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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2007-0842
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 1 82-90
Copyright © 2008 by The Endocrine Society

Asymmetrical Dimethylarginine, Inflammatory and Metabolic Parameters in Women with Polycystic Ovary Syndrome before and after Metformin Treatment

Dennis Heutling, Hasmik Schulz, Ingrid Nickel, Juergen Kleinstein, Petra Kaltwasser, Sabine Westphal, Friedrich Mittermayer, Michael Wolzt, Katarzyna Krzyzanowska, Harpal Randeva, Guntram Schernthaner and Hendrik Lehnert

Departments of Endocrinology and Metabolism (D.H., H.S.), and Reproductive Medicine and Gynaecological Endocrinology (I.N., J.K.), Institute of Clinical Chemistry (S.W.), Otto-von-Guericke-University, D-39120 Magdeburg, Germany; Department of Internal Medicine I (D.H., H.L.), Medical University of Schleswig-Holstein, Campus Luebeck, D-23552 Luebeck, Germany; Department of Obstetrics and Gynaecology (P.K.), Martin-Luther-University, D-06099 Halle, Germany; Department of Clinical Pharmacology (F.M., M.W.), Medical University Vienna, Department of Internal Medicine I (K.K., G.S.), Rudolfstiftung Hospital, A-1030 Vienna, Austria; and Warwick Medical School (H.R., H.L.), University Hospital of Coventry, Coventry CV2 2DX, United Kingdom

Address all correspondence and requests for reprints to: Hendrik Lehnert, M.D., F.R.C.P., Chair of Medicine, Warwick Medical School, University Hospital of Coventry Warwickshire, Clinical Sciences Building, Coventry CV2 2DX, United Kingdom. E-mail: h.lehnert{at}warwick.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Insulin resistance plays a significant role in the pathogenesis of the polycystic ovary syndrome (PCOS) and represents a link to the unfavorable cardiovascular risk profile frequently found in affected patients. The endogenous nitric oxide synthase inhibitor asymmetrical dimethyl-L-arginine (ADMA) is associated with atherosclerosis and represents an independent marker for cardiovascular morbidity and mortality.

Objective: We investigated ADMA levels among other cardiovascular, metabolic, and hormonal parameters in women with PCOS and the effects of metformin treatment on these parameters.

Design: A cross-sectional study and clinical trial were performed.

Patients and Participants: Women with PCOS (n = 83) compared with a control group of healthy women (n = 39) were studied.

Interventions: In a subgroup of patients with PCOS (n = 21), the effect of metformin was assessed after 6 months of treatment.

Main Outcome Measures: ADMA, intima media thickness (IMT), metabolic and hormonal parameters, and markers of inflammation were investigated.

Results: ADMA levels were significantly higher in the PCOS group compared with controls (0.57 ± 0.15 vs. 0.50 ± 0.11; P = 0.024). Androgens, C-reactive protein, fasting C-peptide, area under the curve (AUC) insulin, AUC glucose, homeostatic assessment of insulin resistance, fasting insulin, glycosylated hemoglobin, cholesterol, low-density lipoprotein cholesterol, triglycerides, and IMT were significantly higher in women with PCOS compared with controls. In PCOS patients ADMA was found to be positively correlated with body mass index (BMI), waist to hip ratio, parameters of insulin sensitivity, hyperandrogenemia (free testosterone, free androgen index), and IMT. Treatment with metformin ameliorated hyperandrogenemia and decreased ADMA levels (0.53 ± 0.06 vs. 0.46 ± 0.09, P = 0.013). Decrease in ADMA levels subsequent to metformin treatment did not correlate with change in BMI or metabolic parameters.

