Department of Obstetrics and Gynecology, Baskent University Faculty of Medicine, Turkey
1 To whom correspondence should be addressed at: Obstetrics and Gynecology Department, Yurt Mahalle, 229 Sok, Hakan Burak Apt Kat: 2 Daire: 4, Seyhan Adana, Turkey. Email: esrabulgan1972{at}mynet.com
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Abstract |
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Key words: B-group vitamin/folic acid/hyperhomocysteinaemia/metformin/PCOS
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Introduction |
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Polycystic ovarian syndrome (PCOS) is one of the most common endocrinopathies, affecting 37% of women of reproductive age (Knochenhauer et al., 1998; Asuncion et al., 2000
). It is not known whether women with PCOS have a greater risk of cardiovascular disease compared with appropriately aged controls (Legro et al., 2001
). Although epidemiological studies have not shown an increased incidence of death from cardiovascular events in women with PCOS (Pierpoint et al., 1998
), all current data suggest that PCOS possesses the intrinsic conditions that lead to an increased incidence of factors predisposing to cardiovascular diseases (Loverro, 2004
). More than 30% of lean and 75% of obese women with PCOS have hyperinsulinaemia (Conway et al., 1992
). Insulin resistance, dyslipidaemia, impaired glucose tolerance, type 2 diabetes mellitus, and elevated systolic blood pressures are more prevalent in obese young women with PCOS, which suggests that women with PCOS are at an increased risk of CVD (Wild et al., 1985
; Wild, 2002a
; Conway et al., 1992
; Rajkhowa et al., 1994
; Guzick, 1996
; Dunaif, 1997
). Cross-sectional studies assessing prevalence suggest that women with PCOS are at greater risk for premature development of diabetes mellitus (Wild, 2002b
). Studies on high blood pressure and dyslipidaemia in women with PCOS patients to date offer conflicting results. Sampson et al., (1996)
found no differences in 24 h, daytime and night-time ambulatory blood pressure measurements. However, Holte et al., (1996)
have reported higher daytime systolic and mean ambulatory arterial blood pressures. A significant increase in left ventricular mass index and a diastolic filling have been described in women of normal weight with PCOS by Orio et al., (2004)
. On the other hand, Zimmermann et al., (1992)
suggest that despite profound insulin resistance and hyperinsulinaemia, women with PCOS do not have increased arterial pressure or left ventricular mass. Women with PCOS appear to have lower high-density lipoprotein (HDL) cholesterol and higher triglyceride (TG) levels independent of body weight (Wild, 2002a
). Most, but not all, studies report this profile. Legro et al., (2001)
have reported that elevations in low-density lipoprotein cholesterol (LDL-C) were the predominant lipid abnormality when obese women with PCOS had relatively elevated HDL-C levels. Talbott et al., (1998)
suggest that LDL-C is substantially higher in young women with PCOS than in matched controls, while pre- to peri-menopausal women (aged
40 years) with PCOS have LDL-C and total cholesterol levels similar to those of age-matched controls. Additionally, significantly higher serum Hcy concentrations have been found in women with PCOS compared with controls by several authors (Yarali et al., 2001
; Loverro et al., 2002
; Schachter et al., 2003
; Wijeyaratne et al., 2004
), while others have found no association between PCOS and Hcy (Sills et al., 2001
; Orio et al., 2003
). Women with PCOS appear to be at increased risk of CVD when markers for atherosclerosis, such as intima-media thickness of femoral and carotid arteries (Talbott et al., 2000
; Lakhani et al., 2004
), are examined. Moreover, an increased prevalence of coronary artery (Christian et al., 2003
; Talbott et al., 2004
) and aortic calcification recently has been demonstrated among middle-aged women with PCOS (Talbott et al., 2004) when compared with controls. Whether PCOS is an independent risk for circulatory death remains unclear, but current literature indicates that women with PCOS cluster risk factors for premature morbidity and mortality (Wild, 2002b
).
Recognition of insulin resistance as a principal factor in the pathogenesis of PCOS has led to the use of insulin sensitizers for its treatment (Seli and Duleba, 2002). In the treatment of PCOS, the most extensively studied insulin sensitizer is metforminan oral antihyperglycaemic agent used initially in the treatment of type 2 diabetes mellitus. It has been shown that metformin increases total serum Hcy levels in non-diabetic male patients with coronary heart disease (Carlsen et al., 1997
), and adding metformin to insulin therapy in type 2 diabetes mellitus reduces levels of folic acid and vitamin B12, which results in a modest increase in Hcy levels within 12 weeks (Wulffele et al., 2003
). The Hcy-increasing effect of metformin in patients with PCOS has been demonstrated by Vrbikova et al. (2002)
. Recently, we demonstrated that 3 months of metformin and rosiglitazone therapy resulted in a significant increase in plasma Hcy concentrations in patients with PCOS (Kilicdag et al., 2005
).
