Ufuk University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey; 2

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 24 August 2009 Article Obesity and insulin resistance associated with lower plasma vitamin B 12 in PCOS Cemil Kaya was born in 1972 in Turkey. He graduated from Ankara University School of Medicine and also completed his obstetrics and gynecology specialisation there. He began research into in-vitro fertilization at the Gulhane Military School of Medicine in He is currently working in Ufuk University Faculty of Medicine. His special interests lie in the field of reproductive endocrinology, infertility and endoscopic surgery. Dr Cemil Kaya Cemil Kaya 1,3, Sevim Dincßer Cengiz 2, Hakan Satıroğlu 1 1 Ufuk University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey; 2 Ankara University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey 3 Correspondence: kayacemil000@yahoo.com Abstract Polycystic ovary syndrome (PCOS) shares some or most components of metabolic cardiovascular syndrome, manifested by abdominal obesity, insulin resistance, dyslipidaemia and atherosclerosis. It has been previously demonstrated that folate and vitamin B 12 treatment improved insulin resistance in patients with metabolic syndrome. This study first investigated whether PCOS patients have lower or higher vitamin B 12, folate and homocysteine concentrations when compared with healthy, age and body mass index matched controls, and, then examined associations between vitamin B 12, folate, homocysteine and insulin resistance and obesity in PCOS patients. Homocysteine concentrations and homeostasis model assessment index were higher, whereas concentrations of vitamin B 12 were lower in PCOS patients with insulin resistance compared with those without insulin resistance. Serum vitamin B 12 concentrations were significantly lower in obese PCOS women in comparison with obese control women (P < 0.05). Fasting insulin, insulin resistance and homocysteine are independent determinants of serum vitamin B 12 concentrations in PCOS patients. Insulin resistance, obesity, and elevated homocysteine were associated with lower serum vitamin B 12 concentrations in PCOS patients. Keywords: folate, homocysteine, insulin resistance, obesity, PCOS, vitamin B 12 Introduction Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age, is a multifaceted metabolic disease linked with insulin resistance (IR) (Diamanti-Kandarakis et al., 1999). It is characterized by hyperandrogenism, anovulation and hyperinsulinaemia (Dunaif, 1997). Elevated plasma homocysteine (Hcy) concentrations are an independent cardiovascular risk factor in women with PCOS (Yarali et al., 2001; Loverro et al., 2002; Orio et al., 2003; Schacter et al., 2003; Boulman et al., 2004). It is well known that PCOS patients are more insulin resistant than healthy women, even taking into account body weight (Legro et al., 1999). PCOS shares some or most components of metabolic cardiovascular syndrome, manifested by abdominal obesity, insulin resistance, dyslipidaemia and atherosclerosis (Talbott et al., 1995; Azziz et al., 2005). Hcy is a non-protein-forming, thiol-containing amino acid formed by demethylation of methionine (Fonseca et al., 1999). The importance of vitamin B 12 in the remethylation of Hcy to methionine is well recognized, and hyperhomocysteinaemia is a feature of vitamin B 12 deficiency (Selhub and Miller, 1992; McCarty, 2000). Furthermore, plasma Hcy is a sensitive biomarker of folate deficiency (McCarty, 2000). Insulin resistance in women with PCOS is associated with high plasma Hcy (Loverro et al., 2002; Schacter et al., 2003). It has been previously demonstrated that folate and vitamin B 12 treatment 721 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 722 improved insulin resistance in patients with metabolic syndrome (Setola et al., 2004). Many of the anthropometric and metabolic abnormalities of PCOS overlap with components of metabolic syndrome (Ehrman et al., 2006). Thus, in addition to total Hcy, plasma vitamin B 12 and folate may also be related to the extent of IR in PCOS. Although