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Project Aging Women and the Institute for Cardiovascular Research, Vrije Universiteit, Departments of Obstetrics and Gynecology (M.S.P., M.O.V., M.J.v.d.M., P.K.), Internal Medicine (C.D.A.S.), and Clinical Chemistry (T.T.), VU University Medical Center, 1007 MB Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: T. Teerlink, Ph.D., Department of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: t.teerlink{at}vumc.nl.
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Abstract |
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Introduction |
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Although HRT is no longer prescribed for cardiovascular prevention in postmenopausal women, it is still the most effective treatment for women with climacteric complaints. Discussion is ongoing as to whether or not the HERS and WHI results can be extrapolated to younger, early postmenopausal women without signs of arterial disease. Therefore, it is especially important to study the effect of HRT on cardiovascular risk factors in this population.
Nitric oxide (NO) has a vasodilating effect, inhibits platelet aggregation, and suppresses smooth muscle cell proliferation. In postmenopausal women, its levels have been shown to rise during continuously combined oral estrogen plus progestogen replacement therapy (7, 8), during the estrogen-only phase of sequentially combined oral estrogen replacement therapy (9), but not in the combined phase (9, 10). Possibly the increased endothelium-dependent flow-mediated dilation that has been observed during oral HRT by some investigators (11, 12), but not by others (13, 14), is associated with an increase in NO production or availability. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS) (15) and a risk factor for acute coronary events in middle-aged men (16) and for overall mortality and cardiovascular events in patients with end-stage renal disease (17). In addition, in critically ill patients on a surgical intensive care unit, high plasma concentrations of ADMA were associated with an adverse outcome (18).
Recently, we have found that unopposed conjugated equine estrogens reduce plasma levels of ADMA (19). Because the effect of estrogen replacement therapy on the cardiovascular system depends on the type of estrogen, the dosage, the route of administration and the addition of a progestogen, we decided to investigate whether unopposed oral 17ß-estradiol also lowers ADMA and whether the addition of the progestogens, dydrogesterone or trimegestone, modulates this effect. In addition, the effects on plasma levels of arginine, a precursor of NO, and symmetric dimethylarginine (SDMA), a stereoisomer of ADMA that does not inhibit NOS, were investigated. Measurements were performed in stored plasma samples of a randomized placebo-controlled 12-wk study in healthy early postmenopausal women (20, 21, 22, 23, 24, 25, 26).
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Subjects and Methods |
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Healthy, nonhysterectomized postmenopausal women were recruited through advertisements in local newspapers; 65 women were enrolled in this 12-wk study, which was performed at the outpatient clinic of the Department of Obstetrics and Gynecology (VU University Medical Center, Amsterdam, The Netherlands) (20, 21, 22, 23, 24, 25, 26). The investigation conformed to the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from each participant before entering the study. The Institutional Review Board of the VU University Medical Center approved the protocol.
Participants were between 45 and 60 yr old, smoked fewer than 15 cigarettes per day, were normotensive (<160/90 mm Hg), had a body mass index (BMI) no greater than 30 kg/m2, and had been amenorrheic for 6 months to 5 yr with serum FSH concentrations above 20 IU/liter and estradiol concentrations lower than 150 pmol/liter. None of the women had received HRT for at least 3 months before entering the study, and none took cardiovascular medication. Exclusion criteria included a history of cardiovascular, venous thromboembolic, metabolic, endocrinological, and (pre-) malignant disease, as well as clinically relevant abnormalities in laboratory tests of hematological, renal, and hepatic function. Women with fasting serum levels of cholesterol and triglycerides greater than 8 mmol/liter and greater than 4 mmol/liter, respectively, were also excluded.
Design
Eligible women were randomly assigned to placebo (n = 17), unopposed micronized 17ß-estradiol 2 mg/d (E2 group; n = 18), or sequentially combined HRT consisting of micronized 17ß-estradiol 2 mg/d plus either dydrogesterone 10 mg/d (E2+D group; n = 15; Femoston, Solvay Pharmaceuticals, Weesp, The Netherlands), or trimegestone 0.5 mg/d (E2+T group; n = 15; Hoechst Marion Roussel, Romainville-Cedex, France). The progestogens were given for the last 14 d of each 28-d cycle. The pharmacist of the VU University Medical Center (Amsterdam, The Netherlands) manufactured placebo and estradiol as capsules of identical appearance. The tablets of the sequentially combined HRT were put into capsules of identical appearance by Hoechst Marion Roussel (Paris, France). A computerized randomization list was made. Randomization codes were put into sealed envelopes and stored by the pharmacist of the VU University Medical Center. Medication boxes were numbered, and allocation was done in sequence. Unblinding was done at the end of the study period. Women assigned to treatment with unopposed estradiol were treated with dydrogesterone 10 mg/d for 14 d to induce a withdrawal bleeding.