Previously, folic acid administration has been shown to reduce Hcy levels in healthy subjects (Brattström et al., 1988; Ubbink et al., 1993
), patients with kidney disease (Arnadottir et al., 1993
), and in patients with vascular disease (Brattström et al., 1990
; Landgren et al., 1995
). Furthermore, in patients with non-insulin-dependent diabetes mellitus on long-term metformin treatment, the Hcy-increasing effect of metformin has been shown to be counteracted by folate administration (Aarsand and Carlsen, 1998
).
Folic acid, vitamin B6 and vitamin B12 are all co-factors in Hcy metabolism. Serum vitamin B12 and folic acid levels are known to decrease during metformin therapy. Hence, during metformin therapy, Hcy levels might increase. In view of these considerations, we studied the effects of B-group vitamins and folic acid administration on serum levels of Hcy in patients with PCOS on short-term metformin treatment.
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Materials and methods |
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Patients with oligomenorrhoea (cycle intervals >35 days), amenorrhoea, anovulatory infertility, or a hirsutism score of >7 according to Ferriman and Gallway (1961), who also had at least one of the following criteriaan elevated serum testosterone level (>0.82 ng/ml) or appearance of PCOwere diagnosed as having PCOS, after all other causes of hyperandrogenism had been excluded (Homburg, 2002). Sonographic diagnosis of PCO was confirmed if
10 subcapsular follicular cysts, 28 mm in diameter, arranged around a thickened ovarian stroma were apparent (Adams et al., 1986
). All patients with elevated 17OH-progesterone levels were evaluated for 21-hydroxylase deficiency; none of the patients had 21-hydroxylase deficiency. Subjects treated with hormonal medications within the prior 3 months also were excluded from the study. When evaluating inclusion criteria according to Rotterdam 2003 criteria for PCOS (not published at the start of the study: Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group, 2004
), we found that all patients fulfilled them, that is, at least two of the three criteria: polycystic ovaries, clinical and/or biochemical signs of hyperandrogenism, and oligo- and/or anovulation.
Patients were randomly assigned to one of three treatment groups. The patients were randomized into three groups by an allocation sequence generated from a random number table and assigned through consecutively numbered, opaque, sealed envelopes. Patients in group 1 (n=20) received metformin (850 mg twice daily) (Glucophage®; Merck, Turkey); patients in group 2 (n=20) received metformin (850 mg twice daily) and B-group vitamins [vitamin B1 (250 mg); vitamin B6 (250 mg); vitamin B12 (1000 µg) twice daily (Apikobal®; Santa Farma, Turkey)]; and patients in group 3 (n=20) received metformin (850 mg twice daily) and folic acid [folic acid (174 µg), vitamin D (1200 µg) and calcium (666.670 mg) twice daily (Folic Plus®; Assos, UK)] for 3 months. Sixty patients were included, and 49 patients completed the study. Three patients became pregnant (two in the metformin group and one in the metformin plus folic acid group). Three patients withdrew because of gastrointestinal adverse effects of metformin (two in the metformin group and one in the metformin plus folic acid group). Three patients withdrew from the study owing to lack of motivation, and another two patients left the study without giving any reason. Fourteen patients in the metformin therapy group, 17 patients in the metformin plus folic acid therapy group, and 18 patients in the metformin plus B-group vitamins therapy group completed the study (Figure 1).
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Samples were collected at 08:00 (12 h fasting state) and at 120 min after glucose ingestion. Plasma glucose levels were measured using the glucose oxidase method; plasma insulin concentrations were measured by the microparticle enzyme immunoassay method (AxSYM insulin assay; Abbott, Japan).
Hcy levels are influenced by several variables, including smoking, renal function, vitamin B status, and enzyme dysfunction states. Renal status was examined before the women entered the study. All patients had normal creatinine levels. Vitamin B12 and folate levels were examined before and after treatment. All women were non-smokers. Patients with folic acid and vitamin B12 deficiencies were excluded from the study. None of the patients had used metformin or any type of vitamin before initiation of the trial.
