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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1021
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2218-2224
Copyright © 2005 by The Endocrine Society

Oral Estradiol Decreases Plasma Homocysteine, Vitamin B6, and Albumin in Postmenopausal Women But Does Not Change the Whole-Body Homocysteine Remethylation and Transmethylation Flux

R. G. V. Smolders, K. de Meer, P. Kenemans, C. Jakobs, W. Kulik and M. J. van der Mooren

Project Aging Women, Institute for Cardiovascular Research-Vrije Universiteit, Departments of Obstetrics and Gynecology (R.G.V.S., P.K., M.J.v.d.M.) and Clinical Chemistry (K.d.M., C.J., W.K.), Vrije Universiteit University Medical Center, 1007 MB Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: M. J. van der Mooren, M.D., Ph.D., Vrije Universiteit University Medical Center, Department of Obstetrics and Gynecology, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: mj.vandermooren{at}vumc.nl.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Estrogens, both endogenous and exogenous, lower the fasting levels of the independent risk factor for cardiovascular disease homocysteine. The mechanism behind this observation remains unclear.

In a randomized, placebo-controlled, double-blind study, 25 postmenopausal women with a screening homocysteine concentration above 10 µmol/liter were included. We investigated the influence on homocysteine levels of a 3-month treatment with a daily oral dose of 4 mg 17ß-estradiol (ET) or 4 mg ET combined with 10 mg dydrogesterone (EPT); the comparison group received placebo treatment. We performed primed continuous infusions of L-[2H3-methyl-13C]methionine to assess steady-state flux rates of transmethylation, remethylation, and transsulfuration. Homocysteine concentration relationships with S-adenosylmethionine, S-adenosylhomocysteine, creatinine, albumin, vitamins B6 and B12, and folate status were determined as well. The mean change from baseline in homocysteine concentration by both treatments compared with placebo (ET, –13%; EPT, –10%) was accompanied by a decrease in the concentration of vitamin B6 (ET, –25%; EPT, –38%) and albumin (ET, –7%; EPT, –11%). No significant changes in flux rates were observed. In a multivariate analysis, changes in homocysteine concentration were related to changes in albumin concentration. No relation to other variables was observed.

We conclude that the ET- and EPT-induced homocysteine changes in this study were not accompanied by a significant change in methionine-homocysteine flux rates and hypothesize that an estrogen-induced lowering of homocysteine levels is primarily part of a change in albumin metabolism.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
HYPERHOMOCYSTEINEMIA AND EVEN mildly elevated levels of homocysteine are associated with an increased risk for cardiovascular disease (1). Several studies have shown that endogenous and exogenous female sex hormones in general (2) and postmenopausal hormone therapy in particular (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) can lower homocysteine. This decrease in plasma homocysteine observed with hormone therapy was found to be more pronounced in women with higher levels of homocysteine before treatment (3, 6). This effect may be negatively influenced by the addition of a progestogen, although this seems to depend on the type of progestogen used (10, 14). It is not known which metabolic pathways of homocysteine metabolism are modulated by hormone therapy.

Homocysteine is the transmethylated product of the essential amino acid methionine (Fig. 1Go). Methionine is transmethylated to homocysteine via S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), important regulatory intermediates in the methionine-homocysteine metabolism (15). Homocysteine levels are furthermore controlled by the rate of remethylation to methionine as well as by the rate of transsulfuration to cysteine. An increase in the flux rate of one of these pathways might lead to a lower homocysteine concentration.



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  		FIG. 1. Homocysteine metabolism, showing the three principal metabolic pathways. Right, Transmethylation pathway, the production of homocysteine from methionine. Bottom, Transsulfuration, breakdown of homocysteine and production of cysteine. Left, Remethylation (recycling) of homocysteine to methionine including the folate cycle, which acts as the methyl donor and provides the carboxyl group necessary to convert homocysteine into methionine. THF, Tetrahydrofolate; MS, methionine synthase; BHMT, betaine-homocysteine methyltransferase; CBS, cystathionine ß-synthase; B6, vitamin B6; B12, vitamin B12.

 
Remethylation requires vitamin B12 as a cofactor and 5-methyltetrahydrofolate as methyl donor. 5-Methyltetrahydrofolate is formed from 5,10-methylenetetrahydrofolate in a reaction catalyzed by methylenetetrahydrofolate reductase (MTHFR). Transsulfuration requires vitamin B6 as a cofactor and has cystathionine ß-synthase as the rate-limiting enzyme.

