Article
Obesity and insulin resistance associated with
lower plasma vitamin B
12
in PCOS
Dr Cemil Kaya
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 2004. He is currently working in Ufuk University Faculty of Medicine. His special interests lie in the field of reproductive endocrinology, infertility and endoscopic surgery.
Cemil Kaya1,3, Sevim Dincßer Cengiz2, Hakan Satırog˘lu1
1Ufuk University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey; 2Ankara University, Faculty of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey 3
Correspondence: e-mail: kayacemil000@yahoo.com
Abstract
Polycystic ovary syndrome (PCOS) shares some or most components of metabolic cardiovascular syndrome, man-ifested by abdominal obesity, insulin resistance, dyslipidaemia and atherosclerosis. It has been previously demon-strated that folate and vitamin B12 treatment improved insulin resistance in patients with metabolic syndrome.
This study first investigated whether PCOS patients have lower or higher vitamin B12, folate and homocysteine
con-centrations when compared with healthy, age and body mass index matched controls, and, then examined associa-tions between vitamin B12, folate, homocysteine and insulin resistance and obesity in PCOS patients. Homocysteine
concentrations and homeostasis model assessment index were higher, whereas concentrations of vitamin B12were
lower in PCOS patients with insulin resistance compared with those without insulin resistance. Serum vitamin B12
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 B12
con-centrations in PCOS patients. Insulin resistance, obesity, and elevated homocysteine were associated with lower serum vitamin B12concentrations in PCOS patients.
Keywords: folate, homocysteine, insulin resistance, obesity, PCOS, vitamin B12
Introduction
Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women of reproductive age, is a multi-faceted 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) con-centrations 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 resis-tant than healthy women, even taking into account body
weight (Legro et al., 1999). PCOS shares some or most components of metabolic cardiovascular syndrome, mani-fested by abdominal obesity, insulin resistance, dyslipida-emia 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 ( Fons-eca et al., 1999). The importance of vitamin B12in the
reme-thylation of Hcy to methionine is well recognized, and hyperhomocysteinaemia is a feature of vitamin B12
defi-ciency (Selhub and Miller, 1992; McCarty, 2000). Further-more, 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 B12 treatment
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improved insulin resistance in patients with metabolic syn-drome (Setola et al., 2004). Many of the anthropometric and metabolic abnormalities of PCOS overlap with compo-nents of metabolic syndrome (Ehrman et al., 2006). Thus, in addition to total Hcy, plasma vitamin B12 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 B12, folate and IR and obesity
in PCOS patients are not well characterized.
This study first investigated whether PCOS patients have lower or higher vitamin B12, folate and Hcy concentrations
when compared with healthy, age- and body mass index (BMI)-matched controls, and then examined associations between vitamin B12, 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 bio-chemical 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/m2for age and BMI, respectively. The healthy state of the controls was determined by medical history, physical and pelvic examination and complete blood chem-istry. They were recruited from hospital staff and students. All PCOS subjects had irregular menses, and 61% of partic-ipants had eight or fewer spontaneous cycles per year. The diagnosis of PCOS was made according to the Rotterdam European Society for Human Reproduction and Embryol-ogy (ESHRE)/American Society for Reproductive Medi-cine (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 (hir-sutism, acne) and/or hyperandrogenaemia and (iii) polycys-tic ovaries. The presence of polycyspolycys-tic 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 stimulat-ing 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, thy-roid disorders, Cushing’s syndrome (1 mg dexamethasone suppression test), hypertension, vitamin B12and folate
defi-ciency, hepatic or renal dysfunction or were smokers were excluded from the study. Subjects treated with any hor-monal medications, vitamins or drugs that increase Hcy lev-els within the previous 3 months and those with folate,
vitamin B12or vitamin B6deficiencies 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 modi-fied 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/m2were considered as overweight. BMI values of
30 kg/m2
were considered as obese. Waist circumference was measured at the narrowest level between the costal margin and iliac crest, and the hip circumference was mea-sured 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 measure-ments (serum progesterone measuremeasure-ments <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 B12, folate and glucose
dur-ing 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 electrochemilumines-cence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics, Mannheim, Germany). Impaired glu-cose tolerance (IGT) was defined as a 2-h post-load gluglu-cose 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 varia-tion were <2%.
