Homocysteine and Blood Pressure in the Third National Health and Nutrition Examination Survey, 19881994
Unhee Lim and
Patricia A. Cassano
From the Division of Nutritional Sciences, Cornell University, Ithaca, NY.
Received for publication February 21, 2001; accepted for publication July 22, 2002.
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ABSTRACT
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Studies suggest that homocysteine may elevate blood pressure and increase the risk of hypertension. The association of homocysteine with blood pressure and with the risk of hypertension was investigated using cross-sectional data from the Third National Health and Nutrition Examination Survey (19981994). Homocysteine had an independent positive association with blood pressure after adjusting for cardiovascular risk factors. A 1 standard deviation (
5 µmol/liter) increase in homocysteine was associated with increases in diastolic and systolic blood pressure of 0.5 and 0.7 mmHg, respectively, in men and of 0.7 and 1.2 mmHg in women. Similarly, higher levels of homocysteine were associated with an increased risk of hypertension. In a comparison of the highest and lowest quintiles of homocysteine, women had a threefold increase in the risk of hypertension (95% confidence interval (CI): 1.7, 5.4), and men had a twofold increase (95% CI: 0.7, 5.1). In light of the homocysteine-blood pressure association, the association of homocysteine with prevalent cardiovascular disease was examined with and without adjusting for blood pressure. The results support a mediating role for blood pressure in women and suggest that the full effect of homocysteine on cardiovascular risk may be underestimated when blood pressure is adjusted.
biological markers; blood pressure; cardiovascular diseases; folic acid; homocysteine; hypertension; pyridoxine
Abbreviations:
Abbreviations: CI, confidence interval; DASH, Dietary Approaches to Stop Hypertension; NHANES III, Third National Health and Nutrition Examination Survey.
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INTRODUCTION
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High blood pressure is a major risk factor for cardiovascular disease. Although its etiology has not been fully elucidated mostly because of as yet unknown genetic variation, multiple nonhereditary factors including dietary and other lifestyle factors have been identified that have important and modifiable influences on blood pressure (1). Nutritional advice for the control and prevention of hypertension is already substantial (2), but additional understanding of the causal role of nutritional factors is important given the high prevalence of elevated blood pressure. Recent studies suggest that mild elevations in serum homocysteine may contribute to elevations in blood pressure. Although controversy remains as to whether the relation between homocysteine and cardiovascular disease is causal or not (3, 4), the association of homocysteine with blood pressure deserves attention because blood pressure may mediate part of the cardiotoxic effect of homocysteine. Epidemiologic studies in the past that controlled for blood pressure or hypertension status may have underestimated the total effect of homocysteine on cardiovascular disease risk.
A causal link between homocysteine and blood pressure is supported by experimental studies. Animal studies report an increase in blood pressure in response to induced hyperhomocysteinemia (5, 6). Homocysteine may elevate blood pressure through multiple mechanisms, including its effect on vascular endothelial integrity. Homocysteine administration caused direct endothelial cell injury in vitro (7) and in animals (8). In cell culture studies, homocysteine induced oxidative stress to endothelium (9) and reduced available nitric oxide, a potent vasodilator (10). Confirming these observations in humans, endothelium-dependent vasodilation was impaired in temporary (11, 12) or chronic (13, 14) hyperhomocysteinemia. Homocysteine-induced endothelial dysfunction in humans was improved with administration of folic acid and vitamin B6 (1517).
Folate, vitamin B6, and vitamin B12 are the major nutritional determinants of homocysteine levels (18) and have a protective association with cardiovascular disease (1924). The protective effect of these B vitamins may be partly explained by mechanisms independent of homocysteine, as suggested by several recent studies (25), and the B vitamins may themselves affect blood pressure (2630).
Although the association of homocysteine with blood pressure or hypertension risk has been reported in observational epidemiologic studies (3140), most studies were limited by incomplete consideration of potential confounding factors (3135) or specific characteristics of study subjects, such as age and pregnancy (36, 37). The few studies that considered confounding factors provided little quantitative information on the homocysteine-blood pressure association or considered blood pressure as a determinant of homocysteine (3840).
Thus, the relation of homocysteine to blood pressure and to hypertension was investigated using data from the Third National Health and Nutrition Examination Survey (NHANES III, 19881994). First, the homocysteine-blood pressure association was assessed, with and without consideration of folate and vitamins B6 and B12. Second, the association of homocysteine with prevalent hypertension was investigated. Finally, the association of homocysteine with prevalent cardiovascular disease was assessed to consider whether the association was partly mediated by blood pressure.
