From the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA.
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ABSTRACT |
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cardiovascular diseases; estrogen replacement therapy; homocysteine; menopause; premenopause
Abbreviations: FSH, follicle-stimulating hormone; NHANES III, Third National Health and Nutrition Examination Survey.
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INTRODUCTION |
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Data from the Third National Health and Nutrition Examination Survey (NHANES III) provide a unique opportunity to explore the variation of homocysteine concentration with estrogen status in a large representative sample of the general US population, across a broad spectrum of ages. Our earlier analyses of NHANES III data confirmed in the US population male-female differences in homocysteine concentration from adolescence on, as well as an abrupt increase in homocysteine concentration in women after age 50 years that may indicate an effect of menopause (4). These findings suggest the hypothesis that estrogen status modulates homocysteine concentration, but the sex difference could also be attributable to male-female differences in muscle mass (7
) or nutritional status (12
). In this report, we address the estrogen hypothesis specifically by stratifying women according to available indicators of estrogen status and performing comparisons of homocysteine concentration among women of higher inferred estrogen status, women of lower inferred estrogen status, and men. These comparisons are performed after controlling for alternative explanations for between-group differences, such as serum vitamin levels, markers of muscle mass, and age.
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MATERIALS AND METHODS |
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Homocysteine concentrations were measured as an NHANES III (phase 2), 19911994, surplus sera project on serum samples derived from participants aged 12 years. All sample members were invited to undergo a physical examination that included phlebotomy. Of the 13,635 phase 2 sample members aged
12 years, 10,280 responded to the invitation to be examined, and homocysteine measurements were obtained for 8,585 (64 percent). Missing homocysteine measurements for examined individuals resulted from the failure to obtain a blood sample or from a lack of surplus sera.
Homocysteine measurement
Blood was drawn and processed in mobile examination centers under controlled, constant environmental conditions according to a standard protocol (15). Participants had fasted for varying lengths of time, but analyses demonstrated that length of fast had no measurable effect on homocysteine concentrations. Whole blood, which was not treated with anticoagulant, was collected in serum separator tubes and was held at room temperature for 3060 minutes before centrifugation. Sera were separated, frozen at -20°C, and transferred on dry ice to the Centers for Disease Control and Prevention's central laboratory for priority analyses. Samples went through 14 freeze-thaw cycles. After priority analyses were completed, additional analyses were carried out subject to approval by the Surplus Sera Bank Steering Committee. The surplus sera were stored at -70°C for 8 months to 3 years before being analyzed for total homocysteine concentration. Plasma samples frozen for up to 10 years have been shown to be acceptable for characterizing a person's plasma total homocysteine concentration at the time the samples were drawn (16
). Serum total homocysteine concentration refers to the combined concentration in serum of trace amounts of the free amino acid homocysteine, oxidized disulfides comprising a molecule of homocysteine bound to a molecule of another sulfur-containing amino acid (about 30 percent), and protein-bound homocysteine (about 70 percent) (2
). The serum total homocysteine concentration was measured by the high-performance liquid chromatography method of Araki and Sako (17
) at the US Department of Agriculture Human Nutrition Research Center on Aging after approval by the New England Medical Center Human Investigations Review Committee.
Subject classification
For data analyses, subjects were first stratified by age and then by inferred estrogen status within age group. Age groups corresponded to the years near menarche (ages 1216 years), the childbearing years (ages 1744 years), the perimenopausal years (ages 4554 years), middle age (ages 5569 years), and old age (70 years). The question of interest for each age range concerned whether the geometric mean serum total homocysteine concentration varied according to estrogen status. Estrogen measurements were not available, however, and estrogen status was inferred from sex, serum concentration of follicle-stimulating hormone, where available, and interview responses regarding various aspects of reproductive history (table 1).