The presence of IR was investigated using basal insulin con-centrations, fasting glucose concentrations and homeostasis model assessment (HOMA-IR > 2.1). HOMA-IR was calcu-lated 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 con-centrations were measured using an electrochemilumines-cence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics) with specific chemiluminescence assays
(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 B12 and folate concentrations were
mea-sured using an electrochemiluminescence immunoassay (ECLIA) (ELECSYS 2010 HITACHI; Roche Diagnostics) with specific chemiluminescence assays (ELECSYS 2010 HITACHI; Roche Diagnostics). Mean and intra- and inter-assay coefficients of variation were 5.2% and 3.4%, respec-tively, for vitamin B12and 6.8% and 7.9%, respectively, for
folate. The free androgen index (FAI) was calculated according to the equation FAI (%) = testosterone (ng/ ml) 3.47 100/sex hormone binding globulin (nmol/l).
Statistical analysis
Data are shown as means ± SD, or raw numbers and per-centages. Data analysis was performed using the Statistical Package for Social Sciences (SPSS) for Windows, version 11.5 (SPSS Inc., USA). Groups were compared using Stu-dent’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 para-metric variables and nominal parapara-metric data were assessed
by Pearson correlation coefficients. Multiple linear regres-sion stepwise method was used to determine the indepen-dent predictors that most commonly affected plasma vitamin B12 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 summa-rized inTable 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 B12, 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 B12were lower in PCOS women
with IR compared with in PCOS women without IR (Table 2). To determine the effects of IR on serum vitamin B12,
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 B12were lower in women with IR compared with
those with non-IR (Table 3).
To examine the effects of obesity on serum vitamin B12,
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.
Parameter PCOS (n = 61) Control (n = 61) P-value
Age (years) 27.2 ± 4.1 26.7 ± 3.7 NS BMI (kg/m2) 27.8 ± 1.7 26.6 ± 1.6 NS FSH (IU/l) 4.7 ± 1.5 4.2 ± 1.1 NS LH (IU/l) 7.4 ± 2.1 6.1 ± 1.9 NS Total testosterone (ng/ml) 0.84 ± 0.32 0.48 ± 0.22 <0.01 FAI (%) 12.4 5.2 <0.01 Total-C (mg/dl) 187 ± 39 171 ± 31 NS LDL-C (mg/dl) 102 ± 29 86 ± 24 <0.05 HDL-C (mg/dl) 57.4 ± 19.1 58.6 ± 17.9 NS Triglyceride (mg/dl) 91 ± 29 92 ± 32 NS
Fasting insulin (lIU/ml) 17.8 ± 7.4 10.2 ± 4.3 <0.05
Fasting glucose (mg/ml) 85.6 ± 8.8 81.6 ± 7.4 NS
HOMA >2.1 3.7 ± 1.7 1.8 ± 1.1 <0.01
Homocysteine (lmol/l) 13.9 ± 7.3 9.4 ± 2.1 <0.01
Vitamin B12(pg/ml) 268.2 ± 46.0 308.9 ± 68.4 NS
Folate (ng/ml) 9.2 ± 2.1 9.7 ± 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 < 0.05. Data
BMI. Obesity, defined by a BMI above 30 kg/m2, was pres-ent in 24 PCOS patipres-ents and 20 healthy controls (39.3% and 32.7%, v2= 2.78). The two groups did not differ in terms of obesity. There were no statistically significant differences in terms of serum vitamin B12, folate and Hcy concentrations
between obese and non-obese subjects in both the PCOS and control subjects. When obese PCOS women were com-pared with obese control women, Hcy concentrations and HOMA index were found to be higher, whereas concentra-tions of vitamin B12 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 B12concentrations (Table 4).