Because there may be therapeutic consequences if the estrogenic homocysteine-lowering effect works directly via one of the metabolic pathways or indirectly through modifying factors such as vitamin concentrations, protein metabolism, or renal function, we investigated metabolic rates. To determine whether and how the transmethylation of methionine and/or the remethylation and transsulfuration of homocysteine is modulated by postmenopausal hormone therapy, we used a validated stable isotope tracer technique (primed continuous infusion of L-[2H3-methyl-13C]methionine) (16). In a randomized, placebo-controlled double-blind study in postmenopausal women who had a fasting plasma homocysteine concentration of more than 1.35 µg/liter (10 µmol/liter), we investigated the effect of a 3-month treatment with daily oral 4 mg micronized 17ß-estradiol (ET), with or without daily addition of 10 mg dydrogesterone (PT), on the rate of transmethylation of methionine and remethylation and transsulfuration of homocysteine. Furthermore, we investigated changes in plasma homocysteine levels, vitamin B6, B12, and folate levels, SAM to SAH ratios, creatinine, and albumin levels.


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

To perform a sample size estimation, we extrapolated the differences in homocysteine concentrations (average 12%) as found in previous hormone therapy intervention studies (3, 6) and combined this with the variation in the L-[2H3-methyl-13C]methionine infusion method as applied in healthy volunteers in our clinic (17). This resulted in a sample size of eight in each group to detect a difference in flux rates of 10% among the groups with a power of 80% and {alpha} = 0.05.

Twenty-five postmenopausal women who had a screening fasting plasma homocysteine concentration higher than 1.35 µg/liter (10 µmol/liter) were included. They were recruited through advertisements in local newspapers between November 2000 and November 2001. After screening for homocysteine levels and medical history, eligible women underwent physical and gynecological examination.

The study conformed to the principles outlined in the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board. Written informed consent was obtained from each participant before entry into the study.

Participants had to be between 45 and 70 yr old, smoke less than 10 cigarettes per day, have a blood pressure below 160/100 mm Hg, and have a body mass index of 30 kg/m2 or less. Postmenopausal status was defined as the cessation of menstrual bleeding for more than 12 months and having serum FSH concentrations above 40 IU/liter and estradiol (E2) concentrations lower than 27.2 pg/ml (100 pmol/liter). Participants using drugs known to influence estrogen or progestogen metabolism were excluded. Postmenopausal hormone therapy users were eligible after a 4-month washout period. Use of other concomitant medication including B vitamins had to have remained unchanged for more than 3 months and during the course of the study. Exclusion criteria were a history of cardiovascular, metabolic, endocrinological (except for thyroid disease in a stable phase), and (pre-) malignant disease (except for successfully resected basal cancer of the skin), as well as clinically relevant abnormalities in laboratory tests of hematological, renal, and hepatic function or glucose metabolism or abnormalities on the transvaginal ultrasound, including an endometrial thickness of more than 4 mm.

Study design and intervention

The study consisted of two stable isotope infusion tests, 1 d before treatment onset and one in the 13th wk of treatment. In a double-blind fashion, eligible women were randomly assigned to once daily placebo (n = 9), oral 4 mg micronized ET (n = 8), or oral 4 mg micronized ET continuously combined with 10 mg PT (n = 8) (Zumenon and Femoston 2/10, Solvay Pharma, Weesp, The Netherlands). All tablets (including placebo) for a single dose were put into a capsule by the university hospital pharmacy. These capsules were of identical appearance.

After the last test, the women who were in the unopposed E2 treatment arm and were nonhysterectomized received a 14-d treatment with PT 10 mg daily to induce a withdrawal bleeding (n = 6) and thereby mimicking one 3-month sequentially combined hormone treatment cycle, which is considered not to increase the risk for endometrial dysplasia. Computerized randomization was done in blocks, which had a size of six. Allocation and management of the randomization codes was done by the university hospital pharmacy, keeping both patients and physicians blind to the treatment code. At the end of each individual’s intervention period, the pharmacy determined whether a 2-wk treatment with PT was necessary, thereby partly unblinding the treatment code to the physician. The complete code was broken and revealed to the researchers at the end of the study.