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MATERIALS AND METHODS
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Study population
Data on adults aged 17 or more years from a nationally representative sample of the noninstitutionalized US population were available from NHANES III (41, 42), a large-scale cross-sectional survey. The plan and operation of the survey have been described by the National Center for Health Statistics (43). The Cornell University Committee on Human Subjects approved the use of these public-domain data.
Data collection
Six seated measurements were taken for diastolic and systolic blood pressure on two occasions: three during the household interview and three during the medical examination. Trained interviewers and physicians took measurements using a mercury sphygmomanometer (W. A. Baum Co., Inc., Copiague, New York) according to standardized blood pressure measurement protocols recommended by the American Heart Association (43). The average of all available measurements was used in this study. A person was considered to have hypertension if blood pressure met the definition of stage 2 hypertension: elevated systolic blood pressure (
160 mmHg), elevated diastolic blood pressure (
100 mmHg), both, or use of antihypertensive medications (44, 45).
Blood was collected and sera were stored at 70°C until analysis. Homocysteine was measured only in the second phase of the survey (19911994) by high-performance liquid chromatography (coefficient of variation, <6 percent) at the US Department of Agricultures Human Nutrition Research Center on Aging (46). Other biologic markers of nutritional status, including B vitamins, antioxidants, and lipids, were assayed at the National Center for Environmental Health (46). Questionnaires filled out at the household interview and medical examination provided information on demographics, dietary intake, and lifestyle factors.
Statistical analysis
With the use of linear (continuous blood pressure outcome) and logistic (dichotomous hypertension outcome) regression analyses, men and women were considered in separate models after statistically significant effect modification of the homocysteine-blood pressure association by sex was found in a combined model. Sex-specific models also facilitated the consideration of sex-specific confounding variables. Metabolic (serum homocysteine, log transformed) and nutritional (red blood cell folate, serum vitamin B12, dietary intake of vitamin B6) variables were considered alone and simultaneously to allow for direct and indirect effects. All regression models were adjusted for the complex sampling design by SUDAAN software (Research Triangle Institute, Research Triangle Park, North Carolina), and analyses were weighted according to National Center for Health Statistics guidelines (47) to account for oversampling of certain age, sex, race/ethnicity domains, and differential nonresponse or undercoverage. In all models, potential confounding variables were carefully considered in light of proposed mechanisms.
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RESULTS
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NHANES III data on 7,612 adults with homocysteine and blood pressure measurements comprised the study sample. A total of 376 persons were excluded because of mixed race/ethnicity, as recommended by the National Center for Health Statistics (41). In analyses of blood pressure (continuous outcome), a further 1,258 persons were excluded because they were either pregnant (n = 133) or were taking antihypertensive medications (n = 1,125, 452 had concurrent physician-diagnosed cardiovascular disease), yielding 5,978 persons for study. The analyses of hypertension (categorical outcome) included persons taking antihypertensive medications as cases, yielding 7,103 persons for analyses of the categorical outcome.
The mean level of homocysteine in the adults studied was 9.6 µmol/liter (standard deviation, 4.8), comparable with previous reports (31, 48, 49). The average diastolic and systolic blood pressure measurements increased by 3.7 and 9.3 mmHg, respectively, from the lowest to the highest quintile of homocysteine, unadjusted for age (table 1). Serum homocysteine was negatively associated with red blood cell and serum folate and serum vitamin B12, but the bivariate relation with dietary vitamin B6 intake was inconsistent. Homocysteine was positively associated with age, male sex, postmenopausal status (defined as age
55 years), renal dysfunction (defined by elevated levels of serum creatinine), percentage of current smokers, serum cotinine, and total serum cholesterol. Homocysteine was inversely associated with high density lipoprotein cholesterol and serum vitamin C. In addition, homocysteine had a positive association with caffeine and a negative association with mineral/vitamin supplement use in the past month (data not shown). There was little or no association of homocysteine with serum vitamin E, alcohol intake, serum C-reactive protein, poverty income ratio, education, body mass index (weight (kg)/height (m)2), and current use of estrogen supplements in women (data not shown). The association of homocysteine with demographic and lifestyle variables was similar to previous data (50).