In the PCOS group, Pearson correlation analysis showed that serum Hcy was positively correlated with fasting insu-lin (r = 0.34, P < 0.05) and HOMA index (r = 0.72, P < 0.01). As expected, there was a strong negative correla-tion between plasma Hcy concentracorrela-tions and vitamin B12
(r = 054, P < 0.01) and folate (r = 0.36, P < 0.05). In the Pearson correlation analysis, fasting insulin concentra-tions 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).
Parameter PCOS Control
Obese (n = 24) Non-obese (n = 37) P-valuea Obese (n = 20) Non-obese (n = 41) P-valueb Vitamin B12 (pg/ml) 227.8 ± 99.2c 234.3 ± 43.8 NS 317.6 ± 69.2 274.2 ± 72.8 NS Folate (ng/ml) 9.6 ± 1.6 8.6 ± 1.9 NS 8.0 ± 1.4 8.7 ± 1.6 NS Homocysteine (lmol/l) 11.9 ± 3.2c 10.9 ± 2.4b NS 8.9 ± 3.3 7.8 ± 1.6 NS Insulin (lIU/ml) 17.6 ± 5.1c 10.7 ± 3.6d <0.05 12.7 ± 3.1 7.5 ± 2.2 <0.05 HOMA index 3.6 ± 1.3c 2.1 ± 0.69d <0.001 2.5 ± 0.6 1.4 ± 0.4 <0.001
HOMA, homeostasis model assessment; NS, not statistically significant. Statistical significance was defined as P < 0.05. 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.
d
P < 0.05 between non-obese PCOS and non-obese control.
Table 3. Comparison of serum vitamin B12, folate and homocysteine concentrations of women
irrespective of whether or not they had polycystic ovary syndrome as stratified by insulin resistance (IR).
Parameter IR (n = 29) Non-IR (n = 93) P-value
Vitamin B12(pg/ml) 268.2 ± 46.0 371.9 ± 56.4 <0.01
Folate (ng/ml) 9.2 ± 2.1 9.7 ± 1.3 NS
Homocysteine (lmol/l) 11.7 ± 5.3 7.9 ± 3.1 <0.01
NS, not statistically significant. Statistical significance was defined as P < 0.05. Data are shown as means ± SD. Groups were compared using Student’s t or Mann–Whitney U-test, as appropriate.
Table 2. Comparison of serum vitamin B12, folate and homocysteine concentrations in polycystic
ovary syndrome patients stratified by insulin resistance (IR).
Parameter IR (n = 19) Non-IR (n = 42) P-value
Vitamin B12(pg/ml) 178.4 ± 96.0 324.4 ± 66.4 <0.001
Folate (ng/ml) 9.5 ± 2.1 9.9 ± 1.7 NS
Homocysteine (lmol/l) 12.1 ± 4.8 9.7 ± 3.6 <0.01
NS, not significant. Statistical significance was defined as P < 0.05. Data are shown as means ± SD. Groups were compared using Student’s t or Mann–Whitney U-test, as appropriate.
correlated with serum vitamin B12concentrations (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 (0.02–0.15)], HOMA [(partial coefficient, b = 0.02, P < 0.001, 95% CI (0.04–0.001)], and Hcy [(partial coefficient, b = 0.02, P < 0.001, 95% CI (0.000–0.002)], affected serum vitamin B12 concentrations in women with PCOS. This model
explains 54.3% of variation of serum vitamin B12
concentra-tions in PCOS patients.
Discussion
This study is the first report of the relationship between vitamin B12 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 concentra-tions of vitamin B12in PCOS. Fasting insulin, insulin
resis-tance and Hcy were independent determinants of serum vitamin B12levels in PCOS.