obesity and IR are closely related in patients with PCOS, associations between vitamin B 12, folate and IR and obesity in PCOS patients are not well characterized. This study first investigated whether PCOS patients have lower or higher vitamin B 12, folate and Hcy concentrations when compared with healthy, age- and body mass index (BMI)-matched controls, and then examined associations between vitamin B 12, folate, Hcy and IR and obesity in PCOS patients. Materials and methods Subjects The study group consisted of 61 young PCOS patients and 61 controls. Control group subjects were healthy volunteers with normal menstrual cycles who had no clinical or biochemical features of hyperandrogenism. Each control was defined as age- and BMI-matched with a PCOS case when the differences between the case and control was <2 years and <1 kg/m 2 for age and BMI, respectively. The healthy state of the controls was determined by medical history, physical and pelvic examination and complete blood chemistry. They were recruited from hospital staff and students. All PCOS subjects had irregular menses, and 61% of participants had eight or fewer spontaneous cycles per year. The diagnosis of PCOS was made according to the Rotterdam European Society for Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM)-sponsored PCOS Consensus Workshop Group (ESHRE Rotterdam, 2004). Specifically, all eligible patients presented with at least two of the following three criteria: (i) chronic anovulation, (ii) hyperandrogenism (hirsutism, acne) and/or hyperandrogenaemia and (iii) polycystic ovaries. The presence of polycystic ovarian appearance was determined ultrasonographically (Balen et al., 2003). Oligomenorrhoea (cycle intervals >35 days), amenorrhoea (absence of menstruation for 3 consecutive months), and luteal phase progesterone measurements less than 4 ng/ml in women with regular menstrual cycles were considered indicative of oligo-ovulation. Hirsutism was determined by a modified Ferriman and Gallway (1961) score above 7(Balen et al., 2003). All subjects underwent baseline testing of thyroid stimulating hormone (TSH), prolactin, 17-hydroxyprogesterone, and glucose during a 2-h oral glucose tolerance test (OGTT). Patients and controls who had diabetes mellitus, hyperprolactinaemia, congenital adrenal hyperplasia, thyroid disorders, Cushing s syndrome (1 mg dexamethasone suppression test), hypertension, vitamin B 12 and folate deficiency, hepatic or renal dysfunction or were smokers were excluded from the study. Subjects treated with any hormonal medications, vitamins or drugs that increase Hcy levels within the previous 3 months and those with folate, vitamin B 12 or vitamin B 6 deficiencies were excluded from the study. Patients were also excluded if they had used any confounding medications, including oral contraceptive agents, antilipidaemic drugs and insulin-sensitizing drugs which may affect the metabolic criteria, within 3 months prior to enrollment. None of the subjects contemplated pregnancy during the study period. All subjects were asked to give written consent and the institutional review boards of hospitals approved the study. Clinical and anthropometrical variables, including a modified hirsutism score and BMI, were determined by a single investigator in all subjects. BMI was calculated as weight (kg) divided by height (m) squared. Weight and height were measured in light clothing without shoes. BMI values of 25 kg/m 2 were considered as overweight. BMI values of 30 kg/m 2 were considered as obese. Waist circumference was measured at the narrowest level between the costal margin and iliac crest, and the hip circumference was measured at the widest level over the buttocks while the subjects was standing and breathing normally. The waist-to-hip ratio (WHR) was calculated. A WHR >0.72 was considered abnormal (Ashwell et al., 1982). Blood collections were carried out in the follicular phase of a spontaneous cycle or at any time after a spontaneous luteal phase was excluded by serum progesterone measurements (serum progesterone measurements <3 ng/ml) in patients with delayed