Effect of folic acid in women with and without insulin resistance who have hyperhomocysteinemic polycystic ovary syndrome

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CLINICAL ARTICLE

Effect of folic acid in women with and without insulin resistance who have hyperhomocysteinemic polycystic ovary syndrome

Talieh Kazerooni ⁎ , Nasrin Asadi, Sedigheh Dehbashi, Jaleh Zolghadri

Department of Obstetrics and Gynecology, Shiraz University of Medical Sciences, Shiraz, Iran Received 8 August 2007; received in revised form 19 September 2007; accepted 11 October 2007

Abstract

Objective: To study the effect of folic acid on homocysteine (Hcy) levels in women with insulin resistance and polycystic ovary syndrome (PCOS) in a prospective clinical trial. Method: Of 210 women with PCOS, 70 were hyperhomocysteinemic; and of these, 32 were insulin resistant and 38 were not. The 70 women were treated with folic acid for 3 months. Baseline and serum levels of Hcy and insulin were measured in both groups. Results: In both groups Hcy concentrations were significantly decreased following folic acid supplementation. The mean ± SD levels before and after treatment were 14.03 ± 1.5 µmol/L and 12.53 ± 1.72 µmol/L in group 1 (Pb0.001), and they were 12.07 ± 0.87 µmol/L and 8.83 ± 0.78 µmol/L in group 2 (Pb0.001). Conclusion: The Hcy levels of hyperhomocysteinemic women with PCOS were reduced after 3 months of folic acid supplementation, and the rate of reduction was higher among women without insulin resistance.

No change was found in fasting insulin levels.

KEYWORDS Folic acid;

Insulin resistance;

Hyperhomocysteinemia;

Polycystic ovary syndrome

1. Introduction

Homocysteine (Hcy) is an essential amino acid required for the growth of cells and tissues. The only source of Hcy for humans is methionine, which is present in dietary proteins, mainly of animal origin. Folate and cobalamin (vitamin B12) are involved

in Hcy remethylation, and pyridoxal 5′-phosphate (the active form of vitamin B6) is involved in Hcy transsulfuration[1].

Levels of Hcy are increased in postmenopausal women[1,2]

and in many clinical conditions, among them non-insulin- dependent diabetes mellitus, endometrial dysplasia and carcinoma, hypertension with insulin resistance (IR), polycystic ovary syndrome (PCOS)[3], obesity with IR[4], pre-eclampsia [1,5], placental abruption, recurrent early pregnancy loss, neural tube defects, intrauterine growth restriction, congeni- tal orofacial malformations, and intrauterine fetal death.

Nitrous oxide and drugs such as methotrexate or zaribine can also increase Hcy levels[1]. Hyperhomocysteinemia appears to

⁎ Corresponding author. # 204, No. 18 Ave. Motahari Blv. Shiraz, Fars Iran, 71867-86968. Tel.: +98 711 6262680; fax: +98 711 6288427.

E-mail address:kazeront@yahoo.com(T. Kazerooni).

doi:10.1016/j.ijgo.2007.10.024

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

w w w . e l s e v i e r. c o m / l o c a t e / i j g o

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adversely affect the vasculature by inducing endothelial cytotoxicity and lipid peroxidation, increasing platelet adhe- siveness, enhancing the activation of the coagulation system, and stimulating vascular smooth muscle cell proliferation[6].

Insulin resistance is a fundamental abnormality in the pathogenesis of type 2 diabetes. It may also play a role in the development of atherosclerotic cardiovascular disease (CVD) [7]; be an expression of diffuse arterial endothelial dysfunction contributing to atherosclerosis, and thus directly lead to arterial damage through the toxic effect of hyperinsulinemia [8]; or act indirectly through the atherogenic effect of the constellation of risk factors associated with IR syndrome[3].

Plasma levels of insulin seem to influence Hcy metabolism, possibly by affecting glomerular filtration or influencing the activity of 5, 10-methylenetetrahydrofolate reductase or cystathioneβ-synthase, the key enzymes in Hcy metabolism [9]. Recently, IR and hyperhomocysteinemia have been in- tensively investigated for their possible roles in the pathogenesis of CVD[10].