Conclusions: ADMA amd parameters of insulin sensitivity are elevated in women with PCOS and the degree of insulin resistance confers the greatest influence on ADMA level. Metformin treatment led to improvement of hormonal and metabolic parameters and decreased ADMA levels possibly independent of BMI and metabolic changes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder of women in the reproductive age, affecting 5–10% of all women in this life span (1). Over the last years, it has been established that hyperinsulinemia represents a fundamental disturbance in the majority of women with PCOS (2). Women with PCOS have an unfavorable cardiovascular risk profile and frequently exhibit insulin resistance, obesity, dyslipidemia, and hypertension. An increased risk for the development of type 2 diabetes is known (3). Different surrogate markers of cardiovascular disease (CVD) have been studied in women with PCOS. Inflammatory markers were elevated (4, 5, 6, 7, 8, 9, 10), and endothelial dysfunction, an early sign of cardiovascular damage, has been demonstrated (6, 11, 12, 13, 14). However, epidemiological data demonstrating an increase in clinical cardiovascular events are not available to date.

The mechanisms linking PCOS and increased cardiovascular risk profile are not well understood. Besides insulin resistance, it has been suggested that hyperandrogenemia contributes to vascular damage in women. However, data derived from different studies have shown contradictory results with regard to the impact of androgens on vascular function in women. In women with PCOS, elevated levels of endothelin-1 were positively correlated with testosterone level (15). Endothelial dysfunction appears to be associated with both insulin resistance and androgen level (6, 11, 14, 15). In contrast, some authors (16, 17) suggested beneficial effects of androgens on vascular function in women.

Asymmetrical dimethylarginine (ADMA), a guanidino-substituted analog of L-arginine, is a potent endogenous competitive inhibitor of the endothelial nitric oxide (NO) synthase. Symmetrical dimethyl-L-arginine (SDMA) is produced in equivalent amounts but does not affect NO synthesis. Increased levels of ADMA reduce NO formation and are associated with endothelial dysfunction (18). In healthy individuals the L-arginine to ADMA ratio correlates with endothelial function (19). The plasma levels of ADMA are increased in different pathological conditions, e.g. hypercholesterolemia (20), hypertriglyceridemia (21), and hyperhomocysteinemia (22). ADMA is associated with incident cardiovascular events (23, 24, 25, 26). Furthermore, a close relationship between insulin resistance and plasma concentrations of ADMA exists.

Over the last years, metformin has been shown to exert several beneficial effects in women with PCOS, and improved menstrual problems, hyperandrogenemia, and metabolic profile (27). Metformin action involves the suppression of endogenous glucose production, primarily by the liver, and probably has insulin-sensitizing effects in peripheral tissues, such as muscle and fat (28). Recently, antiinflammatory effects of metformin have been reported in obesity (29).

The aim of our study was to assess metabolic and hormonal profile, including plasma ADMA, in women with PCOS and control individuals. Here, we tested the hypothesis whether women with PCOS have increased levels of ADMA and whether ADMA levels in women with PCOS are associated with particular metabolic and/or hormonal characteristics. In the clinical substudy, the effects of metformin treatment on ADMA, metabolic and hormonal parameters were studied.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

A total of 83 women of Caucasian origin with PCOS was studied. The women with PCOS were outpatients of the Department of Reproductive Medicine and Gynaecological Endocrinology of Magdeburg University and the Department of Obstetrics and Gynaecology of Martin-Luther-University Halle. The metabolic study was performed in the Outpatient Department of Endocrinology and Metabolism of Magdeburg University.

For the control group, 39 premenopausal volunteers were recruited by public advertising. These women had neither signs of hyperandrogenemia nor menstrual irregularities, and had a similar age and body mass index (BMI) as women with PCOS. All women included were nonsmokers and otherwise healthy. None of them was currently taking drugs, nor had they taken hormonal contraceptives or steroids within the previous 6 months. The metabolic study was conducted in the follicular phase of menstrual cycle in all regular cycling women, as well as in women with PCOS and mild oligomenorrhea. In amenorrheic or severe oligomenorrheic women, investigations were done with no specific temporal limitations.

