMER), Puduchey, India. The witten infomed consent was obtained fom patients and contols. The diagnosis of PCOS was based on the ESHRE/ASRM consensus confeence held in Rottedam in Patients with diabetes mellitus, thyoid dysfunction, cushing s syndome, congenital andenalhypeplasia, hype poloctinemia, andogen seceting tumou, enal and live disodes wee excluded fom the study by specific laboatoy tests. Subjects with medication like ovaluation induction agent, antiandogens, antidiabetic, antiobesity, homonal dugs and cuent o pevious use of OC within last 6 month smoking and alcohol intake wee also excluded fom study. Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 392

3 Subjects One hunded and fou patients with PCOS aged between 20 to 35 yeas wee ecuited fom the out patient depatment of Obstetics and Gynecology, JIPMER, Puduchey, India. The contol goup consisted of 95 healthy voluntee females with egula menstual cycles aged between 20 to 35 yeas. Body mass index (BMI) and waist to hip atio (WHR) wee calculated. weight and height of the subjects wee measued in light clothing without shoes. BMI was calculated as weight by height squae (Kg/m 2 ). Waist cicumfeence was measued at naowest level between costal magin and the Iliac cest and hip cicumfeence was measued at the widest level ove the buttocks with subject was standing and beathing nomally. Degee of hisutism was detemined by using Feyman-Gallwey scoing. A scoe 8 was (Feiman-Gallwey, 1961) defined as Hisutism. Afte hous fasting 7ml of anticoagulant venous blood was collected between 8.00 and 8.30am on the 2 nd day of spontaneous o pogesteone (Medoxy pogesteone acetate-10mg/day fo 7 days) induced withdawal bleeding fo estimations of insulin esistance makes, sex steoid homones and oxidative stess paametes, polactin, cotisol, thyoid pofile, live function test and enal function test etc. A standad 75gam glucose toleance test (OGTT) was pefomed afte h fasting between 8.30 and 10.30am. Glucose toleance state was evaluated using the citeia of Ameican Diabetes Association Those patients with DM wee not included in the study. The homeostasis model assessment fo insulin esistance (HOMA-IR) was calculated accoding to the following fomula: fasting glucose (mmol/l) x fasting insulin (µu/ml) / 22.5 (Mathews et al 1985). The quantitative insulin sensitivity check index (QUICKI) was calculated accoding to following fomula : 1/[log fasting insulin (µu/ml) + log fasting glucose (mg/dl)] (Katz et al, 2000). Fasting glucose (mg/dl)-insulin (µiu/ml) atio also calculated (Paa A et al 1994). Plasma glucose (Agappe-India) was estimated by glucose oxidase-eoxidase (GOD-POD) method by clinical chemisty autuanalyse. [Baye Expess plus, USA] Plasma Insulin was (Bio line, Belgium) estimated by ELISA Techniques (Lab system-multiskan Ascent, Finland). Plasma TAS was measued spectophotometically at 593 nm using feic educing antioxidant powe assay (FRAP). The esults wee expessed as µmol of FRAP/ml. Plasma MDA levels wee estimated by spectophotomety at 530nm by Yagi (Yagi-1984). The esults wee expessed µmol/ml, plasma potein cabonylation was estimated by spectophotomety at 366nm by modification of Levines method by chakobothy. The esults wee expessed as nmol/ml. Eythocyte educed glutathione (GSH) was estimated by spectophotomety at 412nm by Beutle et al (Beutle et al 1963). The esults wee expessed as mg/g Hb. Haemoglobin was estimated with Dabkin eagent (Mek, Bombay, India) fo calculation of glutathione assay. RBC-catalase activity was assayed by spectophotomety at 240nm by Aebi (Aebi 1984). The esults wee expessed as K/ml. The RBC glutathione peoxidase (GPx) activity was detemined by spectophotomety at 412 by Wendel method (Wendel et al 1984). The esults wee expessed as U/gmHb. Plasma levels of LH, FSH, Polactin (immuno tech, Czech epublic) and TSH (BRIT, India) wee estimated by immuno adiometic assay (IRMA) (PC-RIA-MAS STRA Tec-Gemany), plasma total testosteone, pogesteone (immune tech-czech epublic), estadiol (RADIM-Italy), T3, T4 (BRIT-India) and cotisol (Diasoia-USA) wee estimated by RIA. (PC-RIA-MAS STRA Tec-Gemany) and fee testosteone, Andostenedione, 17-Hydoxy pogesteone (17-OHP), Pogesteone, sex homone binding globulin (SHBG) and dehydoepiandostenedion sulphate (DHEA-S) (EQUIPAR-Diagnostic, Italy) wee measue by ELISA technique (Stat-Fax, 300 CPC-Gemany). Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 393