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Women 17 years old were classified as pregnant under the following two circumstances: 1) if they said they were pregnant in response to a specific interview item and 2) if a urine pregnancy test (given to examined women aged 2049 years) gave a positive result. Use of estrogen supplements and oral contraceptives was defined as self-report of current use (dose information was not available). Women in the age range 1734 years who provided menstrual histories and did not report either a double oophorectomy or the combination of at least 3 months of amenorrhea and a single oophorectomy were considered premenopausal. Ninety-five percent of those considered premenopausal reported that their last menstrual period had occurred <2 months prior to the interview. Of the 74 women aged 1734 years who were considered premenopausal and had reported that their last menstrual period had occurred
2 months prior to the interview, eight had had surgery to remove the uterus but not the ovaries, 28 had been pregnant during the year prior to the interview or were breastfeeding at interview, 21 had had a menstrual period within 3 months of the interview, and one had apparently unexplained amenorrhea for a year prior to the interview but had been pregnant within 2 years of the interview. Only three of the remaining 16 had a body mass index between 19 and 26 kg/m2 (for six subjects, the body mass index was >40; for two, it was <18), but seven of the 16 had menstruated within 6 months of the interview, and another two had menstruated within a year of the interview.
Serum concentrations of follicle-stimulating hormone were used to classify women aged 3554 years as either "premenopausal" (follicle-stimulating hormone (FSH) 35 IU/liter) or "postmenopausal" (FSH > 35 IU/liter). All women aged 55 years and older were considered postmenopausal unless the follicle-stimulating hormone concentration was available and
35 IU/liter. For 90 of the 1,404 women aged
55 years, neither the reproductive history nor the follicle-stimulating hormone concentration was available. However, the follicle-stimulating hormone concentration was missing for only one woman aged 5560 years. Furthermore, only three of 198 women aged 6165 years reported that they had menstruated in the past 2 years.
Statistical analysis
To account for the complex NHANES III survey design (i.e., a staged sampling scheme and unequal probability of selection and nonresponse), data were analyzed using SUDAAN statistical software (18), and sample weights were incorporated into analytical procedures. Because total homocysteine concentrations were extremely skewed, the values were logarithmically transformed before being used in data analyses. The SAS system (19
) was used to create and manipulate the data files. A p < 0.05 was considered statistically significant for all analyses.
Preliminary data analyses accomplished two goals: 1) the description of subjects stratified by age group and inferred estrogen status as to sociodemographic characteristics, body composition indicators, and status as to vitamin B-12, folate, and cigarette smoking; and 2) the evaluation of univariate relations between these factors and the two main variables of interestthe serum total homocysteine concentration and estrogen status category. All of these variables were considered potential confounders of the relation between serum total homocysteine concentration and estrogen status based on one or more of the following three criteria: 1) relation to total homocysteine concentration demonstrated in previous studies (i.e., age, creatinine, vitamin B-12, folate, and cigarette smoking), 2) consistent consideration as a potential total homocysteine correlate in previous studies (i.e., body mass index), or 3) status as a major sociodemographic characteristic not previously well studied (i.e., race-ethnicity). Depending on the scale of the potentially confounding variable (i.e., continuous or categorical), linear regression (SUDAAN PROC REGRESS) or cross-tabulation (SUDAAN PROC CROSSTAB) procedures were used to perform the two preliminary data analytical functions. Means were compared via F tests; contingency tables were analyzed via chi-square tests.
Although smoking data were collected from all subjects, in the age group <17 years, only seven subjects, none of whom was in the premenarche category, were regular pack-a-day smokers. Consequently, potential confounding by smoking in this age group was handled by the elimination of the seven regular smokers from data analyses. An additional 157 subjects in various age ranges were excluded from data analyses because of missing information on potential confounders. Women were excluded from data analyses if insufficient data were collected to infer their estrogen status (n = 37). NHANES III participants excluded for this reason included one subject aged <17 years, 34 subjects aged 1744 years, and two subjects aged 4554 years. Female NHANES III participants in estrogen status categories that were rare in a particular age range but common in another age range were also excluded. More specifically, we excluded pregnant girls aged <17 years (n = 6), apparently postmenopausal women aged 1744 years (n = 74), and apparently premenopausal women aged 55 years (n = 21). The final proportion of the phase 2 sample members included in data analyses was 61 percent (n = 8,384).