Polycystic ovary syndrome is one of the most common endocrine diseases in women, affecting up to 10% of women of reproductive age[11], and IR may be the major risk factor for the occurrence of CVD in women with PCOS[12]. As elevated levels of Hcy are considered a risk factor for CVD, they may account for the increased risk of CVD found in insulin-resistant women with PCOS[13]. The aim of this study was to investigate the effect of folic acid on hyperhomocysteinemic women with IR and estimate the incidence of hyperhomocysteinemia and IR in women with PCOS.

2. Patients and methods

A total of 210 women with PCOS were enrolled in this study at endocrine clinics of Shiraz University of Medical Sciences, Shiraz, Iran, between October 2005 and November 2006. The study was approved by the institutional review board. The participants were assigned the diagnosis PCOS if they met the consensus definition reached at the 2003 Rotterdam conference[14]. Oligo-ovulation was defined as oligomenorrhea (cycle intervalsN35 days) and amenorrhea. All participants had at least 2 of the 3 following criteria: polycystic ovaries, clinical and/or biochemical signs of hyperandrogenism, and oligo-ovulation and/or anovulation. None of the participants had taken any hormonal, steroid, or psycho- tropic medications in the 90 days before testing, as such medications can affect Hcy levels. None had thyroid abnormalities, hyperprolactinemia, or diabetes. Any history of liver or kidney disease, breast cancer, thromboembolism, or smoking was considered an exclusion criterion.

Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), prolactin, total testosterone, dehydroepiandrosterone sulfate (DHEAS), fasting insulin, and homocysteine were measured by assay in the 210 participants on the third day of the menstrual cycle (whether the menstruation was progesterone induced or occurred sponta- neously), and glucose-insulin ratio. A 75 g oral glucose tolerance test (GTT) was performed at baseline. Body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) was measured at the initial visit and upon completion of the study.

The total fasting Hcy plasma concentration was determined using an enzyme conversion immunoassay kit (Axis-Shield, Dundee, UK). The participants were considered hyperhomocysteinemic if the fasting Hcy concentration exceeded 11 µmol/L[5].

Blood samples were obtained at baseline and at 30-minute intervals for 3 h to measure glucose levels (in mmol/L) after ingestion of a 75 g glucose load.

Insulin resistance was defined as an abnormal level of fasting insulin (≥20 µIU/mL) and a glucose to insulin ratio less than 4.5[5].

Participants with IR were evaluated at baseline and after 3 months of treatment with folic acid.

Fasting insulin levels were measured using an AESKULISA kit (AESKU Diagnostics, Wendelsheim, Germany); levels of total testosterone (in ng/mL) and DHEAS (in µg/mL) were measured using an enzyme-linked immunosorbent assay kit from G-DRG (Diagnosis-Related Groups in Germany); levels of FSH (in mIU/

mL) and LH (in mIU/mL) were measured using a Monobind kit (Costa Mesa, California, USA) and levels of prolactin (in ng/mL) were measured using a Biome Rio kit (USA).

Group 1 (n = 32) consisted of participants with hyperhomocysteinemia and IR and group 2 (n = 38) consisted of participants with hyperhomocysteinemia without IR. To determine the effects of folic acid on Hcy levels and IR in women with PCOS, group 1 and group 2 both received 1 mg per day of folic acid (Rouz Darou, Tehran, Iran) for 3 months. All participants completed and signed the informed consent form before starting the folic acid treatment. After 3 months, Hcy levels as well as levels of insulin and other hormones of interest were reassessed.

Posttreatment and baseline values were compared and the effects of folic acid were evaluated for both groups.

The statistical analysis was performed using the SPSS statistical package for windows, version 11.5 (SPSS, Chicago, IL, USA). Differences in rates were compared by the Fisher exact test and parametric data sets were compared by the t test.

Pearson coefficients were used to calculate correlations between paired data sets. Pb0.05 was considered significant.