menstruation. After a 3-day 300-g carbohydrate diet and 12-h overnight fasting, samples were obtained for the measurement of total testosterone, 17- hydroxyprogesterone, dehydroepiandrosterone-sulphate, prolactin, TSH, serum vitamin B 12, folate and glucose during a 2-h OGTT. Serum biochemistry and lipid profiles were also obtained. Next, all patients underwent a 2-h OGTT with a 75-g glucose load, with determinations of both glucose and insulin at baseline (before glucose load) and after 120 min. Baseline and post-treatment serum levels of insulin were measured using an electrochemiluminescence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics, Mannheim, Germany). Impaired glucose tolerance (IGT) was defined as a 2-h post-load glucose of 140 mg/dl or greater and less than 200 mg/dl (Expert Committee on the Diagnosis and Classification, 2003). Samples were immediately centrifuged at 3500 g, for 15 min, and serum was separated and frozen at 20 C until assayed. Hcy was measured as total Hcy by high performance liquid chromatography technique (Cromosystem, Mannheim, Germany). The intra- and interassay coefficients of variation were <2%. The presence of IR was investigated using basal insulin concentrations, fasting glucose concentrations and homeostasis model assessment (HOMA-IR > 2.1). HOMA-IR was calculated using the formula fasting glucose (mmol/l) fasting insulin (liu/ml) 0.055/22.5 (Matthews et al., 1985; Belli et al., 2004) Serum testosterone, LH, FSH and prolactin concentrations were measured using an electrochemiluminescence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics) with specific chemiluminescence assays

3 (ELECSYS 2010 HITACHI; Roche Diagnostics). Plasma glucose was determined using the glucose hexokinase method, (Cobas Integra 400 Plus; Roche Diagnostics). Levels of total cholesterol (Total-C) and triglycerides (TG) were determined with enzymatic colourimetric assays (Roche Diagnostics). High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were determined by a colorimetric method using a Cobas Integra 400 Plus autoanalyser (Roche Diagnostics). The intra-and interassay coefficients of variation were <5% for all assays performed. Serum vitamin B 12 and folate concentrations were measured using an electrochemiluminescence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics) with specific chemiluminescence assays (ELECSYS 2010 HITACHI; Roche Diagnostics). Mean and intra- and interassay coefficients of variation were 5.2% and 3.4%, respectively, for vitamin B 12 and 6.8% and 7.9%, respectively, for folate. The free androgen index (FAI) was calculated according to the equation FAI (%) = testosterone (ng/ ml) /sex hormone binding globulin (nmol/l). Statistical analysis Data are shown as means ± SD, or raw numbers and percentages. Data analysis was performed using the Statistical Package for Social Sciences (SPSS) for Windows, version 11.5 (SPSS Inc., USA). Groups were compared using Student s t or Mann Whitney U-test, where appropriate. ShapiroWilk test was used in order to detect whether or not the continuous variables were normally distributed. Groups were compared using Student s t or Mann Whitney U-test, where appropriate. Chi-squared test was used to compare differences in rates. Correlations between parametric variables and nominal parametric data were assessed by Pearson correlation coefficients. Multiple linear regression stepwise method was used to determine the independent predictors that most commonly affected plasma vitamin B 12 concentrations. Descriptive statistics were shown as means ± standard deviation for continuous data and percentages for categorical ones. A P-value <0.05 was considered statistically significant. Results Anthropometrical, metabolic and cardiovascular risk (CVR) profiles of PCOS and control groups are summarized in Table 1. Compared with the control group, women with PCOS had significantly higher fasting insulin, HOMA index, total testosterone, FAI, Hcy and LDL cholesterol (for each parameter P < 0.05). To determine the effects of IR on serum vitamin B 12, folate and Hcy concentrations, PCOS