Levels of plasma fasting glucose, total-C, HDL-C and TG were determined by the colorimetric method using a Cobas Mira Plus autoanalyser (Roche Diagnostics, Germany). LDL-C and VLDL-C levels were calculated by the formula of Friedwald (1972). Apo A1, Apo B, and Lp (a) were quantified by the immunoturbidimetric method in a Roche/Hitachi 912 autoanalyser (DP, modular system, Japan). Insulin, LH, FSH, E2, PRL and Hcy concentrations were measured using an AxSYM hormone autoanalyser (Abbott Laboratories, USA) using the microparticle enzyme immunoassay method. Total testosterone and DHEAS were measured in an Immulite One autoanalyser (Bio Diagnostic Products Corp., USA) using the chemiluminescent method. Insulin sensitivity was calculated using the homeostasis model assessment (HOMA) [(formula: fasting glucose (mmol/l) x fasting insulin (µIU/ml)/22.5)] (Matthews et al., 1985). Intra- and inter-assay CV for glucose were 0.4 and 1.2%, 0.6 and 1.3%, and 0.7 and 1.1%, at 82.2, 81.4 and 80.5 mg/dl respectively. Intra- and inter-assay CV of the insulin were 2.6 and 1.8%, 4.1 and 2.5%, and 2.9 and 2%, at 8.7, 42.2 and 126.2 mIU/ml respectively.
Hcy, mixed disulphide, and protein-bound forms of Hcy in the samples were reduced to form free Hcy using dithiothreitol. Free Hcy was converted to S-adenosyl-L-Hcy (SAH) using SAH hydrolase and excess adenosine. SAH and labelled fluorescein tracer compete for sites on the monoclonal antibody molecule. The intensity of polarized fluorescent light was measured using a fluorescence-polarization immunoassay (FPIA) technique optical unit. The coefficient of variation of FPIA was 4.6% at 7.99 µmol/l, 3.1% at 13.71 µmol/l, and 2.8% at 26.67 µmol/l.
Plasma folic acid and vitamin B12 concentrations were measured using the chemiluminescent method with an E170 immunoassay analyser (Roche Diagnostics Corp., USA). The electrochemiluminescence immunoassay (ECLIA) was used on a Roche Modular Analytics E170 immunoassay analyser (Roche Diagnostics).
The primary outcomes were changes in Hcy, folic acid and vitamin B12 levels. Secondary outcomes included changes in body mass index (BMI), lipid profile, HOMA levels and menstrual cycle pattern with metformin, metformin plus B-group vitamins, and metformin plus folate therapy.
Statistical analyses
Sample size calculations, assuming 80% power to detect 2 µmol/l changes in Hcy levels between groups, indicated the need for 15 patients in each group.
Baseline characteristics of the three groups were compared using a one-way analysis of variance (ANOVA). Repeated measures ANOVA were used to analyse changes in variables before and after treatment in all groups. Repeated measures ANOVA was also used to assess significance within and between groups. The Bonferroni post hoc test was applied. Data are expressed as mean±SEM; P<0.05 was considered statistically significant. All statistical procedures were performed using SPSS software (Statistical Package for the Social Sciences, version 10.0; SSPS Inc., USA).
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Results |
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Adverse effects
Seven of the 60 patients on metformin had problems with nausea and vomiting; three patients discontinued therapy owing to nausea and vomiting. At the end of therapy, four of 49 patients complained of gastrointestinal adverse effects, two patients complained of headache, and two patients complained of fatigue.
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Discussion |
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Hcy is an essential amino acid required for the growth of cells and tissues in the human body. The only source of Hcy in the human organism comes from the methionine in dietary proteins, which are mainly of animal origin. Hcy, in turn, uses two pathways for biotransformation: trans-sulphuration and remethylation. Vitamin B6 takes part in the process of trans-sulphuration, whereas folic acid, together with vitamin B12, participates in the most important and more often used pathway for Hcy metabolism: remethylation. A reduction in dietary intake of B-group vitamins leads to an increase in levels of Hcy in plasma. Any drug, such as methotrexate, nitrous oxide or azaribine, that reacts with folic acid, vitamin B12 or B6 respectively can cause hyperhomocysteinemia (de la Calle et al., 2003). It has been previously demonstrated that metformin treatment decreases vitamin B12 levels (Wulffele et al., 2003; Pongchaidecha et al., 2004
), probably due to malabsorption (Tomkin et al., 1971
; Caspary et al., 1977
). Hence, during metformin treatment, Hcy levels may increase (Carlsen et al., 1997
; Hoogeveen et al., 1997
; Aarsand and Carlsen, 1998
). Therefore, it appears that both vitamin B12 and B6 have important roles in Hcy regulation, and supplements with these vitamins may lower Hcy levels.