In this study, PCOS women with IR as well as women with IR regardless of whether or not they had PCOS were com-pared with non-IR women. Interestingly, Hcy concentra-tions were higher whereas concentraconcentra-tions of vitamin B12
were lower in women with IR. Decreased serum vitamin B12concentrations were associated with elevated Hcy
con-centrations in PCOS women with IR. Serum vitamin B12
concentrations were significantly lower in PCOS patients with IR than in those with non-IR patients. This data shows that serum vitamin B12concentrations are negatively
associated with PCOS women with IR. In the Pearson cor-relation analysis, serum vitamin B12 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 B12. This
indi-cates that IR is one of the most important factors affecting serum concentrations of vitamin B12in women with PCOS,
regardless of whether or not they are obese or non-obese. Plasma vitamin B12concentrations are also related to
pro-tein intake. Vegetarianism and low milk intakes contribute to low vitamin B12status, although adequate serum folate
concentrations indicate adequate dietary intake. The die-tary habits of the patients in IR and non-IR groups were not different. The results presented here indicate that low vitamin B12concentrations were independent of the energy
intake in the PCOS series.
So far, serum concentrations of vitamin B12and the
asso-ciations with fasting insulin and IR have not been reported in PCOS patients. The low concentrations of vitamin B12 found in PCOS women with IR suggest the
involvement of vitamin B12 in hyperinsulinaemia, insulin
resistance and hyperhomocysteinaemia. The importance of vitamin B12 in the remethylation of Hcy to methionine
is well recognized, and hyperhomocysteinaemia is also a feature of vitamin B12 deficiency (Selhub and Miller,
1992; Fonseca et al., 1999; McCarty, 2000). There is a strong negative correlation between plasma Hcy
concen-trations and vitamin B12. The mechanism by which fasting
insulin and IR reduces serum vitamin B12 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 B12to
increase in PCOS women (Kaya et al., 2009). In that study, vitamin B12 increases were related to decreases in
IR by the effect of statins. In the present study, low serum vitamin B12 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 B12 concentrations. The results of this study support the
relationship between vitamin B12 and insulin resistance
in PCOS.
In the present study, serum vitamin B12 concentrations
were significantly lower in obese PCOS women in compar-ison with obese control women (seeTable 4). Low vitamin B12 concentrations were associated with a greater BMI in
PCOS. Therefore, it was speculated that low vitamin B12
will trap folate as 5-methyltetrahydrofolate, prevent the generation of methionine from homocysteine, and there-fore reduce protein synthesis and lean tissue deposition in PCOS women. As a result, low vitamin B12
concentra-tions may be affect adiposity in PCOS women. It was therefore postulated that low vitamin B12 concentrations
would predict greater adiposity and IR in PCOS women. Therefore, if these findings could be confirmed in prospec-tive cohort studies, low serum vitamin B12may be a
mar-ker 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 con-centrations 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 concentra-tions 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 B12metabolism, where high or low
vitamin B12concentrations in the follicular fluid may occur
(Steegers-Theunissen et al., 1993). More recently, it was demonstrated that preconception folic acid supplementa-tion significantly alters folate and total Hcy concentrasupplementa-tions in follicular fluid (Boxmeer et al., 2008). It is possible that IR and obesity may be associated with low follicular vita-min B12 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 stimula-tion for assisted reproducstimula-tion very difficult (Crosignani et al., 2002; Pasquali and Gambineri, 2004). Weight loss
725
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 B12
associ-ated with IR and obesity may affect fertility in PCOS. In conclusion, the present results suggest that hyperinsuli-naemia, IR and elevated Hcy are associated with low serum vitamin B12 concentrations in PCOS patients. The data
raise the important possibility that low serum vitamin B12
may be a marker for possible adipocity, insulin resistance or hyperhomocystenaemia in young PCOS patients. Fur-ther studies will be necessary to confirm these results.
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Declaration: The authors report no financial or commercial conflicts of interest.
Received 30 November 2008; refereed 6 January 2009; accepted 8 June 2009.