Stable isotope infusion technique

The infusion technique used includes L-[2H3-methyl-13C]methionine as a tracer and has been previously described by Storch et al. (16). This compound labeled with stable isotopes has a molecular mass of +4 Da compared with natural methionine; therefore, its relative abundance compared with natural methionine can be determined by gas chromatography (GC)-mass spectrometry (MS). When L-[2H3-methyl-13C]methionine is transmethylated (Fig. 1Go), [2H3]methyl is removed, which forms [1-13C]homocysteine. Remethylation of [1-13C]homocysteine results in [1-13C]methionine. If [1-13C]homocysteine is transsulfurated, the 13C atom leaves the cycle and reappears as 13CO2 in the expired air by oxidation of {alpha}-ketobutarate in the Krebs cycle.

Measuring the (un)labeled compounds in a steady state (during the continuous infusion in a fasting patient), the rate of disappearance is equal to the rate of appearance. Therefore, the whole-body protein synthesis/breakdown, remethylation, transsulfuration, and subsequently transmethylation can be determined. To reach steady states in the enrichments of L-[2H3-methyl-13C]methionine, L-[1-13C]methionine, and 13CO2 in a reasonable time, patients were given a priming dose of 13C-bicarbonate and L-[2H3-methyl-13C]methionine at the start of the experiment.

Stable isotope infusion protocol

One day before and in wk 13 of the treatment period, the infusion experiment was performed. Infusions were done after an overnight fast and started between 0730 and 0930 h. Patients were only allowed to drink small amounts of tap water. Throughout the experiment, the patients were kept on a bed. In each hand, an iv catheter was placed in a dorsal hand vein, one for blood sampling and one for the isotope infusion. By placing the hand used for blood sampling in a heating box for 5 min before the sampling, arterialized blood samples could be obtained (18). L-[2H3-methyl-13C]methionine and NaH13CO3 tracers (Mass Trace, Woburn, MA) were prepared in sterile water, as described previously (17). The infusion was started with a priming dose of 13C-bicarbonate (5.9 µmol) and L-[2H3-methyl-13C]methionine (2.9 µmol/kg) and then continued for 6 h (1.8 µmol/kg·h) with a calibrated precision infusion pump (Teruma, Tokyo, Japan).

Blood was collected in heparinized glass tubes (Becton Dickinson, Plymouth, UK), immediately placed on ice, and within 15 min centrifuged for 10 min at 3000 rpm at 4 C. End-tidal expired breath air samples were obtained by instructing the patients to exhale through a straw. The air was collected in a glass tube (plane venoject tube, Becton Dickinson), the straw was withdrawn from the tube during the last 3 sec of expiration, after which the investigator immediately closed the tube.

Samples (both blood and breath air) were taken before the start of the experiment (three samples), after 180 and 220 min and then every 20 min until 360 min of infusion. Furthermore, at baseline, separate blood samples were taken to determine the plasma concentrations of homocysteine, vitamins B6 and B12 and folate, albumin, creatinine, cholesterol, SAM, SAH, and MTHFR phenotype.

Laboratory analyses

MS. Isotopic enrichments in plasma and breath air were determined as described elsewhere (19). In short, the enrichments of [1-13C]methionine and [2H3-methyl-1-13C]methionine were measured by gas GC-MS with negative ion chemical ionization (HP5989B quadrupole GCMS, Hewlett Packard, Palo Alto, CA) from the acetyl-3–5 bis, trifluoromethylbenzyl derivate prepared from 500 µl plasma samples. Enrichments were calculated on the basis of the abundance relative to all measured methionine species: m + 0, m + 1, and m + 4 (16, 20). Calibration curves obtained by measurement of calibrators, containing weighted amounts of tracer and trace, were used to correct for minor instrumental variation. 13C-enrichment of carbon dioxide in breath samples was measured on a gas-chromatographic isotope-ratio mass spectrometer (BreathMATplus; Finnigan, Bremen, Germany). To establish the 13CO2 enrichment as an atom percent excess (APE), the isotope enrichment of the geometric mean of the three baseline samples was subtracted from each sample.

Clinical chemistry. Plasma homocysteine is defined as plasma total homocysteine measured as the sum of all homocysteine subfractions in plasma, including free and protein-bound forms. Total and free homocysteine were both measured using HPLC with fluorescence detection according to Ubbink et al. (21). The lower limit of detection was 0.2 µmol/liter, and the intra- and interassay coefficients of variation were 1.8 and 3.5%, respectively.