PCOS was diagnosed after exclusion of other causes of hyperandrogenemia and menstrual irregularities according the revised Rotterdam criteria (Rotterdam European Society for Human Reproduction & Embryology/American Society for Reproductive Medicine-sponsored PCOS Consensus Workshop Group 2004). All patients had amenorrhea or oligomenorrhea, and/or clinical (hirsutism with a Ferriman-Gallwey score ≥ 7, acne, alopecia) and/or biochemical signs of hyperandrogenism [increased circulating level of total or free testosterone (FT), androstendione, or dehydroepiandrosterone sulfate (DHEAS)]. In addition, all women had polycystic ovaries on ultrasound scans. Transvaginal ultrasound was used to detect polycystic ovaries, defined as the presence of at least one ovary more than 10 ml or with at least 12 follicles of 2- to 9-mm diameter.

The study design was approved by the local ethics committee of the University of Magdeburg, and written informed consent was obtained from all participants.

Study protocol

Clinical features, including anthropometric variables, and the degree of hirsutism, family history, menstrual cyclicity, as well as endocrine and biochemical parameters were recorded. Blood pressure was measured in sitting position after a rest of at least 5 min. The average of three measurements was obtained. Hirsutism was assessed using the Ferriman-Gallwey Score. A score of seven or greater was considered to represent hirsutism. BMI was calculated as body weight (kg) divided by height squared (m2). Waist to hip ratio (WHR) was calculated as waist circumference divided by hip circumference.

Blood samples were collected between 0800 and 0900 h, after a 3-d normal carbohydrate diet and an overnight fast for at least 10 h. A 120-min 75-g oral glucose tolerance test (OGTT) was performed in all women, and blood samples were drawn for the determination of glucose and insulin before and 30, 60, 90, and 120 min after glucose ingestion. Blood samples for testing of all other parameters were drawn before the OGTT. The samples were immediately cooled, and serum and plasma were prepared within 1 h and stored at –80 C until assayed.

The free androgen index (FAI) was calculated using the equation: FAI = (testosterone/SHBG) x 100. To estimate insulin sensitivity, homeostatic assessment of insulin resistance (HOMA-IR) was calculated according the following formula: HOMA-IR = [fasting insulin (IU/liter) x fasting glucose (mmol/liter)]/22.5 (30). In addition, insulin sensitivity was assessed using the values for glucose and insulin derived from the OGTT. For this, the area under the curve (AUC) for insulin and glucose was calculated from the values obtained during the OGTT using the trapezoid method. As a measure for post-load insulin sensitivity, the ratio between the AUC for glucose and insulin was calculated as follows: AUC glucose (mg/dl)/AUC insulin (pmol/liter) (31).

Baseline data of women with PCOS were compared with those of 39 control subjects.

A treatment with metformin in an "off label use" within a study was offered to all women diagnosed with PCOS independently from the results of insulin sensitivity testing. Sole women accepting a treatment period for 6 months with metformin alone and a repetition of the basal assessment after that time were enrolled. In the end a subgroup of 34 women participated in the metformin protocol. In these women therapy was initiated after basal assessment, and the dose of metformin was increased according to the tolerability to a maintain dose of 850 mg twice a day. Women included were closely followed up for the period of the study. Although no specific diet or exercise regimen was advised, all women were informed about the relationship between PCOS, body weight, and insulin sensitivity, and standard advice concerning the beneficial effects of lifestyle modification was given.

Assays

DHEAS, SHBG, FSH, LH, and C peptide assays were performed with the Immulite system (Diagnostic Products Corp., Los Angeles, CA; distribution in Germany, Diagnostic Products Corp., Biermann GmbH, Bad Nauheim), a fully automatic random access chemiluminescence-enhanced enzyme immunoassay system. Testosterone, FT, and androstendione were analyzed by commercial RIAs (Diagnostic Products Corp Biermann GmbH). Homocysteine was determined as total homocysteine by HPLC with fluorescence detection (32), with samples from the same patient being measured in one run. The assay has a between-run coefficient of variation (CV) of 5%. The analysis of fibrinogen was performed with commercial coagulometric method (Technoclone GmbH, Vienna, Austria) on STA EVOLUTION (Roche Diagnostics, Mannheim, Germany).