Sixty-five participants were initially enrolled. Five women dropped out before the measurement at 4 wk and were therefore excluded from the analysis (placebo group, n = 1; E2 group, n = 2; E2+D group, n = 1; E2+T group, n = 1). Another three women dropped out between 4 and 12 wk (placebo group, n = 1; E2+D group, n = 1; E2+T group, n = 1). In these three cases, the last-observation-carried-forward procedure was applied for the missing values at 12 wk. Therefore, the analyses were based on 60 participants. Reasons for dropout have been published previously (22, 23, 24, 25).
Blood collection
At baseline and after 4 wk (cycle 1) and 12 wk (cycle 3) of follow-up, venous blood samples were taken between 0800 and 1000 h. Blood sampling was performed between d 24 and 28 of these cycles, i.e. at the end of the combined estrogen-progestogen phase of the sequential regimens. The subjects had fasted and had refrained from smoking for at least 10 h and from consuming alcohol for at least 24 h before sampling. After 20 min of rest, blood was collected into tubes containing EDTA (K3) (Becton Dickinson, Meyren-Cedex, France). The blood samples were immediately placed on ice and centrifuged within 1 h of collection at 3000 x g and 4 C for 30 min. Plasma was snap-frozen and stored at -70 C until analysis.
Laboratory methods
ADMA, arginine, and SDMA were measured by HPLC with fluorescence detection (27). Briefly, 0.2 ml of plasma was mixed with 0.1 ml of a 40-µmol/liter solution of the internal standard monomethylarginine and 0.7 ml PBS. This mixture was applied to Oasis mixed-mode cation-exchange solid-phase extraction columns (Waters, Milford, MA). The columns were consecutively washed with 1.0 ml of 0.1 mol/liter HCl and 1.0 ml methanol. Basic amino acids were eluted with 1.0 ml of concentrated ammonia/water/methanol (10:40:50). After evaporation of the solvent under nitrogen, the amino acids were derivatized with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid. The derivatives were separated by isocratic reversed-phase chromatography on a Symmetry C18 column (3.9 x 150 mm; 5-µm particle size; Waters, Milford, MA) at a column temperature of 30 C. Potassium phosphate buffer (50 mmol/liter; pH 6.5), containing 8.7% acetonitrile, was used as mobile phase at a flow-rate of 1.1 ml/min. After elution of the last analyte, strongly retained compounds were quickly eluted by a strong solvent flush with acetonitrile. Fluorescence detection was performed at excitation and emission wavelengths of 340 and 455 nm, respectively. All samples from individual patients were analyzed in the same analytical series. The intra-assay coefficients of variation for ADMA, arginine, and SDMA were 1.2, 0.4, and 0.8%, respectively. In each participant the plasma arginine/ADMA ratio was determined.
Serum total cholesterol was measured with an autoanalyzer (Roche Molecular Biochemicals, Mannheim, Germany). Serum FSH was determined with a specific immunometrical (luminescence) assay (Amerlite, Amersham, Little Chalfont, UK). Serum total 17ß-estradiol was quantified using a double-antibody RIA (Sorin Biomedica, Saluggia, Italy) with a lower limit of detection of 18 pmol/liter.
The major route of elimination of ADMA is hydrolysis by dimethylarginine dimethylaminohydrolase (DDAH), an enzyme of which the activity may be inhibited by homocysteine. Plasma homocysteine concentrations were determined earlier (21) by HPLC with fluorescence detection according to Fiskerstrand et al. (28).
Statistical analysis
Statistical analysis was performed using the Statistical Package for the Social Sciences 9.0 (SPSS Inc., Chicago, IL). Baseline characteristics (Table 1
) are given as mean ± SD when normally distributed or as median (25th-75th percentile) when the distribution was skewed. Concentrations of the variables investigated are given as mean ± SD. The mean of the individual percentage changes from baseline to 4 and 12 wk are given as mean and 95% confidence interval (CI). Standard parametric tests were performed (for variables with a skewed distribution after log transformation). At baseline, BMI differed slightly among the groups (Table 1), and therefore a general linear model for repeated measurements, with the baseline values of the variable under consideration and BMI as constant covariates [analysis of covariance (ANCOVA)], was used for comparisons among and between the groups. Correlations between variables were calculated with Pearson’s correlation coefficient. A two-tailed P < 0.05 was accepted as the level of significance.