The relation between the serum total homocysteine concentration and the estrogen status category was analyzed via multiple linear regression analysis using SUDAAN PROC REGRESS. Point estimates and 95 percent confidence intervals of the geometric mean (inverse of the logarithmic mean) total homocysteine concentration were calculated for various estrogen status categories from the least-square means and standard errors generated by the regression procedure. Three separate models were run to adjust for potentially confounding factors. The basic model included age (to adjust for any residual confounding by age within age strata) and race-ethnicity. The intermediate model included the basic model variables plus cigarette smoking, body mass index, and serum creatinine. The full model included the intermediate model variables plus serum concentrations of vitamin B-12 and folate. We explored possible effect modification by race-ethnicity, cigarette smoking, and vitamin status by including appropriate interaction terms individually in the full model. Initially, means and 95 percent confidence intervals were reported without regard to possible effect modifiers. However, after the serum folate concentration was identified as a modifier of the effect of sex on the serum total homocysteine concentration at ages consistent with women's childbearing years, the folate distribution for that age range was divided into fifths, and confounder-adjusted mean serum total homocysteine concentrations and associated 95 percent confidence intervals were generated for premenopausal women and men separately for each of the five serum folate categories.
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RESULTS |
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In the age range 1744 years, pregnant women (n = 135) and users of oral contraceptives (n = 366) were younger than subjects in other estrogen status categories. Men in this age range (n = 1,764) were more likely to smoke than were women in all subgroups. Oral contraceptive users had a lower body mass index than subjects in other subgroups and, compared with other nonpregnant women (n = 1,751) and men, they were of lower vitamin B-12 status and more likely to be non-Hispanic White. Pregnant women were of higher folate status than subjects in the other subgroups.
In the age range 4554 years, women who were postmenopausal (n = 168) or receiving estrogen replacement therapy (n = 87) were older than other women (n = 219) and men (n = 354). Women in this age range tended to smoke less than the men, but only the premenopausal women smoked statistically significantly less. Finally, estrogen users were of somewhat higher folate status than were members of other subgroups.
In the age range 5569 years, estrogen users (n = 103) differed from members of other subgroups; they were statistically significantly younger than non-estrogen-using postmenopausal women (n = 562), and they were of higher folate status and more likely to be non-Hispanic White than both the other women and the men (n = 638).
Among NHANES III participants aged 70 years, non-estrogen-using postmenopausal women (n = 670) were older than the men (n = 524), but estrogen-using women (n = 48) were not. Elderly estrogen users were of somewhat higher folate status than the other women and the men, but in this age range, it was the non-estrogen-using women who had the highest serum vitamin B-12 concentrations. Estrogen users in this age range were statistically significantly less likely to smoke than were members of the other subgroups and, similar to the middle-aged estrogen users, they were more likely than members of the other subgroups to be non-Hispanic White.
To summarize, each one of the potentially confounding factors was statistically significantly related to inferred estrogen status in at least one age stratum, and all but body mass index were statistically significantly related to serum total homocysteine concentration in at least one age stratum.
The basic multivariate model (table 3) revealed no statistically significant difference in the serum total homocysteine concentration between girls who had and had not passed menarche, but the menstruating girls alone had lower total homocysteine concentrations than the boys. In the age range 1744 years, the total homocysteine concentrations of women, whether or not they were oral contraceptive users, were significantly lower than those of men, and the total homocysteine concentrations of pregnant women were significantly lower than those of not only the men but also the nonpregnant women.
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Changes from the basic to the intermediate model resulted mainly from the inclusion of a term for serum creatinine in the model, which tended to bring the means for men and women closer together. Nevertheless, the total homocysteine concentrations of premenopausal women, pregnant women, and women receiving estrogen replacement therapy continued to be significantly lower than those of the men.
Addition of terms for serum concentrations of folate and vitamin B-12 to the multivariate model resulted in somewhat higher mean total homocysteine estimates for premenarcheal girls, pregnant women, estrogen users, and all categories of elderly subjects. However, the between-group differences noted with simpler models tended to be maintained. To summarize, the serum total homocysteine concentrations of menstruating women in all age ranges were lower than those of comparably aged men. Furthermore, in those age ranges that included postmenopausal women, the means of postmenopausal women not receiving estrogen replacement therapy were comparable with those of men and higher than those of women taking estrogen supplementation. Finally, among women in their childbearing years, pregnant women had serum total homocysteine concentrations that were much lower than those of subjects in all other estrogen status categories, but the serum total homocysteine concentrations of oral contraceptive users were indistinguishable from those of other nonpregnant women of reproductive age.