Triglycerides and cholesterol were determined by commercial enzymatic methods in a random-access analyzer (Hitachi 911; Roche). All reagents and calibrators were also from Roche. Free fatty acids were measured by a commercial enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany). High-sensitivity IL-6 (R & D Systems GmbH, Wiesbaden, Germany) was measured using ELISAs and leptin with a coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories, Sinsheim, Germany). All samples were assayed in duplicate, with samples from the same patient being measured in a single run at the end of the study. The intraassay CVs for the determination of leptin and IL-6 were 3.2 and 6.9%, respectively. The interassay CVs were 5.7 and 5.5%, respectively. Glucose and high-sensitive C-reactive protein (hs-CRP) were determined by the glucose oxidase method or immunoturbidimetrically on a Modular-System random-access analyzer (Roche), insulin by commercial RIA (BI-Insulin IRMA; Bio-Rad, Marnes la Coquette, France), and hemoglobin A1c by HPLC (Bio-Rad Laboratories GmbH, Munich, Germany).

Determination of ADMA, SDMA, and L-arginine

For measurement of L-arginine, ADMA, and SDMA, plasma was subjected to cation exchange solid-phase extraction and analyzed by HPLC (33). The CVs for intersample and intrasample variations tested with pooled plasma sample were less than 3% for all analytes. The detection limit for dimethylarginines was 0.04 µmol/liter (34).

Intima media thickness (IMT)

Measurement of carotid IMT is a widely accepted tool to provide information about preclinical vascular disease. B-mode sonography of the proximal part of the carotid bulb was conducted on both sides, and the segments of the common carotid arteries 1.0-cm proximal were scanned longitudinally with Hitachi EUB-5000 plus-G (Hitachi, Ltd., Tokyo, Japan) using a 10-MHz linear-array transducer. All women were investigated in supine position with the head slightly hyperextended and turned away from the side being scanned. The image was focused on the posterior wall, and the resolution function was used to magnify the arterial far wall. When an optimal image was obtained, it was frozen, and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end-diastolic phase, to minimize variability during the cardiac cycle. All images were photographed. Five measurements were conducted on each side, and the mean IMT was calculated as the average of these measurements. All measurements were performed by an experienced ultrasound sonographer (intraobserver CV was 6.8%) who was blinded to the diagnosis of subjects.

Statistics

Results are expressed as mean ± SD. P values less than 0.05 were considered significant. Because some variables, including ADMA, were not distributed normally as checked by histograms and the Kolmogorov-Smirnoff test, nonparametric testing for further analysis was used. The differences between the PCOS and control groups and between the PCOS group and subgroup of women treated with metformin were assessed using the Mann-Whitney-U-test. In addition, analysis of covariance (ANCOVA) for ADMA with BMI as covariate was performed. Relations between variables were investigated using Spearman’s bivariate correlation in PCOS women and control individuals. Furthermore, correlation analysis of the changes in variables after metformin therapy was performed. Subsequently, if individual bivariate correlations achieved statistical significance, variables were entered into a linear regression model, and stepwise (probability of F to enter ≤ 0.05; probability of F to remove ≥ 0.10) multiple regression analysis with ADMA as a dependent variable was performed to test the joint effect of different parameters on ADMA. Within-group differences (metformin trial) were analyzed with the Wilcoxon test. All statistical analyses were done using SPSS version 12.0 (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Cross-sectional study

Clinical characteristics and androgen status Table 1Go shows anthropometric data and androgen status of women with PCOS and controls. Mean values for age, BMI, and WHR were not different between the two groups.