Vitamin B6 levels were determined by fluorescence HPLC (22). Vitamin B12 levels were measured by radioassay (ICN Pharmaceuticals, Costa Mesa, CA). The methionine concentration of the infusate was measured in each experiment using a standard amino acid analyzer equipped with a high-pressure analytical column packed with Utrapac 8 resin (Biochrom 20, Pharmacia Biochrom Ltd., Cambridge, UK).

SAM and SAH levels, both in plasma and in whole blood, were determined using tandem MS as described by Struys et al. (23) with intra- and interassay coefficients of variation of 4 and 7%, respectively. Plasma total cholesterol, folate, creatinine, and albumin were measured using standard laboratory methods.

A common less active variant of the enzyme MTHFR has been described (24). This thermolabile variant is the result of a C677T mutation and, depending on the expression, can lead to an impaired remethylation and thereby to a higher homocysteine level. Therefore, MTHFR status (MTHFR C677T polymorphism) was assessed in DNA obtained from the buffy coat of EDTA blood. The method described by Frosst et al. (24) was used but with a different sense primer (GCC AGC CTC TCC TGA CTG TC) to obtain a better separation of the fragments.

Serum FSH was determined with a specific immunometrical (fluorescence) assay (DelfiaWallac, Turku, Finland). Serum E2 was quantified by a double-antibody RIA (Sorin Biomedica, Saluggia, Italy) with a lower limit of detection of 18 pmol/liter.

Calculations

The calculations to determine the transmethylation, remethylation, and transsulfuration rates in steady state from the isotopic enrichments in methionine and breath CO2 during L-[2H3-methyl-13C]methionine infusion have been described previously (16, 17, 25).

Carbon dioxide production rate of each subject was estimated from the literature (26) using the subjects’ lean body mass (LBM). LBM was calculated from four skinfolds (Holtain, caliper, accuracy 0.1 mm), according to Durnin and Womersley (27).

The calculations and the variables are summarized in Table 1Go. All metabolization (flux) rates are expressed in micromoles per hour per kilogram, using measured weight or calculated LBM.


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  		TABLE 1. Parameters with explanations and equations

 
Statistics

Statistical analysis was performed using the Statistical Package for the Social Sciences 9.0 (SPSS Inc., Chicago, IL). Values are given as mean ± SD. We compared baseline measurements between groups using standard tests where applicable. Correlations between variables were calculated with Pearson’s or Spearman’s correlation coefficient. For the different variables, one-way ANOVAs, with Tukey post hoc test, were conducted for comparisons among and between the groups. A linear regression model was used to perform post hoc analyses to elucidate the influence of the different parameters on the subsequent outcomes. A two-sided P < 0.05 was accepted as the level of significance, except for the correlation analyses, where a two-sided P < 0.01 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
After screening 246 women on homocysteine levels and medical history, 28 underwent a physical examination screening. Twenty-five subjects were found eligible and were included. Main reasons for screening dropout were: homocysteine lower than 10 µmol/liter, blood pressure higher than 160/100 mm Hg, or not being postmenopausal.

One participant (EPT group) dropped out after 5 wk due to vaginal bleeding problems. All other randomized participants completed the study (see Fig. 2Go). Four participants used vitamin B supplements at onset of the study and continued their use during the study (one in the placebo group, two in the ET group, and one in the EPT group). Excluding these participants from the analyses provided similar results, both in mean values as well as in significance of the statistical comparisons (data not shown).



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  		FIG. 2. ET, Estrogen therapy; EPT, estrogen-progesterone therapy.

 
Baseline characteristics

Baseline characteristics are given in Table 2Go. Treatment groups were comparable for the main baseline characteristics except for weight and LBM. Therefore, the influence of these differences on all outcome parameters was tested by including weight or LBM as a (co-)variable in an ANOVA or linear regression models. It did not influence the results of the other variables or the result of the comparison among the groups. Therefore, we used a one-way ANOVA to do the final analysis of the main outcome parameters. Two subjects in the ET group were homozygous for the MTHFR C677T polymorphism. To test whether this influenced the outcome, we repeated the analyses without these two participants. This did not change the significance of the differences nor did this substantially change the mean values of the outcome parameters. Therefore these subjects were included in the final analyses. In the outcome parameters at baseline, there were small differences among the groups that were not statistically significant.