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TABLE 1. General characteristics of subjects by quintiles of serum homocysteine in adults* surveyed in phase 2 of NHANES III, 19911994
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Homocysteine-blood pressure association
First, the homocysteine-blood pressure association was assessed. Much of the crude positive association of homocysteine with blood pressure was explained by age and sex in model 0 of table 2. In sex-stratified analyses including sex-specific confounding variables, homocysteine was positively associated with both diastolic and systolic blood pressure as shown in model 1 of table 2. An increase of 5 µmol/liter in homocysteine (about 1 standard deviation) was associated with increases in diastolic and systolic blood pressure of 0.5 and 0.7 mmHg, respectively, in men. The association of homocysteine with blood pressure was stronger in women than in men: 0.7 and 1.2 mmHg in diastolic and systolic blood pressure, respectively. The homocysteine-sex interaction was statistically significant for systolic blood pressure (p = 0.04), and there was little or no evidence of effect modification by age in either men or women. In both men and women, further adjusting for red blood cell folate and dietary vitamin B6 intake only slightly attenuated the relation of homocysteine to blood pressure as shown in model 3 of table 2. Excluding adults who reported a physician diagnosis of cardiovascular disease (n = 224) had little or no effect on the findings, with the exception of strengthening the homocysteine-systolic blood pressure association in men (coefficient changed from 0.73 to 0.95).
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TABLE 2. Multiple regression analysis of the association of homocysteine and folate with blood pressure among 5,978 adult participants (2,775 men and 3,203 women) in phase 2 of NHANES III, 19911994
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Red blood cell folate and dietary vitamin B6 were negatively associated with blood pressure in models that did not adjust homocysteine, as in model 2 of table 2. The red blood cell folate-blood pressure association was statistically significant only in women and only for systolic blood pressure (model 2). Among women, a 1 standard deviation increase in red blood cell folate (100 ng/ml) was associated with about a 0.5-mmHg decrease in diastolic (95 percent confidence interval (CI): 1.00, 0.14) and systolic (95 percent CI: 1.24, 0.02) blood pressure. The regression coefficient for red blood cell folate was reduced by about a third with homocysteine in the model, as shown in models 2 and 3 of table 2. The dietary vitamin B6-blood pressure association was stronger in men, and there was little or no change in the coefficients in models including homocysteine, as shown in models 2 and 3 of table 2. Among men, 1 standard deviation increase in dietary vitamin B6 intake (1.3 mg/day) was associated with a 1 mmHg decrease in both diastolic (95 percent CI: 1.40, 0.06) and systolic (95 percent CI: 1.82, 0.26) blood pressure. Serum vitamin B12 had little or no independent association with blood pressure and had little or no effect on the homocysteine-blood pressure association once folate and vitamin B6 were adjusted (data not shown).
Other potential effect modifiers and confounders were considered but not retained in the final models. Serum measurements of vitamin C and vitamin E were considered to test the hypothesis that homocysteine-related endothelial dysfunction is mitigated by these antioxidants (11, 12), but no effect modification was found (data not shown). Serum total and high density lipoprotein cholesterol were neither effect modifiers nor confounders of the homocysteine-blood pressure association. Alcohol intake (questionnaire data on "had 12 or more drinks past year"-yes/no) slightly strengthened the homocysteine-systolic blood pressure association in a subgroup of women who responded to the alcohol questionnaire (data not shown). There was no evidence of effect modification by alcohol intake. Similarly, the homocysteine-blood pressure association was not confounded by coffee intake or supplement use, and it was not modified by supplement use. Serum C-reactive protein, an indicator of the acute-phase response, and serum creatinine, an indicator of renal function, had little or no effect on the association of homocysteine with blood pressure.
Homocysteine-hypertension association
Second, the association of homocysteine with prevalent hypertension was investigated using logistic regression (table 3). A 1 standard deviation increase (5 µmol/liter) in homocysteine (continuous variable) was associated with a 50 percent increase in the risk of hypertension in women only (95 percent CI: 1.12, 2.00). There was little or no association of the continuous homocysteine variable with prevalent hypertension in men (p = 0.006 for the homocysteine-sex interaction). To allow for nonlinearity, homocysteine was categorized into quintiles: The trend in the risk of hypertension with increasing homocysteine quintiles was significant only in women (p for trend = 0.0001). Women in the highest quintile of homocysteine had three times (95 percent CI: 1.7, 5.4) the risk of hypertension compared with those in the lowest quintile. There was a positive trend in men, with men in the highest quintile at twofold higher risk than men in the lowest quintile (95 percent CI: 0.71, 5.14). Extending the outcome definition and/or refining the comparison group, for instance by excluding adults who had a physician diagnosis of hypertension but no elevation in blood pressure and no use of antihypertensive medication, made little or no difference in the odds ratios. Overall, there was little or no change in these estimates when adults with physician-diagnosed cardiovascular disease were excluded from consideration (data not shown).