4 STATISTICAL ANALYSIS Data was expessed as mean ± S.D. Fo compaison of all quantitative vaiables between contol and PCOS goups, the unpaied t-test was used and association between the paametes wee assayed by Peason coelation analysis. Statistical significance was accepted at P<0.05. All statistical analysis wee pefomed using SPSS Pogam (Chicago, IL). RESULTS Mean age, BMI and basal clinical and endocinological paametes of PCOS patients and contol subjects wee shown in Table I. Basal Insulin esistance and oxidative stess paametes wee shown in Table II. TABLE I : BASAL CLINICAL AND HORMONAL PARAMETERS IN CONTROL AND PATIENTS WITH OBESE PCOS SUBJECTS Vaiables Contols (n=95) PCOD (n=104) Age (Ys) 27± ±3.30 BMI (Kg/m 2 ) 22.08± ±1.45* W/H atio 0.82± ±0.01* F-G scoe ( 8) 0/75 17/87* LH (µiu/ml) 5.98± ±7.00* FSH (µiu/ml) 5.74± ±1.85 Total Testosteone (ng/dl) 36.60± ±16.65* Fee Testosteone (Pg/mg) 2.01± ±0.74 Andostnedione-A4 (ng/dl) 1.47± ±0.87* Pogesteone (ng/dl) 0.45± ± Hydoxy pogesteone (ng/dl) 0.53± ±0.18 Estadiol-E2 (Pg/ml) 58.92± ±11.51 Sex homone binding globulin 62.39± ±11.44* SHBG-(nmol/L) Dehydo epiandostenedion sulphate ± ±72.33* DHEA-S (µg/dl) * P<0.05 compaed to contols. Table I shows baseline clinical and endocinological paametes of contol subjects and obese PCOS patients. Mean age between contol and obese PCOS goups wee simila. Compaed with healthy women, those with obese PCOS had inceased BMI, WHR atio and F-G scoe (P<0.05). Homonal Paametes : Compaed to contol, subjects with obese PCOS exhibited highe level of LH (P<0.05), total testosteone (P<0.05), and andostenedione (P<0.05) and DHEA-S (P<0.05), whee as deceased levels of sex homone binding globulin (P<0.05). Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 394

5 TABLE II : BASAL INSULIN RESISTANCE AND OXIDATIVE STRESS PARAMETERS IN CONTROL AND PATIENT WITH OBESE PCOS SUBJECTS Vaiables Contols (n=95) PCOD (n=104) Fasting glucose (mg/dl) 83.76± ±12.56** Fasting insulin (µiu/ml) 14.27± ±5.3** HOMA-IR 2.92± ±2.36** QUICKI 0.33± ±0.09** Fasting G:I atio 6.20± ±0.33** MDA (µmol/l) 3.20± ±1.63* PC (µmol/ml) 3.21± ±1.61* TASC (µmol/ml) ± ±17.82* Eythocyte GSH (mg/g Hb) 7.37± ±17.82* Eythocyte catalese (K/ml) 23.44± ±4.3* Eythocyte GPx (U/g Hb) 25.19± ±7.22* **P<0.005 compaed to contols. * P<0.05 compaed to contols. Insulin esistance Table II shows baseline insulin esistance and oxidative stess paametes of contol and obese PCOS patients. Insulin esistance makes like fasting glucose, fasting insulin and HOMA-IR ee significantly inceased in obese patients than the contol subjects (P<0.005) whee as QUICKI and fasting G/I atio wee deceased (P<0.005). Oxidative stess Women with obese PCOS had significantly highe plasma MDA, Plasma potein cobonylation and inceased activity of eythocyte glutathione peoxidase (P<0.05) than the contol subjects. Whee as plasma TAS, eythocyte GSH levels and eythocyte catalase activity wee loweed in obese PCOS than the contol (P<0.05). Coelation Analysis The coelation between oxidative stess and antioxidant paametes in PCOS cases ae shown in table III. Both MDA and potein cabonyl wee significantly coelated with TAS, GSH, Catalase and GPx (P<0.01). TABLE III: CORRELATION BETWEEN OXIDATIVE STRESS AND ANTIOXIDANT PARAMETERS IN PCOD CASES Vaiables MDA Potein cabonyl p p TAS < < 0.01 GSH (mg/g Hb) < < 0.01 Catalase (K/ml) < < 0.01 GPx (U/g Hb) < < 0.01 Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 395