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  		TABLE 2. Baseline characteristics

 
Homocysteine, methionine, and SAM/SAH

In both treatment groups, total homocysteine decreased, with mean individual percentage reduction (SD): 13% (5%) in the ET and 10% (14%) in the EPT group (for absolute values, see also Table 3Go). The decrease was significant in both groups compared with placebo (ANOVA, P < 0.01; Tukey, ET vs. placebo, P < 0.01; EPT vs. placebo, P < 0.05). Differences between the two treatment groups were not statistically significant.


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  		TABLE 3. Baseline and posttreatment values

 
In unbound homocysteine concentrations, no significant differences were observed between the groups.

SAM, SAH, and SAM to SAH ratios both in plasma and in whole blood did not change significantly, nor were there any significant differences among the groups.

Vitamins

There were two women in the EPT group that had baseline vitamin B6 levels more than three times the SD above the baseline mean. For this analysis, they were considered outliers and were excluded. During treatment, vitamin B6 levels decreased significantly (ANOVA P = 0.03) in both treatment groups compared with placebo (ET, –25.4%; EPT, –38.9%; placebo, –2.6%). Including the outliers gave similar statistical comparisons, although the baseline mean in the EPT group was higher (68.7 pmol/liter; SD 60.4). Mean percentage change from baseline in vitamin B12 levels were similar to changes in vitamin B6 levels, but the difference was not statistically significant. Folate levels did not change significantly as well.

Proteins

Albumin levels changed statistically significantly different among the groups (ANOVA P < 0.01). Translating to a mean individual percentage change (SD) of –11.1% (8.3%) in the ET group and of –6.8% (8.7%) in the EPT group compared with 3.8% (7.7%) in the placebo group. The Tukey post hoc test showed this difference to be attributable to the difference between each intervention group compared with the placebo group (ET vs. placebo, P < 0.01; EPT vs. placebo, P < 0.05) and not to the difference between the treatment groups (P = 0.55).

Flux rates

Mean flux rates are shown in Table 3Go. Small nonsignificant changes compared with baseline were found in all groups. Significant differences among the three different groups were not found in any of the five calculated flux rates, neither in posttreatment values nor in absolute or relative changes.

Post hoc tests

To evaluate the parameters that most likely can explain the changes in homocysteine concentrations in this study, we performed a regression analysis. We used posttreatment homocysteine concentration as dependent variable, and in the regressive (backward) method, we used the independent variables baseline homocysteine, a (combined) treatment dummy, flux rates, B vitamins, and the proteins as well as albumin and creatinine levels. In this analysis, beside the treatment dummies, albumin proved to be the strongest predictor (P < 0.001). Moreover, the effect of ET or EPT on homocysteine was (statistically) absent when corrected for albumin change, i.e. albumin change was the only variable significantly predicting homocysteine levels.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
This study has three major findings. First, in postmenopausal women, fasting homocysteine concentrations decrease by more than 10% after a 12-wk treatment with daily oral 4 mg ET, which is not changed by the addition of EPT. Second, these homocysteine changes are not explained by a significant change in methionine-homocysteine metabolism flux rates. Third, the levels of vitamin B6 and albumin are lowered by both hormone treatments. Together, this might lead to the hypothesis that if estrogen-induced lowering of the homocysteine concentration has a cardioprotective effect, this may result from actually decreasing the exposure to homocysteine and not from altering a common underlying cause of elevated homocysteine levels.

Mechanisms by which homocysteine induces cardiovascular disease remain a topic of interest. If the underling cause of (mildly) elevated homocysteine proves to be important in that respect, the mechanism by which it is lowered will very likely be important as well (28, 29). In that context, the L-[2H3-methyl-13C]methionine infusion method is an elegant but elaborate test to investigate the actual flux rates of the methionine-homocysteine metabolism. Nevertheless, this study has a few characteristics that limit the possibility to extrapolate the results. It investigated short-term therapy (3 months), and it was limited to women with a moderately elevated basal homocysteine level. Furthermore, we cannot exclude the possibility that we missed a smaller but real difference in flux rate changes. However, the flux rates did not show any statistical trend toward a treatment effect, nor did they correlate with the change in homocysteine. Finally, the nonstatistically significant flux rate increases observed in the ET group were not present in the EPT group. This makes it even more unlikely that a change, if any, in the flux rates of one of the metabolic pathways can explain the differences in estrogen-induced changes in homocysteine.