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TABLE 3. Logistic regression analysis of the association of homocysteine with hypertension prevalence* in 7,103 adult participants in phase 2 of NHANES III, 19911994
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Homocysteine-cardiovascular disease association
Finally, the association of homocysteine with prevalent cardiovascular disease was assessed. We first replicated the recent findings of Morris et al. (49) and produced results close to those reported (data not shown). We excluded adults using antihypertensive medications because their medicated blood pressure is not a fair indicator of their usual blood pressure, and the homocysteine-blood pressure association is attenuated in this subgroup. Next, we examined the homocysteine-cardiovascular disease association in models with and without blood pressure as a covariate (table 4). When blood pressure was omitted from the model, as in models 2 and 4 of table 4, the homocysteine-cardiovascular disease association was slightly strengthened in women, but there was little or no change in men. When women having more than 12 µmol/liter were compared with women having less than or equal to 12 µmol/liter, effect sizes were about 20 percent higher in models without blood pressure.
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TABLE 4. Logistic regression analysis of the association of homocysteine with cardiovascular disease prevalence in adult participants in phase 2 of NHANES III,* 19911994: comparison among models with and without consideration of a potential mediating effect of blood pressure
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DISCUSSION
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An increase of 5 µmol/liter in homocysteine (about 1 standard deviation) was associated with increases in diastolic and systolic blood pressure of 0.5 and 0.7 mmHg, respectively, in men and of 0.7 and 1.2 mmHg in women. Among the B vitamins, red blood cell folate and dietary vitamin B6 attenuated the homocysteine-blood pressure association. In women, there was an independent association of red blood cell folate with blood pressure. Dietary vitamin B6 was inversely associated with blood pressure in men and with systolic blood pressure in women. Higher levels of homocysteine were associated with an increased risk of hypertension. In a comparison of the highest and the lowest quintiles of homocysteine, women had a threefold increase in the risk of hypertension, and men had a twofold increase. In light of the homocysteine-blood pressure association, the association of homocysteine with prevalent cardiovascular disease was examined with and without adjusting for blood pressure. When blood pressure was omitted, the homocysteine-cardiovascular disease association was slightly strengthened in women, but there was little or no change in men.
The findings herein are consistent with past reports that, although limited for various reasons, considered the relation of homocysteine to blood pressure (3840). Araki et al. (38) reported that average homocysteine levels were higher in hypertensive compared with normotensive subjects. Sutton-Tyrrell et al. (39) found an increased risk of isolated systolic hypertension in the elderly among those with higher homocysteine. Neither study (38, 39) provided a quantitative estimate of the strength of the association of homocysteine with blood pressure throughout its continuous range. One previous study (40) reported that blood pressure was a significant positive predictor of homocysteine after adjusting for age, sex, smoking status, physical activity, heart rate, and serum total cholesterol.
Recently, a randomized, placebo-controlled, homocysteine-lowering trial reported a decrease in blood pressure in response to treatment (51). Trial participants were siblings of patients with both premature atherothrombotic disease and abnormal postmethionine hyperhomocysteinemia. After 2 years of treatment with folic acid (5 mg/day) and pyridoxine (250 mg/day), diastolic blood pressure decreased 1.9 mmHg (95 percent CI: 3.7, 0.02), and systolic blood pressure decreased 3.7 mmHg (95 percent CI: 6.8, 0.6). The demonstration of blood pressure lowering in response to a homocysteine-lowering intervention in an at-risk population supports a causal association. Furthermore, the trial results suggest that the magnitude of the homocysteine-blood pressure relation may be larger than is evident from the NHANES III data.
The association of homocysteine with blood pressure and with hypertension was stronger in women than in men. This was not due to a difference in the range of homocysteine or blood pressure, and there was no evidence for differential nonlinearity in the homocysteine-blood pressure association in men and women. A recent meta-analysis of prospective and case-control studies on homocysteine and coronary heart disease reported a higher summary odds ratio of 1.92 (95 percent CI: 1.25, 2.93) in women compared with 1.46 (95 percent CI: 1.32, 1.62) in men (52). Further studies need to be done to delineate the reasons for the gender difference.
Contrary to two clinical studies where homocysteine-induced endothelial dysfunction was attenuated by administration of antioxidants (11, 12), there was no evidence in the NHANES III data of effect modification by serum vitamin C or vitamin E. The antioxidant effect may be limited to an acute pharmacologic dose administered in the clinical studies (1 mg of vitamin C and 800 IU of vitamin E/day). Whether the effect modification would be observed in populations with a different range of these antioxidants should be examined in future studies.