6 The coelation between oxidative stess and antioxidant paametes insulin esistance in PCOD cases ae shown in table IV. MDA, PC, Catalase and GPx wee positively coelated with insulin and HOMA-IR and negatively coelated with QUICKI (P<0.01). TAS and GSH wee negatively coelated with insulin and HOMAIR and positively coelated with TAS and GSH (P<0.01). Table 22: Coelation between oxidative stess and insulin esistance in PCOD cases DISCUSSION Vaiables Insulin HOMA-IR QUICKI MDA (µmol/l) 0.778* 0.763* * PC (nmol/ml) 0.787* 0.789* * TAS (µmol/ml) * * 0.724* GSH (mg/g Hb) * * 0.764* Catalase (K/ml) 0.685* 0.699* * GPx (U/g Hb) 0.740* 0.707* * * p < 0.01 Insulin esistance with esultant hypeinsulinaemia is a pominent featue of PCOS, and it is seen both in obese and nomal weight woman (Chang et al 1983, Dunaif et al 1989). Moeove, obese women develop a geate degee of insulin esistance as thei body mass inceases (Rittmaste et al 1993, Naveed Satton BHR, 2009). Recent studies have-shown that PCOS is not only a gynalcological condition affecting women of epoductive age but also a compehensive syndome with a vaiety of associated metabolic disodes such as insulin esistance, dyslipidaemia (Wild et al 2000). The pesent study evaluated insulin esistance and moe ecently emeging isk factos such as oxidative stess. Although the mechanism leading to the development of PCOS ae still not completely undestood. Howeve insulin esistance and hypeinsulinaemia may play an impotant ole in pathophysiology of PCOS (Dunaif et al 1989 and 1997). Insulin esistance is associated with obesity.(azziz et al 2004 and Diamanti-kandaakis 2008). In the pesent study, insulin esistance, as detected by the makes HOMA-IR highe in obese PCOS than in healthy contols and QUICKIand fating G/I atio wee loweed. Oxidative stess is an accepted isk facto fo the development of CVD (Betteidge, 2000). PCOS is also associated with an inceased isk of CVD. In PCOS, oxidative stess in esponse to hypeglycemia may be capable of diectly stimulating hypeandogenism. This is suggested by the association between plasma testosteone o andostedione and eactive oxygen species (ROS) geneation. In vito studies have shown that the ovaian steoidogenic enzymes esponsible fo andogen poduction ae stimulated by oxidative stess and inhibited by antioxidants such as statins (Piotowski et al 2005 and R Zepczynska IJ et al 2005 and Elizabeth H et al 2008). In ou study, plasma TAS levels, eythocyte GSH levels and eythocyte catalase activity wee significantly lowe in obese women with PCOS compaed with healthy contols. Highe plasma MDA levels, plasma potein cobonylation, and activity of eythocyte glutathione eductase wee found in patients than contols. These findings implicate the pesence of inceased oxidative stess in obese patients with PCOS. In conclusion, in the pesent study, we have evaluated insulin esistance and oxidative stess and thei elationship in obese PCOS patients. Insulin esistance and oxidative stess and accepted isk factos fo CVD. The pesent study confimed that such isk factos ae pesent moe in obese patient with PCOS in ou population. Futhe moe we found that inceased levels of andogens in obese PCOS patients. Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 396

7 REFERENCES Ameican Diabetes Association Sceening fo Diabetes. (2002) Diabetes Cae; 25: Aebaeda M, Rodigue Z-Espinosa, Muugo M, de Leyvaa and cocoy R. (2000) Assessment of insulin sensitivity and beta cell function fom measuement in the fasting state and duing and oal glucose toleance test. Diabetitogia; 43: Atiomo WU, EL Mahdi and Hadiman. (2003) Family association in women with polycystic ovay syndome. Fetile Steil; 80: Azziz R, Woods KS, Reyna R, Key TJ, Knochenhaue ES and Ylldiz BO. (2004) The pevalence and featues of polycystic ovay syndome in an unselected population. J din Endocinol Metab; 89: Batteide DJ. (2000) What is the oxidative stess? Metabolism;49(1): Bjontop P. (1996) The andoid woman-a isky condition. J Inten Med; 236: Bughen GA, Givens JR and Kitabchi HE. (1980) Coelation of hype andogenism with hypeinsulinemia in polycystic ovaian disease. J Clin Endocinol Metab; 50: Caimi G, Gaollo C and Deshi RL. (2003) Diabetes mellitus: oxidative stess and wine. Cu Med Res Opinion; 19: Chamukuttan Shehalatha, Vijay Viswanathan and Ambady Ramachandan. (2003) Cut off values fo nomal anthopometic vaiables in Asian Indian a dults-diabetes Cae; 26: Cibula D. (2004) Is insulin esistance an essential component of PCOS? Hum Repod; 19: Diamanti-Kandaa Kis E and Papaxastiline AG. (2006) Molecula mechanisms of insulin esistance in polysystic ovay syndome. Teads Mol. Med; 12: Dunaif A. (1997) insulin esistance and polycystic ovay syndome : Mechanism and implications fo pathogenesis. Endoc Res; 18: Ehmann DA, Banes RB, Rosenfield RL and Caaghan MK and Impeial J. (1999) Pevalence of impaied glucose toleance and diabetes in women polycystic ovay syndome. Diabetes Cae; 22: Elizabeth H, Rude, Teyl J, Hatman, Jeffey Blumbey and Melene B Goldman (2008) oxidative stess and antioxidant: exposue and impact on female fetility. Human Repoduction update Expet Committee on the diagnosis and classification of diabetes mellitus. (2003) Follow-up epot on the diagnosis of diabetes mellitus. Diabetes cae; 26: Fenkci V, Fenkci S, Yilmaze M and Setese M.