Abnormal fasting Hcy levels tend to reflect an impaired remethylation pathway, whereas an abnormal response to a methionine load points to a disturbed trans-sulphuration pathway. In our study, fasting Hcy levels increased significantly with metformin treatment. Although not statistically significant, we observed a 19.78% decrease in vitamin B12 levels in patients treated with metformin, while decreases in folic acid levels were minimal (0.6%). Therefore, it seems likely that a rise in Hcy levels may be related to a decrease in vitamin B12 levels. On the other hand, in the current study, Hcy levels decreased significantly with both metformin plus B-group vitamins and with metformin plus folic acid. Although there was no significant difference between the two groups with respect to the amount of Hcy reduction observed, decreases in Hcy levels were greater in patients treated with metformin plus B-group vitamins than they were in patients treated with metformin plus folic acid (2.77 ± 0.69 versus 1.26 ± 0.52 µmol/l). One explanation for this is that vitamin B6 and vitamin B12 are two factors that act separately in Hcy metabolism and probably potentiate each other. We used a combination of vitamins B1, B6 and B12 because in Turkey there is no oral preparation that includes only vitamin B12.
The strongest evidence to date that an elevated plasma Hcy concentration may be a risk factor for vascular disease comes from the study of individuals with homocystinuria. If left untreated, 25% of these individuals suffer a vascular event by the age of 16 years and 50% by the age of 29 years (Mudd et al., 1985). Treatment of homocystinuria is directed at lowering Hcy concentrations by administering vitamin B6, folate, betaine (a methyl donor), and vitamin B12. This treatment has been shown to have a marked effect on preventing the occurrence of vascular events. Evidence from long-term follow-up of 158 patients with homocystinuria, with 2822 patient-years of treatment, has demonstrated that Hcy lowering, even to concentrations several times higher than the normal reference range, significantly reduces the incidence of vascular events (Yap et al., 2001
). Lowering Hcy levels in PCOS is important when one considers the elevated levels of Hcy and possible increasing effects of metformin on Hcy levels in patients with PCOS. Treatment of PCOS is symptomatic, but lifestyle measures, such as diet and exercise, could play an important role (Powell et al., 1987
). Although the exact mechanism is not known, regular exercise significantly lowers plasma Hcy in young, overweight or obese women with PCOS (Randeva et al., 2002
). Observational epidemiological studies, both case-controlled and cohort, demonstrate that patients with elevated Hcy levels have low to moderately increased risks of CVD. Folic acid (Jacobsen, 1998
) and B-group vitamins (Woodside et al., 1998
) effectively lower Hcy concentration in plasma. However, at present, because of the lack of data from large-scale randomized trials, it remains unclear whether lowering Hcy levels will reduce the risk of CVD (Splaver et al., 2004
).
Because there is strong evidence for the use of metformin in the regulation of cycle and ovulation induction among patients with PCOS who desire fertility (Stadtmauer et al., 2002), it should be remembered that this medication may interfere with vitamin B12 and folic acid pathways and lead to an increase in plasma Hcy levels (Desouza et al., 2002
). The association between increased levels of Hcy and neural tube defects and other congenital defects, spontaneous miscarriages, intrauterine growth retardation, pre-eclampsia, and intrauterine fetal death have been previously described (de la Calle et al., 2003
). Many authors suggest not only administration of folic acid but also vitamin B12 administration for prevention of neural tube defects (Czeizel and Dudas, 1992
; Mills et al., 1995
; Nelen et al., 1997
; Brouwer, 2000
; Nelen et al., 2000
), because vitamin B12 fosters cell intake of folic acid, so that even though concentrations of folic acid are normal, a deficiency of vitamin B12 also could increase the risk of neural tube defects (Smithells et al., 1983
; Czeizel and Dudal, 1992
; Eskes, 1998
; Leeda et al., 1998
; Brouwer, 2000
). In our study, both B-group vitamins and folic acid decreased Hcy levels. B-Group vitamins are likely more effective than folic acid for lowering Hcy levels. Both vitamin B12 and folic acid should be offered to patients, especially those using metformin, who desire to become pregnant.
Although the sample size in the current study was small, these findings suggest that daily administration of folic acid or B-group vitamins may be effective in reducing elevated Hcy levels in patients with PCOS undergoing short-term metformin therapy.
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Submitted on December 8, 2004; resubmitted on January 27, 2005; accepted on February 4, 2005.