It has been suggested that homocysteine is an indicator of nephrosclerosis and is elevated because of insufficient renal excretion of homocysteine (53). Thus, the findings may arise from reverse causality if high blood pressure aggravates renal dysfunction, which in turn causes elevations in homocysteine. Alternatively, renal dysfunction may be a confounding variable related to both blood pressure and homocysteine. In the NHANES III data, including renal dysfunction (defined by proxy as elevated serum creatinine) in the models had little or no effect on the homocysteine-blood pressure association. Similarly, the results were unchanged when we excluded subjects with prevalent cardiovascular disease or when we adjusted for serum levels of C-reactive protein. Thus, although cross-sectional studies are unable to ensure the correct temporal sequence between putative cause and effect, these findings suggest that the homocysteine-blood pressure association was not due to an elevation of homocysteine as a result of underlying disease.
Homocysteine may elevate blood pressure by causing arterial stiffness due to impaired vascular endothelial integrity and/or by reducing the efficiency of vasodilation (54). Reduced methylation potential due to elevated homocysteine and S-adenosylhomocysteine may compromise the growth and integrity of the endothelial cells (55). In addition, homocysteine generates free radicals during its autooxidation to thiolactone (56) and inhibits the expression of antioxidant enzymes, such as glutathione peroxidase (9). Other as yet unidentified mechanisms also may be at work (51).
In women, a small red blood cell folate-blood pressure association remained after adjusting for homocysteine levels, suggesting an independent effect of folate. An independent role of folate is supported by evidence that it regenerates tetrahydrobiopterin, an essential cofactor for the synthesis of nitric oxide (2527). Dietary vitamin B6 was inversely associated with blood pressure in men and with systolic blood pressure in women. The association was independent of homocysteine. This is consistent with previous literature that reported an independent vitamin B6-cardiovascular disease association (20, 22, 24, 57, 58).
The findings herein support an association of homocysteine with blood pressure. Given within-subject variability in a single homocysteine measurement, the observed association may underestimate the true association by about 15 percent due to regression-dilution bias (59). Thus, the true effect size for systolic blood pressure may be about a 1.2 mmHg increase per 5 µmol/liter increase in homocysteine (effect size corrected for regression dilution: 0.8 and 1.5 mmHg, respectively, for men and women). The Dietary Approaches to Stop Hypertension (DASH) Study clearly demonstrates the importance of dietary factors in lowering blood pressure. The DASH Study diet lowered systolic blood pressure by 5 mmHg compared with the usual American diet when both diets had the same intermediate level of sodium intake (60). Further work (61) suggested that folate intake comprises part of the difference between the DASH Study diet and the usual American diet (168 µg/day on an American diet vs. 418 µg/day on the DASH Study diet). These results suggest the potential contribution of folate, perhaps mediated by homocysteine lowering, in blood pressure reduction. Thus, the current finding of an association of homocysteine and blood pressure, if causal, would mean that homocysteine lowering leads to reduced blood pressure with attendant public health benefits (1).
All prospective epidemiologic studies that examined the relation of homocysteine to coronary heart disease or stroke controlled for either blood pressure (6265) or hypertension status (24, 35, 6671) in their fully adjusted models. Recent studies using the NHANES III data also controlled for blood pressure in estimating the association of homocysteine with cardiovascular disease (49) and stroke (31). However, if the effect of homocysteine on cardiovascular outcomes is partly mediated by blood pressure, statistical adjustment for blood pressure or hypertension status would lead to an underestimation of the total effect of homocysteine on cardiovascular disease (72). The cross-sectional NHANES III data, which comprise prevalent cases, are of limited use for this kind of analysis. Although the results suggest a stronger association of homocysteine with cardiovascular outcomes when blood pressure was omitted as a covariate, particularly among women, this issue deserves to be addressed with prospective data.
This study found an independent association of homocysteine with blood pressure. Serum homocysteine levels were positively associated with blood pressure and with the risk of hypertension, confirming reports from animal experiments (5, 6) and a small intervention trial in an at-risk population (51). Considering that these cross-sectional findings may be affected by lifestyle or dietary changes that lower homocysteine in people with high blood pressure, the homocysteine-blood pressure association should be investigated further in a prospective study. In addition, estimation of the magnitude of the association of homocysteine with cardiovascular outcomes should consider the mediating role of blood pressure. Finally, a direct effect of folate supplementation on blood pressure should be considered in examining the benefit of folate intervention and/or food fortification programs relative to the prevention of cardiovascular morbidity.
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ACKNOWLEDGMENTS
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Supported by a grant from the US Department of Agriculture (99-34324-8120) and by a grant from the Bronfenbrenner Life Course Center at Cornell University.
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NOTES
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Correspondence to Dr. Patricia A. Cassano, Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853 (e-mail: pac6{at}cornell.edu). 
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