(2003) Deceased total antioxidant status and inceased oxidative stess in women with polycystic ovay syndome may contibute to the isk of cadiovascula disease. Fetil Steil; 80: Gaibaldi S, Valantini S, Aagno I, Ponzato MA, Taveso N and Odethi P. (2001) Plasma potein oxidation and antioxidant defense duing aging. Int. J Vitam Nut Res; 7: Hombug (R). (2003) What is polysystic ovay syndome? Hum Repoduction; 17: Jayagobal, Kilpatick ES, Holding S, Jennings Pe and Atkin SL. (2002) The biological vaiation of insulin esistance in polycystic ovay syndome. J Clin Endocinol Metab; 87: Kaen Elkind-Hisch, Oy Maioneaux, Madhu Bhushan, Denise Veno and Raj Bhushan. (2008) Compaison of single and combined teatment with exenatide and metfomin on mensual cyclicity in ove weight women with polysystic ovay syndome. J Clin Endocin Metab; 10: 1210/JC: Katz A. Nambi SS, Mathe K. Baon AD, Follmann DA, Sullivan G and Quon MJ. (2000) Quantitative insulin sensitivity check index: a simple, accuate method fo assessing insulin sensitivity in humans. J Clin Endocinol Metabolism; 85: Knight JA, Smith SE, Kinde VE and Anstall (HB). (1987) Refeence intevals fo plasma lipopeoxides age, sex and specimen-elated vaiations. Clin Chem; 33: Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 397

8 Lego R. (2003) Polycystic ovay syndome and cadiovascula disease: a pematue association. Endoco Rev; 24: Loscalzo J. (2003) Oxidative stess; a key deteminant of atheothombosis. Biochem soc Tans; 31: Nelson VL, Qin K, Rosenfied RL, Wood JR, Penning TM, Lego RS, Stauss JF III and McAlliste JM. (2001) The biochemical basis fo inceased testosteone poduction in theca cells popagated fom patients with polycystic ovay syndome. J Clin Endocinol Metan; 86: Noman RJ, Mastes SC, Hague W, Beng C, Pannall P and Wang Jx. (1995) Metabolic appoaches to the subclassification of polycystic ovay syndome. Fetile Steil; 63: Paa A, Ramiez A and Espinosa de los Monteos A. (1994) Fasting glucose / insulin atio. An index to diffeentiate nomo-fom hypeinsulinemic women with polycystic ovay syndome. Rev Inest Clin; 46: Radziuk J. (2000) Insulin sensitivity and its measuement: Stuctual commonalities among the methods. J Clin Endocinol Metab; 85: Richadson MR. (2003) Cuent pespectivites in polycystic ovay syndome. Am Fam Physician; 68: Rottedam wokgoup (2004): Revised 2003 consensus on diagnostic citeia and long-tem health isks elated to polycystic ovay syndome. Fetile Steile; 81: Sabuncu T, Vual T, Hama M and Hama M. (2001) Oxidative stess in polycystic ovay syndome and its contibution to the isk of cadiovascual disease. Clin Biochem; 34: Salley KES, Wickham ED, Cheang KL, Essah PH, Kajane NW and Nestle JE. (2007) Position statement. Glucose intoleance in polycystic ovay syndome. A position statement of the Andogen Excess Society. J. Clin. Endocinol Med; 92: Solomon CA, Hu FB, Dunaif A, Rich-Edwads J, Willett WC, Hunte DJ, Colditz GA, Speize FE and Manson JE. (2001) Long o highly iegula menstual cycles as a make fo isk of type 2 diabetes mellitus JAMA; 286; Wild S, Piepoint T, Mc Keigue P and Jacobs H. (2000) Cadiovascula disease in women with polycystic ovay syndome at long-tem follow up: a estospective cohet study. Clin Endo Cinol (oxf); 52: Yildiz BO, Yaali H, Oguz H and Bayakta M. (2003) Glucose intoleance, insulin esistance and hypeandogenemia in fist degee elative of women with polycystic ovay syndome. Clin Endocinol Meta; 88: 2031, ************************** Intenational Jounal of Applied Biology and Phamaceutical Technology Page: 398 Now indexed in Schola, DOAG, Open-J-gate, Biology Bowse