The between-group differences we found were not modified by race-ethnicity, cigarette-smoking status, or vitamin B-12 status. Serum folate status did affect the total homocysteine advantage of premenopausal women over men, however. More specifically, when men of low folate status aged 1744 years were compared with premenopausal women who were also of low folate status, the serum total homocysteine concentrations of the men and women were very similar (figure 1). When men and premenopausal women of higher folate status were compared, however, their serum total homocysteine concentrations differed.
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DISCUSSION |
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Unlike estrogen supplementation, oral contraceptive use appeared to have no effect on the serum total homocysteine concentration, a finding that has been obtained by others (20, 21
). van der Mooren et al. (22
) have pointed out that contemporary oral contraceptives usually contain low doses of synthetic estrogens, whose effects may be different from those of natural estrogens. Another factor to keep in mind is that oral contraceptives are used by women of childbearing age, whose estrogen status is already high. Whether oral contraceptive use would offer an advantage to elderly women is unknown.
That pregnant women have very low circulating homocysteine concentrations has long been known (8). Walker et al. (23
) recently found that folate supplementation in pregnancy contributed to the low levels, but we showed that the high folate status of pregnant women did not entirely explain the difference between pregnant and nonpregnant women. Although pregnant women have higher estrogen levels than men and nonpregnant women, their condition is unique in many other ways that might account for their low serum total homocysteine concentrations. Walker et al. (23
) suggested that homocysteine levels may decrease in pregnancy because of utilization by the fetus, a hypothesis raised by the observation of a decreasing plasma total homocysteine gradient from the maternal vein to the umbilical vein to the umbilical artery. On the basis of animal studies, Kim et al. (24
) hypothesized that cortisol, which rises during pregnancy, might also contribute.
Our findings regarding a possible effect of menopause on total homocysteine concentrations were consistent with those of Wouters et al. (25), who found higher plasma homocysteine concentrations in postmenopausal versus premenopausal women in a comparison that could not exclude age as a possible explanation for the difference. That study also demonstrated a negative association between serum concentrations of 17ß-estradiol and postmethionine plasma homocysteine among the 46 premenopausal subjects. A Swedish population-based study of men and women aged 3595 years (7
) revealed stable plasma total homocysteine concentrations in women from the age range 3549 years to the age range 5064 years but rapidly increasing concentrations thereafter. Men's total homocysteine concentrations, on the other hand, increased with each successively higher age range. These results were similar to those reported from our group's earlier analyses of NHANES III data (4
). Brattstrom et al. (7
) concluded that homocysteine values did not increase with age in women in a manner that suggested an effect of menopause. However, our results and those of Brattstrom et al. could be interpreted as indicating that women's total homocysteine concentrations, unlike men's, are kept from rising until an age range when all of the women would have passed menopause. Both the data from Brattstrom et al. (7
) and our results from the current investigation showed that, after the age of menopause, any difference in total homocysteine concentration between men and at least non-estrogen-using women was accounted for by the higher serum creatinine concentrations of men. An analysis of the Framingham study data attributed a male-female difference in total homocysteine concentration observed among elderly cohort members to a sex difference in B-vitamin status (12
), but neither the serum creatinine concentration nor postmenopausal estrogen use was considered in that study.
Both a descriptive study recently completed by our group (26) and a study of Belgian school-age children (21
) sought to provide reference ranges for circulating total homocysteine concentration in children and adolescents. To our knowledge, ours was the first attempt to specifically link menarche to the onset of male-female differences, however. Authors of the Belgian study reported a sex difference in plasma total homocysteine concentration among subjects aged 1519 years but not in younger age ranges. The NHANES III data revealed statistically significant male-female differences in all age ranges, but the two studies were not completely comparable. The next younger age range of Belgian children to the age range 1519 years included boys and girls as young as 10 years, 2 years younger than the youngest NHANES III participants whose total homocysteine concentrations were measured. While the age range 1014 years certainly included girls who had not passed menarche, we identified only 68 female NHANES III participants who had not begun to menstruate, and some of those were undoubtedly on the verge of menarche. Taken together, results of the Belgian study and our finding of a clear male-female difference only between boys and menstruating girls aged <17 years remain suggestive of an effect of menarche, but further study is needed.