Table 1 Characteristic of 210 women with PCOS, using the upper limit of normal for plasma Hcy levels as a threshold^a

Variable Baseline

Hcy level

≤11 µmol/L^b (n = 140)

Baseline Hcy level N11 µmol/L^b (n = 70)

P value

Age, y 21.4 ± 3.0 22.9 ± 7.6 0.043

BMI 27.8 ± 1.4 28.2 ± 1.5 0.056

LH/FSH ratio 2.2 ± 0.3 2.5 ± 0.5 0.002 Prolactin, ng/mL 327.6 ± 83.4 291.6 ± 77.0 0.003 Insulin, mIU/L 14.6 ± 3.0 17.4 ± 5.0 0 Glucose to

insulin ratio

7.4 ± 2.4 5.8 ± 3.2 0

Hcy, µmol/L 8.5 ± 1.3 13.0 ± 1.4 0

DHEAS, µg/mL 253.0 ± 62.2 254.4 ± 60.5 0.873 Testosterone, ng/mL 0.6 ± 0.1 0.7 ± 0.1 0.228 Abbreviations: BMI, body mass index; DHEAS, dehydroepiandrosterone sulfate; FSH, follicle-stimulating hormone; Hcy, homocysteine; IR, insulin resistance; LH, luteinizing hormone; PCOS, polycystic ovary syndrome.

aValues are expressed as mean ± SD.

b Participants with baseline Hcy levels of 11 µmol/L or less constituted the control group for Hcy levels. Overall, there were 165 women without IR, 127 in the control group for Hcy and 38 in the study group for Hcy. Overall, there were 45 women with IR, 13 in the control group for Hcy and 32 in the study group for Hcy (χ²= 36.7, P= 0.000).

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3. Results

All participants completed the study and the primary out- come was analyzed. The mean age was 21.5 ± 3.1 years (range, 16–29 years). Clinical and demographic data are shown in Table 1. Of the 210 women with PCOS, 70 (33.3%) had hyperhomocysteinemia, 32 with IR and 38 without IR. In both groups, Hcy levels were significantly decreased after 3 months of folic acid supplementation. The mean ± SD levels before and after treatment were 14.03 ± 1.5 µmol/L and 12.53 ± 1.72 µmol/L in group 1 (Pb0.001), and they were 12.07±

0.87 µmol/L and 8.83 ±0.78 µmol/L in group 2 (Pb0.001) (Table 2). As the decrease rate was significantly higher in group 2, we concluded that IR affected Hcy response to folic acid.

No correlation was found between elevated BMI and Hcy level (R = 0.05, Pb0.47), and no correlation was found between Hcy level and age (r = 0.04, P = 0.73) (Table 1).

Of the 210 women with PCOS, 45 (21.4%) had IR (defined as a fasting insulin level≥20 µIU/mL and a glucose to insulin ratio of 4.5 or less). Of the 165 remaining women with PCOS but no IR, only 38 (23.0%) had elevated Hcy levels (defined as N11 µmol/L); but of the 45 women with IR, 32 (71.1%) had elevated Hcy levels (χ²= 36.78, Pb0.001) (Table 1). Thus, Hcy concentration was significantly associated with IR, and IR was more common in women who were hyperhomocysteinemic.

There was no correlation between IR and age (r =−0.03;

P = 0.77). When the 210 participants were categorized according to BMI, 175 (83.3%) were obese (BMI≥27). A high BMI, however, was not associated with IR in these women with PCOS (BMI was 28.2 ± 0.2 in those with IR and 27.8 ± 0.1 in those without IR (τ=1.30, P=0.20). No correlation was found between BMI and age (r = 0.03, P = 0.77).