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Results |
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shows demographic characteristics of the four groups at baseline. In each group, except for the E2+T group, one woman had an estradiol level above 150 pmol/liter. However, because this occurred in combination with a FSH level above 35 IU/liter, we did not exclude these women from the analysis. Table 2 summarizes the values of the variables investigated during the study period. At baseline, there were no significant differences among the groups for any of the variables.
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). For arginine and the arginine/ADMA ratio, ANCOVA showed significant differences among groups. This could be attributed, both for arginine and the arginine/ADMA ratio, to significant differences between the E2+T and the other groups. The decreases in arginine and arginine/ADMA ratio observed after 4 wk of treatment persisted after 12 wk.
In addition, ANCOVA showed differences in SDMA among the groups (overall, P = 0.05). This was the result of a significant decrease in comparison to placebo in the E2+D group and nearly significant decreases in the E2 and E2+T groups (Table 2). Figure 1 shows percentage changes from baseline of ADMA, arginine, the arginine/ADMA ratio, and SDMA at 12 wk.
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. As described before in more detail (21), fasting plasma homocysteine concentrations were reduced in all active treatment groups but not in the placebo group. ADMA levels at baseline were not associated with homocysteine. In addition, there were no associations between treatment-induced changes of homocysteine and changes in ADMA levels (data not shown). |
Discussion |
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These results indicate that the effect of unopposed oral 17ß-estradiol 2 mg on ADMA is somewhat smaller than the 8% reduction observed earlier with unopposed oral conjugated estrogens 0.625 mg in a 2-yr trial (19). A possible explanation for this may be that the full effect of estrogen was not reached, given the relatively short duration of the present study. The finding that the reduction in ADMA was more pronounced at 12 wk than at 4 wk in all active treatment groups also points in that direction. Although both progestogens enhanced the effect of 17ß-estradiol, this effect was most pronounced for trimegestone. In the population studied here, a difference in effect of dydrogesterone and trimegestone has also been observed with regard to levels of soluble intercellular adhesion molecule-1 (22), soluble vascular cell adhesion molecule-1 (22), and procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) (24). Both progestogens have a high affinity for progestogen receptors; however, dydrogesterone has no estrogenic or androgenic activity (29), whereas trimegestone has potent antiestrogenic (30) and antiandrogenic activity (31). This might explain part of the observed differences in their effects on various cardiovascular variables.
ADMA and SDMA are derived from the catabolism of proteins containing methylated arginine residues. When these proteins undergo hydrolysis, their methylated arginine residues are released. The observed decrease in ADMA might be the consequence of reduced protein methylation or a diminished catabolism of these proteins. The major pathway for elimination of ADMA is hydrolysis by DDAH, whereas renal clearance is the main mechanism for elimination of SDMA. Because changes in ADMA appeared to be larger than the changes in SDMA in the E2+T group, increased renal clearance is not the most likely explanation for the observed decrease in ADMA, whereas changes in DDAH activity seem a more plausible explanation. A decline in DDAH activity appears to be related to oxidative stress (32). When cultured endothelial cells are exposed to oxidized low-density lipoprotein (LDL), ADMA accumulates in the medium at a faster rate than during exposure to a vehicle or native LDL (33). Estrogens have been reported to increase the resistance of LDL to oxidation (34), and therefore the decrease in ADMA observed in our study could be the result of an increase in DDAH activity. On the other hand, recent data suggest that plasma ADMA levels are not influenced by aggressive lowering of LDL cholesterol by statin treatment (35, 36), making the link between DDAH activity and LDL less plausible. In the present study, baseline plasma cholesterol and ADMA concentrations were not correlated. We did not measure on-treatment cholesterol concentrations, and therefore it was not possible to correlate changes of ADMA with changes of cholesterol. However, in a previous study on the long-term effects of estrogen replacement therapy we did not observe such an association (19). Taken together, it is not likely that the reduction of plasma cholesterol plays a causal role in the ADMA-lowering effect of HRT. Another potential mechanism is based on the observation that DDAH activity in endothelial cells is inhibited by homocysteine (37). This mechanism could potentially provide a link between the homocysteine- and ADMA-lowering effects of HRT. However, in the present study no associations between baseline values of ADMA and homocysteine were found, and treatment-induced changes in these variables were also not related. Thus, it seems that this mechanism plays no substantial role in the present study. It should be noted, however, that our subjects had fasting homocysteine levels within the normal reference range, and therefore we cannot exclude an inhibitory effect of homocysteine on DDAH activity at pathological homocysteine levels.