patients were allocated into two groups based on HOMA index. IR was defined as an abnormal results in HOMA index >2.1 (Matthews et al., 1985; Belli et al., 2004). IR was present in 31.1% (19/61) of PCOS patients. Hcy concentrations were higher, whereas concentrations of vitamin B 12 were lower in PCOS women with IR compared with in PCOS women without IR (Table 2). To determine the effects of IR on serum vitamin B 12, folate and Hcy concentrations, the women (PCOS patients and controls) were allocated into two groups based on HOMA index irrespective of whether or not they had PCOS. Hcy levels were higher, whereas concentrations of vitamin B 12 were lower in women with IR compared with those with non-ir (Table 3). To examine the effects of obesity on serum vitamin B 12, folate and Hcy concentrations, PCOS patients and healthy control women were divided into two groups based on Table 1. Clinical, biochemical and endocrinological parameters in polycystic ovary syndrome (PCOS) patients and controls. PCOS (n = 61) Control (n = 61) P-value Age (years) 27.2 ± ± 3.7 NS BMI (kg/m 2 ) 27.8 ± ± 1.6 NS FSH (IU/l) 4.7 ± ± 1.1 NS LH (IU/l) 7.4 ± ± 1.9 NS Total testosterone (ng/ml) 0.84 ± ± 0.22 <0.01 FAI (%) <0.01 Total-C (mg/dl) 187 ± ± 31 NS LDL-C (mg/dl) 102 ± ± 24 <0.05 HDL-C (mg/dl) 57.4 ± ± 17.9 NS Triglyceride (mg/dl) 91 ± ± 32 NS Fasting insulin (liu/ml) 17.8 ± ± 4.3 <0.05 Fasting glucose (mg/ml) 85.6 ± ± 7.4 NS HOMA > ± ± 1.1 <0.01 Homocysteine (lmol/l) 13.9 ± ± 2.1 <0.01 Vitamin B 12 (pg/ml) ± ± 68.4 NS Folate (ng/ml) 9.2 ± ± 1.3 NS BMI, body mass index; C, cholesterol; FAI, free androgen index; HDL, high density lipoprotein; HOMA, homeostasis model assessment; LDL, low-density lipoprotien; NS, not statistically significant. Statistical significance was defined as P < Data are shown as means ± SD. Groups were compared using Student s t or Mann Whitney U-test, as appropriate. 723

4 Table 2. Comparison of serum vitamin B 12, folate and homocysteine concentrations in polycystic ovary syndrome patients stratified by insulin resistance (IR). IR (n = 19) Non-IR (n = 42) P-value Vitamin B 12 (pg/ml) ± ± 66.4 <0.001 Folate (ng/ml) 9.5 ± ± 1.7 NS Homocysteine (lmol/l) 12.1 ± ± 3.6 <0.01 NS, not significant. Statistical significance was defined as P < Data are shown as means ± SD. Groups were compared using Student s t or Mann Whitney U-test, as appropriate. Table 3. Comparison of serum vitamin B 12, folate and homocysteine concentrations of women irrespective of whether or not they had polycystic ovary syndrome as stratified by insulin resistance (IR). IR (n = 29) Non-IR (n = 93) P-value Vitamin B 12 (pg/ml) ± ± 56.4 <0.01 Folate (ng/ml) 9.2 ± ± 1.3 NS Homocysteine (lmol/l) 11.7 ± ± 3.1 <0.01 NS, not statistically significant. Statistical significance was defined as P < Data are shown as means ± SD. Groups were compared using Student s t or Mann Whitney U-test, as appropriate. BMI. Obesity, defined by a BMI above 30 kg/m 2, was present in 24 PCOS patients and 20 healthy controls (39.3% and 32.7%, v 2 = 2.78). The two groups did not differ in terms of obesity. There were no statistically significant differences in terms of serum vitamin B 12, folate and Hcy concentrations between obese and non-obese subjects in both the PCOS and control subjects. When obese PCOS women were compared with obese control women, Hcy concentrations and HOMA index were found to be higher, whereas concentrations of vitamin B 12 were lower in obese PCOS patients. For the comparison of non-obese PCOS women versus non-obese control women, Hcy concentrations and HOMA index were also higher in PCOS women compared with controls, whereas no significant difference was found in vitamin B 12 concentrations (Table 4). In the PCOS group, Pearson correlation analysis showed that serum Hcy was positively correlated with fasting insulin (r = 0.34, P < 0.05) and HOMA index (r = 0.72, P < 0.01). As expected, there was a strong negative correlation between plasma Hcy concentrations and vitamin B 12 (r = 054, P < 0.01) and folate (r = 0.36, P < 0.05). In the Pearson correlation analysis, fasting insulin