An LH/FSH ratio higher than 2.5 was found only in 42 (20%) of the 210 participants. No correlation was found between LH/

FSH ratio and IR (r = 0.04, P = 0.75) or Hcy level (r = 0.19, P = 0.08). Three participants (8.6%) with a BMI of 27 or greater had an LH/FSH ratio greater than 2.5, whereas 39 participants (22.3%) with a BMI less than 27 had an LH/FSH ratio greater than 2.5. An LH/FSH ratio greater than 2.5 was more common among the lean participants. No changes in BMI or in levels or FSH, LH, total testosterone, prolactin, or DHEAS were re-

corded in either group following the 3 months of folic acid treatment (Table 2). The mean level of fasting insulin before and after treatment was 22.22 ± 1.52 µIU/mL and 22.11 ± 1.59 µIU/mL. Folic acid appears to have no effect on IR (τ=0.68, P=0.50).

4. Discussion

The results of this study provide some information regarding effects of folic acid supplementation on total Hcy and insulin concentrations, and regarding the relation between hyperhomocysteinemia and hyperinsulinemia in women with PCOS.

Because of the relation between insufficient folate intake and higher circulating Hcy concentrations[15,16], elevated fasting total Hcy concentrations are amenable to treatment with folic acid[4,5].

Kilicdag et al.[17]showed that treatment with metformin hydrochloride increased Hcy levels and suggested that both vitamin B12 and folic acid should be prescribed to women with PCOS, especially those using metformin and planning to become pregnant.

Significant positive correlations between Hcy levels and IR have been reported in patients with type 1 and type 2 diabetes, pre-eclampsia[5], and vascular disease[18]. The present study confirmed other results indicating that hyperhomocysteinemia was more common in women with IR, including those who were obese or who had PCOS[3].

Hyperinsulinemia may influence Hcy metabolism through its effects on glomerular filtration, or by influencing the activity of key enzymes in Hcy metabolism[19]. We may there- fore hypothesize that endothelial dysfunction and other cellular causes of oxidative stress in skeletal muscle, liver, and adipose tissue caused by hyperhomocysteinemia have a direct influence on IR[13]. Homocysteine can directly damage endothelial cells, impairing the release of nitric oxide (NO) and leading to a net increase in the production of superoxide (O2−).

It has been shown that folic acid can prevent the disruption of NO synthesis [20]. Although in the present study Hcy levels were significantly decreased in both groups following folic acid supplementation, the rate of decrease was significantly higher among women without IR. It can be concluded that IR impedes Hcy responses to folic acid. Laivuori et al.[5]reported

Table 2 Clinical and endocrine features of hyperhomocysteinemic women with PCOS and IR (group 1) and without IR (group 2), before and after 3 months of folic acid supplementation^a

Variable Group 1(n = 32)^b Group 2(n = 38)^b

Before After Before After

BMI 28.37 ± 1.59 27.96 ± 1.81 28.08 ± 1.57 27.87 ± 1.64

FSH, mIU/mL 6.4 ± 1.27 6.28 ± 1.55 5.98 ± 1.67 5.86 ± 1.63

LH, mIU/mL 14.94 ± 1.63 14.7 ± 1.6 14.7 ± 1.52 14.7 ± 1.67

Total testosterone, ng/mL 0.72 ± 0.11 0.72 ± 0.11 0.7 ± 0.09 0.7 ± 0.09

Prolactin, ng/mL 308.19 ± 77.07 316.57 ± 84.69 287.42 ± 60.95 284.38 ± 59.79

DHEAS, µg/mL 259.04 ± 68.87 273.27 ± 77.76 247.56 ± 51.25 246 ± 51.43

Insulin, µIU/mL 22.22 ± 1.52 22.11 ± 1.59 13.1 ± 2.4 13.4 ± 1.9

Hcy, µIU/mL 14.05 ± 1.52 12.52 ± 1.75 12.07 ± 0.87 8.83 ± 0.78

Abbreviations: BMI, body mass index; DHEAS, dehydroepiandrosterone sulfate; FSH, follicle-stimulating hormone; Hcy, homocysteine; IR, insulin resistance; LH, luteinizing hormone; PCOS, polycystic ovary syndrome.

a Values are expressed as mean ± SD.

b Intergroup and intragroup differences were not significant.