Strikingly, estradiol combined with trimegestone lowered not only ADMA but also arginine levels, leading to a significantly reduced arginine/ADMA ratio. It is tempting to speculate that arginine levels were reduced by increased consumption of arginine for production of NO as a result of diminished inhibition of NOS by ADMA. There are indications that arginine imported by cells is preferentially used as a substrate for NOS. It has been shown that the arginine transporter and endothelial NOS are colocalized in plasma membrane caveolae, suggesting a direct transfer of extracellular arginine to NOS (38). Estrogen receptor 
has also been shown to be partly associated with caveolae in a functional complex with NOS (39, 40). At the moment, it is not clear whether reduced arginine levels cause a reduction of NO production by limiting substrate availability or are a reflection of increased NO production.
One limitation of the study is the partially blinded design. Investigators knew whether women were randomized to placebo or unopposed estradiol on the one hand or to one of the combined arms on the other hand. However, it is very unlikely that this seriously influenced the results, because none of the investigators or the laboratory personnel or the participants were aware of the exact medication.
In conclusion, oral E2, whether unopposed or sequentially combined with dydrogesterone or trimegestone, reduces plasma levels of the cardiovascular risk factor and NOS inhibitor ADMA. This effect was especially pronounced in the E2+T group and already apparent after 4 wk. Whether the reduction of the NOS substrate arginine in the E2+T group counteracts the potentially beneficial effect of ADMA reduction or reflects increased NO production remains to be investigated.
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Acknowledgments |
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Footnotes |
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Abbreviations: ADMA, Asymmetric dimethylarginine; ANCOVA, analyses of covariances; BMI, body mass index; CI, confidence interval; D, dydrogesterone 10 mg; DDAH, dimethylarginine dimethylaminohydrolase; E2, oral micronized 17ß-estradiol 2 mg; HERS, Heart and Estrogen/Progestin Replacement Study; LDL, low-density lipoprotein; NO, nitric oxide; NOS, nitric oxide synthase; SDMA, symmetric dimethylarginine; T, trimegestone 0.5 mg; WHI, Women’s Health Initiative.
Received April 3, 2003.
Accepted June 2, 2003.
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localized in caveolae. Biochem Biophys Res Commun 263:257–262[CrossRef][Medline]
and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87:e44–e52This article has been cited by other articles:
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  M. Zeller, C. Korandji, J.-C. Guilland, P. Sicard, C. Vergely, L. Lorgis, J.-C. Beer, L. Duvillard, A.-C. Lagrost, D. Moreau, et al. Impact of Asymmetric Dimethylarginine on Mortality After Acute Myocardial Infarction Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 954 - 960. [Abstract] [Full Text] [PDF]
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F. Mittermayer, K. Krzyzanowska, M. Exner, W. Mlekusch, J. Amighi, S. Sabeti, E. Minar, M. Muller, M. Wolzt, and M. Schillinger Asymmetric Dimethylarginine Predicts Major Adverse Cardiovascular Events in Patients With Advanced Peripheral Artery Disease Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2536 - 2540. [Abstract] [Full Text] [PDF]
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 D. Cevik, O. Unay, F. Durmusoglu, T. Yurdun, and A S. Bilsel Plasma markers of NO synthase activity in women after ovarian hyperstimulation: influence of estradiol on ADMA Vascular Medicine, February 1, 2006; 11(1): 7 - 12. [Abstract] [PDF]
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M. Reimann, J. Dierkes, A. Carlsohn, D. Talbot, M. Ferrari, J. Hallund, W. L. Hall, K. Vafeiadou, U. Huebner, F. Branca, et al. Consumption of Soy Isoflavones Does Not Affect Plasma Total Homocysteine or Asymmetric Dimethylarginine Concentrations in Healthy Postmenopausal Women J. Nutr., January 1, 2006; 136(1): 100 - 105. [Abstract] [Full Text] [PDF]
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R. Maas Pharmacotherapies and their influence on asymmetric dimethylargine (ADMA) Vascular Medicine, May 1, 2005; 10(2_suppl): S49 - S57. [Abstract] [PDF]
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T. Teerlink ADMA metabolism and clearance Vascular Medicine, May 1, 2005; 10(2_suppl): S73 - S81. [Abstract] [PDF]
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 H. Kirchherr and W. N. Kuhn-Velten HPLC-Tandem Mass Spectrometric Method for Rapid Quantification of Dimethylarginines in Human Plasma Clin. Chem., January 1, 2005; 51(1): 249 - 252. [Full Text] [PDF]
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 J. P. Cooke Asymmetrical Dimethylarginine: The Uber Marker? Circulation, April 20, 2004; 109(15): 1813 - 1818. [Full Text] [PDF]
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K. Tsuda, I. Nishio, D. P. Holden, J. E. Cartwright, G. St.J. Whitley, and S. S. Nussey Estrogen and Asymmetric Dimethylarginine in Postmenopausal Women * Response Circulation, March 30, 2004; 109(12): e174 - e174. [Full Text] [PDF]
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