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  		TABLE 1. Clinical characteristics and androgen levels of the PCOS and control groups

 
Levels of testosterone (2.21 ± 1.44 vs. 1.32 ± 0.62; P < 0.001), FT (2.41 ± 1.3 vs. 1.10 ± 0.6; P < 0.001), and androstendione (3.61 ± 1.43 vs. 2.69 ± 1.10; P < 0.001) were significantly higher in PCOS women. In contrast, DHEAS was lower in the PCOS group (5.0 ± 2.7 vs. 5.8 ± 2.2; P = 0.035). Typically for PCOS we found an elevated level of LH in our study population of PCOS women in comparison with healthy women (9.5 ± 7.7 vs. 4.5 ± 3.7; P < 0.001). As suspected, the Ferriman-Gallwey score was higher in PCOS women (6.3 ± 3.8 vs. 1.3 ± 0.7; P < 0.001).

SHBG levels were lower in the PCOS group (36.2 ± 21.4 vs. 49.0 ± 25.8; P < 0.001). On the basis of SHBG and total testosterone values, the FAI was calculated, which was higher in the PCOS group (8.51 ± 7.93 vs. 3.18 ± 1.68; P < 0.001).

Metabolic parameters Impaired fasting glucose with levels above or equal to 6.1 mol/liter was found in five women within the PCOS group and in two women within the control group. Whereas fasting glucose did not differ significantly between both groups, AUC glucose values were higher in the PCOS group (820.8 ± 171.1 vs. 750.8 ± 165.0; P = 0.038). Glycosylated hemoglobin (HbA1c) was slightly but significantly higher in the PCOS group (5.41 ± 0.39 vs. 5.25 ± 0.32; P = 0.021). In women with PCOS fasting insulin (77.8 ± 49.9 vs. 47.4 ± 21.7; P = 0.001), AUC insulin (71,885 ± 57,549 vs. 32,050 ± 16,329; P = 0.001) and HOMA-IR (2.48 ± 1.90 vs. 1.44 ± 0.81; P = 0.001) were significantly higher. Furthermore, levels of cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, free fatty acids, and fibrinogen were significantly higher, and high-density lipoprotein (HDL)-cholesterol was lower in women with PCOS compared with controls. There was no difference for homocysteine between the groups (Table 2Go).


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  		TABLE 2. Metabolic, inflammatory, and cardiovascular parameters, including ADMA, in the PCOS and control groups

 
Inflammatory parameters Among the inflammatory markers, only C-reactive protein (CRP) was higher in women with PCOS (3.26 ± 2.33 vs. 1.61 ± 1.36; P < 0.001). There was no difference for IL-6 between the groups (Table 2Go).

Cardiovascular parameters and ADMA ADMA levels were significantly higher (0.565 ± 0.150 vs. 0.502 ± 0.105; P = 0.024) and the arginine to ADMA ratio lower (159.9 ± 43.6 vs. 195.1 ± 74.7; P = 0.028) in PCOS women compared with the control group. Because BMI was slightly higher in the PCOS group, ANCOVA with BMI as covariate was performed. Even after adjustment for BMI, ADMA remains significantly higher in women with PCOS (P = 0.035) (Table 2Go).

Furthermore, there was a significant difference in IMT between the groups (0.48 ± 0.07 vs. 0.42 ± 0.05; P < 0.001). In all cases blood pressure was less than or equal to 140/90 mm Hg. Women with PCOS had higher systolic (123.4 ± 9.0 vs. 117.1 ± 8.8; P < 0.001) and diastolic blood pressure (76.9 ± 7.4 vs. 74.6 ± 6.9; P = 0.028) compared with controls (Table 2Go).

Correlation and regression analysis for ADMA The results of correlation analysis for ADMA are shown in Table 3Go. There were some differences in correlation analysis in PCOS and controls. In the PCOS group, ADMA plasma concentrations were positively correlated with BMI (r = 0.353; P = 0.001), fasting insulin (r = 0.405; P < 0.001), and fasting C peptide (r = 0.348; P = 0.001). From the androgens, FT (r = 0.2600; P = 0.018) and FAI (r = 0.331; P = 0.002) were positively correlated in PCOS and testosterone in controls (r = 0.364; P = 0.023). Weak correlations were found for HbA1c (r = 0.267; P = 0.015) and IMT (r = 0.249; P = 0.023) within the PCOS group.