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a positive association between IR and elevated Hcy levels, and found that Hcy levels were higher in women with PCOS who also had IR. Giltay et al.[19]found a significant increase in plasma levels of Hcy in healthy subjects with IR, and Gallisth et al.[9] demonstrated fasting insulin level to be an independent determinant of total Hcy level in 84 obese children and adolescents. Schachter et al.[21]found a significant correlation between IR and Hcy levels regardless of body weight in women with PCOS.

Using the modified insulin suppression test in a study with 55 healthy subjects with a wide BMI range, Abbasi et al.[22]

found no direct association between total plasma Hcy level and IR. On the other hand, although Sills et al.[23]found no association between the diagnostic of polycystic ovary and plasma levels of Hcy, they noted significantly higher insulin levels in women with PCOS.

Limitations to our study include the lack of direct IR measurements. Elevated fasting insulin levels are highly correlated with IR when they are assessed by more direct measurements, especially in healthy glucose-tolerant individuals, and in this study a predictor of IR was the fasting insulin level.

In the present study, supplementation with folic acid had no effect on IR. Villa et al.[2]found no change in fasting insulin levels after treatment with folic acid, but by ascertaining an increased hepatic clearance of insulin they demonstrated a significant improvement of Hcy metabolism and insulin sensitivity. The controversy concerning IR, total Hcy level, and the effect of folic acid on IR may be due to the heterogeneity of study subjects and differences in the methods used to assess IR in humans. In a recent study of women with PCOS, Badawy et al. [24] found significantly higher homocysteine levels among those with IR than those without IR (Pb0.05). Although this demonstrates the role of IR, this condition is not the sole cause of hyperhomocysteinemia in women with PCOS. This elevation might be due to other factors such as hyperandro- genemia or the influence of other sex steroid hormones[24].

One of the significant findings of our study was in the definition of IR. The participants were considered as having IR if they had both a fasting insulin level of≥20 µIU/mL and a fasting glucose to insulin ratio less than 4.5. These criteria ought to be further studied.

In conclusion, a 3-month folic acid supplementation reduced the level of Hcy in hyperhomocysteinemic women with PCOS irrespective of whether they had IR, and the rate of decrease was higher among those without IR. Although we did not measure Hcy levels with different doses of folic acid, women with IR probably should receive a higher dose. Additional studies with a much larger number of participants and different doses of folic acid are needed to determine optimal treatments for hyperhomocysteinemic women with both PCOS and IR.

Acknowledgment

We thank Shiraz University of Medical Sciences Institutional Review Board for its financial support.

References

[1] De la Calle M, Usandizga R, Sancha M, Magdaleon F, Herranz A, Carillo E. Homocysteine, folic acid and B-group vitamins in obstetrics and gynecology. Eur J Obstet Gynecol Reprod Biol 2003;107:125–34.

[2] Villa P, Perr C, Suriano R, Cucinelli F, Panunzi S, Ranieri M, et al.

Folic acid supplementation in healthy postmenopausal women:

effect on homocysteine and glycolipid metabolism. J Clin Endocrinol Metab 2005;90:4622–9.

[3] Meigs JB, Jacques PF, Selhub J, Singer DE, Nathan DM, Rifai N, et al. Fasting plasma homocysteine levels in the insulin resistance syndrome: the Framingham offspring study. Diabetes Care 2001;24:1403–10.

[4] Fonseca VA, Fink KL, Kern PA. Insulin sensitivity and plasma homocysteine concentrations in nondiabetic obese and normal- weight subjects. Atherosclerosis 2003;167:105–9.

[5] Laivuori H, Kaaja R, Turpeinen V, Vinikka L, Yli korkala D. Plasma homocysteine levels elevated and inversely related to insulin sensitivity in preeclampsia. Obstet Gynecol 1999;93:489–930.

[6] Audelin MC, Genest Jr J. Homocysteine and cardiovascular disease in diabetes mellitus. Atherosclerosis 2001;159:497–511.