The rate of methylation, and thereby the flux rate of the transmethylation pathway, was one of the processes thought to be influenced by estrogen. This hypothesis was not supported by changes in SAH or SAM levels or ratios, which further excludes a specific role of estrogen in these metabolic pathways. Furthermore, the few papers cited in which a direct estrogen- or progestogen-induced methionine-homocysteine metabolism enzyme induction is hypothesized are already over 15 yr old (30, 31). Moreover, although data are limited, steady-state flux rates in transmethylation, transsulfuration, and remethylation do not seem to differ between men and women when corrected for body mass (17, 25).

An alternative hypothesis might be that estrogen-induced plasma homocysteine changes are secondary to other estrogen-induced biological changes, such as changes in vitamin B levels and general anabolic/catabolic effects including changes in albumin levels (32, 33, 34, 35). The post hoc analyses suggested that all three might be important here. First, vitamin B6 levels decreased during treatment, an effect that has also been shown in oral contraceptive users (36). This might seem contradictory because vitamin B6 is a cofactor in the transsulfuration pathway, which thereby could be impaired as well. Nevertheless, because it is merely a c-factor and levels stayed well within the normal range, it is unlikely that it was a rate-limiting factor. However, it remains uncertain whether it does not lead to increased homocysteine levels after high methionine intake, i.e. by a oral methionine loading test as suggested in earlier studies (37, 38).

Second, albumin levels decreased. This might suggest a general change in protein synthesis. Alternatively, a decrease can be the result of a local or systemic proinflammatory response to E2 similar to the effect on C-reactive protein because albumin is a negative acute-phase reactant (39, 40). Finally, the decrease in albumin might be a dilutional effect, although this is less likely because the packed cell volume to hemoglobin ratio is not reported to change during hormone treatment. In contrast to other studies (35, 41, 42), creatinine was not a predictor of homocysteine or, more importantly, homocysteine changes. Although in part this may be a power issue (41, 42), it may also be caused by the fact that in our study, subjects only underwent an increase in estrogen concentrations and not a decrease in androgen concentrations comparable with the transsexual subjects studied by Giltay et al. (35).

Third, the strongest indication for an indirect effect of estrogen on homocysteine levels is the correlation of albumin levels both with hormone treatment and homocysteine levels or homocysteine changes. Moreover, when corrected for albumin changes, the treatment effect of hormone therapy disappeared completely. Alongside a possible general protein turnover effect of estrogen, this may be further explained by the fact that a large portion of homocysteine (> 80%) is protein bound, mainly to albumin, and only 1–2% is completely unbound (43, 44). Furthermore, albumin can bind exactly one thiol (homocysteine or cysteine) molecule per albumin molecule, and the mean percentage change of both variables is comparable in our study (45). Thereby, a decrease of albumin levels might result in a similar decrease in total homocysteine levels as observed in our study. This is supported by the fact that levels of unbound homocysteine did not change.

Whether or not these results provide a part of the explanation why no decrease in cardiovascular risk is observed in clinical trials such as the women’s health initiative (46) depends on the role homocysteine plays in the cardiovascular pathogenesis. If a high homocysteine concentration is only an indirect marker of a pathophysiological process that is not influenced by estrogen, the benefit will very likely be limited. If the mere presence of homocysteine in itself induces cardiovascular disease than it is plausible to expect a benefit of lowering homocysteine concentrations with postmenopausal hormone therapy.

In conclusion, this study shows that an estrogen-induced decrease in homocysteine, albumin, and vitamin B6 levels is not accompanied by a significant change in steady-state flux rates when investigated with the L-[2H3-methyl-13C]methionine primed continuous infusion technique. This provides no support for a direct effect of postmenopausal hormone therapy on the cellular metabolism of homocysteine. The data do support the hypothesis that the ET- and EPT-induced homocysteine changes result from a change in albumin metabolism and not from an alteration in methionine-homocysteine turnover.


   Acknowledgments
 
We thank D. E. C. Smith, laboratory technician, for her valuable laboratory assistance and T. E. Vogelvang, M.D., for her assistance in screening the patients and performing the tests.


   Footnotes
 
This work was supported by Research Grant 99-043 from the Biocare Foundation.

First Published Online January 25, 2005

Abbreviations: APE, Atom percent excess; E2, estradiol; EPT, ET plus PT; ET, 17ß-estradiol; GC, gas chromatography; LBM, lean body mass; MS, mass spectrometry; MTHFR, methylenetetrahydrofolate reductase; PT, dydrogesterone; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethione.

Received May 29, 2004.

Accepted January 14, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG 1995 A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 274:1049–1057[Abstract]
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