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  		TABLE 3. Spearman’s correlation coefficients between plasma ADMA and hormonal and metabolic parameters

 
In the control group, but not in the PCOS group, a positive correlation between ADMA and hs-CRP (r = 0.423; P = 0.007) was found. Furthermore, a negative correlation for HDL-cholesterol (r = –0.344; P = 0.032) was found. In women with PCOS, a stepwise multiple linear regression analysis revealed a direct relationship with fasting insulin level and IMT. In controls, homocysteine was the main predictor of plasma ADMA concentration. The results of regression analysis are shown in Table 4Go.


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  		TABLE 4. Models of stepwise multiple linear regression analysis in PCOS patients and control individuals to test the influence of the main independent variables against ADMA (dependent variable)

 
Metformin treatment

Metformin treatment was initiated in 34 women with PCOS. This subset of patients was representative for the population of women with PCOS from the cross-sectional part regarding clinical, biochemical, and hormonal parameters. From these, 21 women completed the protocol and were investigated after 6-month metformin treatment. The baseline data of this subgroup were not statistically different compared with the PCOS group from the cross-sectional part. Causes for dropouts were gastrointestinal side effects and nausea (n = 4), pregnancies (n = 5), incompliance (n = 2), and loss of contact (n = 2). The results before and after metformin treatment are shown in Table 5Go.


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  		TABLE 5. Endocrine, metabolic, and cardiovascular parameters, including ADMA, of women with PCOS (n = 21) before and after metformin treatment

 
The mean BMI decreased after 6 months on metformin (32.8 ± 4.2 vs. 31.4 ± 5.9; P = 0.003). Total testosterone (1.96 ± 0.86 vs. 1.38 ± 0.47; P = 0.003), FT (2.15 ± 1.10 vs. 1.41 ± 0.60; P = 0.002), and FAI (7.35 ± 4.69 vs. 4.92 ± 2.42; P = 0.006) improved significantly at 6 months. The decrease of androstendione level achieved no significance, and DHEAS was slightly increased. ADMA levels decreased significantly after metformin treatment (0.53 ± 0.06 vs. 0.46 ± 0.09; P = 0.013).

Improvements of parameters related to hyperinsulinemia (AUC insulin, HOMA-IR) did not achieve statistical significance. Furthermore, fasting glucose was decreased after metformin treatment. IMT marginally decreased after 6 months (0.49 ± 0.07 vs. 0.48 ± 0.07; P = 0.044). Metformin treatment did not exert a significant effect on CRP, IL-6, and fibrinogen.

Because there were no significant correlations between changes in ADMA to changes in metabolic and endocrine parameters investigated, including insulin sensitivity (data not shown), regression analysis for ADMA as a dependent variable was not performed.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present study is the first to our knowledge investigating ADMA in relation to hormonal and metabolic parameters in women with PCOS and after metformin treatment.

We have demonstrated that ADMA is increased in women with PCOS when compared with controls and decreased significantly after metformin therapy. In our study mainly parameters reflecting insulin resistance and BMI and, to a lesser extent, parameters of hyperandrogenemia were associated with plasma ADMA levels in women with PCOS. The regression analysis revealed fasting insulin as the most important predictor of ADMA level in women with PCOS.

Similarly, in other investigations ADMA has been shown to be associated with degree of insulin resistance and BMI. Plasma ADMA concentrations were positively correlated with impairment of insulin-mediated glucose disposal in nondiabetic, normotensive subjects and with BMI in patients with a high risk of atherosclerosis (35, 36). Plasma ADMA levels are higher in obese, insulin-resistant women than in equally obese, insulin-sensitive women and decrease in response to weight loss when associated with enhancement of insulin sensitivity (37). Our data support the concept of ADMA as an important link between impaired insulin action and endothelial dysfunction (38).