concentrations and HOMA index were negatively and significantly Table 4. Comparison of biochemical parameters in polycystic ovary syndrome (PCOS) patients and controls as stratified by body mass index (BMI). PCOS Obese (n = 24) Non-obese (n = 37) Control P-value a Obese (n = 20) Non-obese (n = 41) P-value b Vitamin B ± 99.2 c ± 43.8 NS ± ± 72.8 NS (pg/ml) Folate (ng/ml) 9.6 ± ± 1.9 NS 8.0 ± ± 1.6 NS Homocysteine 11.9 ± 3.2 c 10.9 ± 2.4 b NS 8.9 ± ± 1.6 NS (lmol/l) Insulin 17.6 ± 5.1 c 10.7 ± 3.6 d < ± ± 2.2 <0.05 (liu/ml) HOMA index 3.6 ± 1.3 c 2.1 ± 0.69 d < ± ± 0.4 <0.001 HOMA, homeostasis model assessment; NS, not statistically significant. Statistical significance was defined as P < Data are shown as means ± SD. Groups were compared using Student s t or Mann Whitney U-test, as appropriate. a P < 0.05 between obese and non-obese in the PCOS group. b P < 0.05 between obese and non-obese in the control group. c P < 0.05 between obese PCOS and obese control. 724 d P < 0.05 between non-obese PCOS and non-obese control.

5 correlated with serum vitamin B 12 concentrations (r = 0.29, P < 0.05, r = 0.48, P < 0.01, respectively). Multiple linear regression analysis showed that fasting insulin [(partial coefficient, b = 0.09, P < 0.001, 95% CI ( )], HOMA [(partial coefficient, b = 0.02, P < 0.001, 95% CI ( )], and Hcy [(partial coefficient, b = 0.02, P < 0.001, 95% CI ( )], affected serum vitamin B 12 concentrations in women with PCOS. This model explains 54.3% of variation of serum vitamin B 12 concentrations in PCOS patients. Discussion This study is the first report of the relationship between vitamin B 12 and insulin resistance and obesity in PCOS patients. The novel finding of the present study was that IR and obesity were associated with lower serum concentrations of vitamin B 12 in PCOS. Fasting insulin, insulin resistance and Hcy were independent determinants of serum vitamin B 12 levels in PCOS. In this study, PCOS women with IR as well as women with IR regardless of whether or not they had PCOS were compared with non-ir women. Interestingly, Hcy concentrations were higher whereas concentrations of vitamin B 12 were lower in women with IR. Decreased serum vitamin B 12 concentrations were associated with elevated Hcy concentrations in PCOS women with IR. Serum vitamin B 12 concentrations were significantly lower in PCOS patients with IR than in those with non-ir patients. This data shows that serum vitamin B 12 concentrations are negatively associated with PCOS women with IR. In the Pearson correlation analysis, serum vitamin B 12 concentrations were negatively and significantly correlated with fasting insulin and HOMA index. Multivariate analysis showed that the presence of IR (according to HOMA index) (b = 0.02, P < 0.001) and fasting insulin (b = 0.09, P < 0.001) are independent determinants of plasma vitamin B 12. This indicates that IR is one of the most important factors affecting serum concentrations of vitamin B 12 in women with PCOS, regardless of whether or not they are obese or non-obese. Plasma vitamin B 12 concentrations are also related to protein intake. Vegetarianism and low milk intakes contribute to low vitamin B 12 status, although adequate serum folate concentrations indicate adequate dietary intake. The dietary habits of the patients in IR and non-ir groups were not different. The results presented here indicate that low vitamin B 12 concentrations were independent of the energy intake in the PCOS series. So far, serum concentrations of vitamin B 12 and the associations with fasting insulin and IR have not been reported in PCOS patients. The low concentrations of vitamin B 12 found in PCOS women with IR suggest the involvement of vitamin B 12 in hyperinsulinaemia, insulin resistance and hyperhomocysteinaemia. The importance of vitamin B 12 in the remethylation of Hcy to methionine is well recognized, and hyperhomocysteinaemia is also a feature of vitamin B 12 deficiency (Selhub and Miller, 1992; Fonseca et al., 1999; McCarty, 2000). There is a strong negative correlation between plasma Hcy concentrations and vitamin B 12. The mechanism