We found that the total homocysteine advantage to premenopausal women versus similarly aged men was restricted to a subgroup with higher folate status. On the other hand, the total homocysteine benefit enjoyed by elderly estrogen users over other postmenopausal women did not seem to be affected by folate status. Data graphed in figure 1 showed that the benefit of being a premenopausal woman was realized at serum folate concentrations other than those consistent with folate deficiency (serum folate concentration < 6.8 nmol/liter) or very low folate status. The 20th percentile of the folate distribution of women aged 70 years (the age range for which estrogen users had lower serum total homocysteine concentrations than non-estrogen users) was 10.2 nmol/liter, and only five estrogen users had concentrations that low. Our data thus did not permit an analysis of the effect of folate deficiency or very low folate status on the relation between estrogen supplementation and the serum total homocysteine concentration.
Several mechanisms have been hypothesized for estrogenic effects on homocysteine metabolism. Boers et al. (5) have suggested that methionine may be catabolized more in premenopausal women than men by a methionine transamination pathway that does not result in homocysteine accumulation. In support of their hypothesis, this team of investigators reported higher concentrations of transamination metabolites in premenopausal women versus young men (6
). How estrogen per se might stimulate the transamination pathway (as opposed to the well-known transsulfuration pathway) has yet to be suggested nor has transamination activity been demonstrated to differ between pre- and postmenopausal women or between estrogen-supplemented and nonsupplemented postmenopausal women. Animal studies have supported the notion that estrogen might prevent hyperhomocysteinemia through increased methionine synthase activity in the kidney (24
), and Boers et al. (5
) have pointed out that the noted variation in homocysteine concentration with estrogen status could also be explained by variation in transsulfuration or renal excretion rates.
This study had many strengths. Among them were the very large sample size, the broad age range of the subjects, the representation by the sample of the US population, the several estrogen status categories that could be distinguished, the data on follicle-stimulating hormone (used as an indicator of menopause in the perimenopausal age range), and our ability to control for several potentially confounding factors.
The chance for information bias to have affected the results was minimal given our infrequent reliance on other than blood data for assessment of status as to the dependent variable, independent variables, and possible confounders of the relation between the two. We used menstrual histories provided by female subjects for assessment of menopausal status, but only in an age range when being postmenopausal would be unlikely. We included as "premenopausal" some young women with unexplained amenorrhea who may have been of low estrogen status; however, excluding these women from analyses had no effect on results. Hormone use was entirely self-reported, but reports were likely to be accurate, especially since current use was of interest. Among potential confounders, only smoking status, age, and race-ethnicity were reported by the subjects.
Despite the study's strengths, we cannot definitively attribute the observed differences to estrogen without actual estrogen measurements. On the other hand, no known homocysteine correlate was left uncontrolled. Three variables we considered in addition to those controlled for in the full multivariate model were region of the country, urban-rural character, and season when the examinations and interviews took place. Adding terms for these factors to the full multivariate model had no effect on the results.
The response rate for homocysteine measurements was 64 percent, and a small number of additional subjects were lost because of missing information on estrogen status indicators or potentially confounding factors. Comparisons between subjects included in the data analyses and interviewed subjects excluded for missing information revealed few differences; however, included subjects were younger and less likely to have had a heart attack than subjects who were merely interviewed. Both heart attacks and age are related to total homocysteine concentration (1, 2
, 4
), and physicians' prescription of estrogen could be related to heart attack history. It is thus reassuring that results did not change when analyses were restricted to those who had not had heart attacks or strokes. Also relevant to the difference we found between estrogen users and nonusers is the consistency of that finding with results of small clinic investigations (22
, 27
), one large uncontrolled study (28
), and a controlled trial (29
). After reviewing these studies, van der Mooren et al. (11
) recently concluded that homocysteine concentrations can be reduced by postmenopausal estrogen replacement therapy. Oral estrogen was also found to be effective in reducing the circulating homocysteine concentrations of 22 elderly men (30
).
This study was the first to look at indicators of estrogen status through the majority of the life cycle in a large population-based study, and results were generally consistent with the hypothesis that estrogen status is inversely related to the circulating total homocysteine concentration. Future investigators of this relation should attempt to link actual estrogen measurements to total homocysteine measurements and to specific metabolic pathways that might result in decreased homocysteine production or increased homocysteine clearance from the circulation.
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ACKNOWLEDGMENTS |
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The authors would like to acknowledge the expert assistance of Dr. Andrew Bostom, who reviewed and commented on the final manuscript.
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NOTES |
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REFERENCES |
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