The role of androgens in the pathogenesis of endothelial dysfunction and development of atherosclerosis in women with PCOS is not clarified to date. As mentioned previously, we found a positive correlation between ADMA and androgens in women with PCOS and in controls. Because ADMA is closely related to endothelial dysfunction, our data are generally consistent with previous reports demonstrating a strong association between endothelial dysfunction, androgen levels, and insulin resistance in women with PCOS (11, 14). In addition, endothelin-1 and the endothelial activation marker soluble vascular cell adhesion molecule-1 (sVCAM-1) were positively correlated with androgen levels and insulin resistance (6, 15).

However, disadvantageous associations between androgens and the vasculature of women were not confirmed in all investigations. Conflicting results have been reported regarding endothelial dysfunction and carotid IMT and their relation to androgens in women with PCOS. Especially for DHEAS, positive effects on endothelial function were noticed (16, 17). Despite preclinical atherosclerosis in young women with PCOS, revealed by increased IMT, DHEAS was negatively associated with IMT. Thus, it has been suggested that hyperandrogenemia could attenuate the consequences of the unfavorable cardiovascular risk profile in PCOS (39).

In our study women with PCOS had a higher IMT compared with controls, indicating early structural changes of vasculature. This result is consistent with investigations demonstrating an increase in carotid IMT in women with PCOS (6, 15, 39, 40). Neither in women with PCOS nor in controls did we find androgens to be correlated with IMT (data not shown).

In the regression analysis, IMT appeared as an independent positive determinant of ADMA level in women with PCOS. Although ADMA may rather represent a causative factor for IMT increase than vice versa, this finding underlines a strong relationship between ADMA and progression of atherosclerosis. Similarly to our findings, it has been reported that ADMA concentration is independently associated with IMT in patients with mild-to-moderate renal failure (41). Furthermore, in a study investigating a large number of subjects without overt cerebro-CVDs, the plasma level of ADMA was associated with IMT of the carotid artery (42).

In the control group, we failed to demonstrate a relationship between ADMA, BMI, and parameters of insulin sensitivity. This may partly be due to the wider range of these variables in the PCOS group compared with the control group, e.g. parameters of insulin sensitivity were in a smaller range with mostly normal values in controls. ADMA was inversely correlated with HDL-cholesterol in control subjects. This might reflect the relevant protective role of HDL-cholesterol on the vasculature. Furthermore, our data are in line with the described association between homocysteine and ADMA (43). However, an interventional study with vitamin B and folic acid supplementation in patients with peripheral vascular disease and hyperhomocysteinemia did not result in the lowering of ADMA, despite a significant decrease in homocysteine levels (44).

Inflammatory markers, such as CRP and IL-6, have been associated with an increased risk of CVD. We found an elevation of hs-CRP in women with PCOS but no difference for IL-6 between PCOS and controls. Our finding of an elevated CRP is in accordance with a number of studies (4, 6, 45, 46). However, an increase of CRP independently of obesity was not confirmed in all investigations in PCOS (47, 48). In the control group, the correlation and regression analysis revealed an association between hs-CRP and ADMA. This is in accordance with the findings of Krzyzanowska et al. (49) in a population of morbidly obese women. However, we could not demonstrate a relationship between ADMA and inflammatory markers in women with PCOS.

In our current study, ADMA levels decreased significantly after 6-month treatment with metformin. IMT only marginally decreased in this time, which is conversely to the greater IMT reduction after metformin treatment reported by Orio et al. (50) in nonobese PCOS patients.

Furthermore, metformin therapy was accompanied by a reduction of BMI, hyperandrogenemia, fasting glucose, systolic blood pressure, and homocysteine. However, changes in these parameters were not significantly correlated with the observed decrease in ADMA levels. ADMA lowering effects of insulin sensitizers in different insulin resistant states have previously been reported. In hypertensive, insulin-resistant individuals, treatment with rosiglitazone improved insulin sensitivity and decreased plasma ADMA concentrations (36). In addition, metformin has decreased the ADMA level in poorly controlled diabetic patients. Metformin was effective as monotherapy as well as in combination with sulfonylurea treatment (51). A decrease of ADMA levels after weight loss after gastroplastic surgery was previously shown in morbidly obese women (49).