[7] Mcfarlane SI, Banerji M, Sowers JR. Insulin resistance and cardiovascular disease. J Clin Endocrinol Metab 2001;86:713–8.

[8] Stout RW. Insulin and atheroma: a 20-yr perspective [abstract].

Diabetes Care 1990;13:631–54.

[9] Gallisth S, Sudi K, Mangge H, Erwa W, Borkenstein M. Insulin is an independent correlate of plasma homocysteine levels in obese children and adolescents. Diabetes Care 2000;23:1348–52.

[10] Godsland IF, Rosankiewicz JR, Proudler AJ, Johnston DG. Plasma total homocysteine concentrations are unrelated to insulin sensitivity and components of the metabolic syndrome in healthy men. J Clin Endocrinol Metab 2001;86:719–23.

[11] Scarpitta AM, Sinagra D. Polycystic ovary syndrome: an endocrine and metabolic disease [abstract]. Gynecol Endocrinol 2000;14:392–5.

[12] Kely CJ, Speirs A, Gould GW, Petvie JR, Lyall H, Kelly JM.

Altered vascular function in young women with polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:742–6.

[13] Lergo RS, Kunselman AR, Dunaif A. Prevalence and predictors of dyslipedemia in women with polycystic ovary syndrome. Am J Med 2001;111:607–13.

[14] The Rotterdam ESHRE/ASRM-sponsored PCOS consensus work- shop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod 2004;19(1):41–7.

[15] Woo KS, Chook P, Chan LL, Cheung AS, Fung WH, Oiao M, et al.

Long-term improvement in homocysteine levels and arterial endothelial function after one-year folic acid supplementation.

Am J Med 2002;112:535–9.

[16] De Leo V, La Marea A, Morgante G, Ciani F, Zammarchi, Steacci C. Low-dose folic acid supplementation reduces plasma levels of the cardiovascular risk factor homocysteine in postmenopausal women. Am J Obstet Gynecol 2000;183:945–7.

[17] Kilicdag EB, Bagis T, Tarim E, Aslan E, Erkanli S, Simsek E, et al.

Administration of B-group vitamins reduces circulating homocysteine in polycystic ovarian syndrome patients treated with metformin: a randomized trial. Hum reprod 2005;20:1521–8.

[18] Hrnciar J, Gabor D, Hrn Ciarova M, Okapcova J, Szentivanyi M, Kurray P. Relation between cytokines (TNF-alpha, IL-1 and 6) and homocysteine in android obesity and the phenomenon of insulin resistance syndrome. Vnitr Lek 1999;45:11–6.

[19] Giltay EG, Hoogeveen EK, Elbers JM, Gooren LJ, Asscherman H, Stehouwer CD. Insulin resistance is associated with elevated plasma total homocysteine levels in healthy, nonobese subjects. Atherosclerosis 2001;139:197–8.

[20] Gori T, Buvotein JM, Ahmed S, Miner SE, Kelly S, Parker JD, et al.

Folic acid prevents nitroglycerin-induced nitric oxide synthase dysfunction and nitrate tolerance: a human in vivo study.

Circulation 2001;104:119–23.

[21] Schachter M, Raziel A, Friedler S, Strassburger D, Bern O, Ron-El R. Insulin resistance in patients with polycystic ovary syndrome with elevated plasma homocycsteine. Hum Reprod 2003;18:721–7.

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[22] Abbasi F, Facchini F, Humphreys MH, Reaven GM. Plasma homocysteine concentrations in healthy volunteers are not related to differences in insulin-mediated glucose disposal.

Atherosclerosis 1999;146:175–8.

[23] Sills ES, Genton MG, Perloe M, Schattman GL, Bralley JA, Tucker MJ. Plasma homocysteine, fasting insulin, and androgen patterns

among women with polycystic ovaries and infertility. J Obstet Gynecol Res 2001;27: 163–8.

[24] Badawy A, State O, El Gawad SSh, El Aziz OA. Plasma homocysteine and polycystic ovary syndrome: the missed link.

Eur J Obstet Gynecol Reprod Biol 2007;131:68–72.