by which fasting insulin and IR reduces serum vitamin B 12 concentrations in PCOS women is beyond the scope of the present study and awaits future investigations. More recently, it was demonstrated that statin treatment caused vitamin B 12 to increase in PCOS women (Kaya et al., 2009). In that study, vitamin B 12 increases were related to decreases in IR by the effect of statins. In the present study, low serum vitamin B 12 concentrations were shown in PCOS patients with IR when compared with PCOS patients without IR. Fasting insulin concentrations and HOMA index were negatively and significantly correlated with serum vitamin B 12 concentrations. The results of this study support the relationship between vitamin B 12 and insulin resistance in PCOS. In the present study, serum vitamin B 12 concentrations were significantly lower in obese PCOS women in comparison with obese control women (see Table 4). Low vitamin B 12 concentrations were associated with a greater BMI in PCOS. Therefore, it was speculated that low vitamin B 12 will trap folate as 5-methyltetrahydrofolate, prevent the generation of methionine from homocysteine, and therefore reduce protein synthesis and lean tissue deposition in PCOS women. As a result, low vitamin B 12 concentrations may be affect adiposity in PCOS women. It was therefore postulated that low vitamin B 12 concentrations would predict greater adiposity and IR in PCOS women. Therefore, if these findings could be confirmed in prospective cohort studies, low serum vitamin B 12 may be a marker for possible prospective identification of young PCOS women prone to develop insulin resistance and obesity in the future. This study found that the concentrations of serum folate were not different between PCOS women with IR and PCOS women without IR. In addition, serum folate concentrations were not different in obese and non-obese PCOS women in comparison with obese and non-obese control women. Yet, changes in the levels of folate may not have been detected, probably on account of its shorter biological half-life (Adams, 1963). When PCOS obese women and non-obese PCOS women were compared, folate concentrations were not different between the groups. This study did not indicate an association between Hcy, folate and IR in PCOS. The composition of follicular fluid to some extent seems to reflect systemic vitamin B 12 metabolism, where high or low vitamin B 12 concentrations in the follicular fluid may occur (Steegers-Theunissen et al., 1993). More recently, it was demonstrated that preconception folic acid supplementation significantly alters folate and total Hcy concentrations in follicular fluid (Boxmeer et al., 2008). It is possible that IR and obesity may be associated with low follicular vitamin B 12 and high follicular Hcy concentrations in PCOS patients, although this is highly speculative. Ebisch et al. (2006) demonstrated an inverse correlation between the FF Hcy concentrations and embryo quality. Excessive body weight can make induction of ovulation or ovarian stimulation for assisted reproduction very difficult (Crosignani et al., 2002; Pasquali and Gambineri, 2004). Weight loss 725

6 reduces insulin concentrations, improves insulin sensitivity and restores normal menses cycles, ovulation and fertility in a large number of obese PCOS women (Pasquali and Gambineri, 2004). Therefore, low serum vitamin B 12 associated with IR and obesity may affect fertility in PCOS. In conclusion, the present results suggest that hyperinsulinaemia, IR and elevated Hcy are associated with low serum vitamin B 12 concentrations in PCOS patients. The data raise the important possibility that low serum vitamin B 12 may be a marker for possible adipocity, insulin resistance or hyperhomocystenaemia in young PCOS patients. Further studies will be necessary to confirm these results. References Adams JF 1963 Biological half-life of vitamin B 12 in plasma. Nature 198, 200. Ashwell M, Chinn S, Stalley S, Garrow JS 1982 Female fat distribution a simple classification based on two circumference measurements. 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