The effect of metformin treatment on homocysteine levels is controversial (52). In the present study, metformin treatment decreased homocysteine levels despite no difference in the homocysteine levels between PCOS women and controls. In contrast, other authors found elevated homocysteine levels in women with PCOS, but no effect of metformin treatment (53).

Chronic subclinical inflammation is closely related to obesity, and a BMI reduction results in a decrease of inflammatory markers (54, 55). However, despite a significant decrease in BMI, we did not find a reduction in hs-CRP and IL-6 after metformin treatment. These result are in disagreement with other studies (5, 9) but might be explained by a lesser reduction of BMI (–1.3) compared with the other studies (–2.7 and –1.9).

Considering the BMI reduction, the lack of a placebo group is a limitation of the present study. However, the observed weight reduction under metformin is in close agreement with two large controlled intervention studies performed in type 2 diabetic patients (56, 57). Recent studies may explain why patients treated with metformin show weight loss; metformin enhances glucagon-like peptide-1 secretion in experimental animal studies (58) and inhibits dipeptidyl peptidase IV activity in type 2 diabetic patients (59).

The mechanisms whereby metformin improves ADMA in women with PCOS are not quite clear. Inflammatory markers such as CRP and IL-6 were not significantly correlated with ADMA in women with PCOS and did not change in response to metformin in our study. Thus, the reduction in ADMA after metformin is not due to antiinflammatory effects. Although amelioration of hyperandrogenemia, improvements of insulin sensitivity, and body weight may be suspected as contributing factors, our data failed to elucidate a main driver for the ADMA- lowering effect of metformin.

Metformin has a variety of effects beyond glucose control and, for example, exerts direct vascular antiinflammatory effects by inhibiting the nuclear factor (NF)-{kappa}B (60). It has been shown that activation NF-{kappa}B triggers insulin resistance and inflammation in PCOS (61). Furthermore, inhibition of NF-{kappa}B decreases the ADMA level in cultured endothelial cells (62). Considering these previous findings, a more direct effect of metformin on the vasculature could contribute to ADMA decrease. However, this study was not designed to address this interesting question.

In conclusion, women with PCOS have elevated ADMA levels and increased IMT, suggesting an increased risk for an early onset CVD. In PCOS, ADMA mainly depends on the degree of insulin resistance and, to a lesser extent, on hyperandrogenemia. Metformin treatment improved hyperandrogenemia and decreased body weight and ADMA levels, probably independently of a change in insulin sensitivity. Further studies are required to clarify the role of androgens in the pathogenesis of atherosclerosis in PCOS.


   Acknowledgments
 
We thank Mrs. Rosalie Ludewig for her great help during the metabolic study and Dr Daniel Pittasch for his help in intima media thickness measurement. We also thank Dr. Bernd-Wolfgang Igl, Institute of Medical Biometry and Statistics, University of Luebeck.


   Footnotes
 
First Published Online November 6, 2007

Abbreviations: ADMA, Asymmetrical dimethylarginine; ANCOVA, analysis of covariance; AUC, area under the curve; BMI, body mass index; CRP, C-reactive protein; CV, coefficient of variation; CVD, cardiovascular disease; DHEAS, dehydroepiandrosterone sulfate; FAI, free androgen index; FT, free testosterone; HbA1c, glycosylated hemoglobin; HDL, high-density lipoprotein; HOMA-IR, homeostatic assessment of insulin resistance; hs-CRP, high-sensitive CRP; IMT, intima media thickness; LDL, low-density lipoprotein; NF, nuclear factor; NO, nitric oxide; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; SDMA, symmetrical dimethyl-L-arginine; WHR, waist to hip ratio.

Disclosure Information: The authors have nothing to declare.

Received April 13, 2007.

Accepted October 26, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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