Serum Total Homocysteine Concentrations in the Third National Health and Nutrition Examination Survey (19911994): Population Reference Ranges and Contribution of Vitamin Status to High Serum Concentrations

Homocysteine, a non-protein-forming sulfur amino acid, has attracted attention because elevated concentrations of circulating total homocysteine are associated with an increased risk for vascular disease (1, 2). Homocysteine is also a sensitive functional marker of inadequate cellular folate and vitamin B12 concentrations (3). Inadequate status of these vitamins has important health consequences that may be independent of their role in homocysteine metabolism. Low folate concentrations increase a woman's risk for having a baby with a neural tube defect (4, 5), and an inadequate vitamin B12 concentration is known to produce various neurologic and cognitive effects (6, 7). Persons with low circulating folate or vitamin B12 concentrations have higher fasting total homocysteine concentrations (8-10), and elevated fasting total homocysteine concentrations are usually normalized by treatment with folic acid and vitamin B12 (6, 11-14). However, less is known about the importance of these vitamins as risk factors for high homocysteine concentration in the general population. Only three studies have examined the relation between homocysteine concentration and its vitamin determinants in samples that were designed to be representative of U.S. national (8) or regional (9, 10) populations. One of these studies (9) reported that approximately two thirds of all cases of moderately elevated total homocysteine concentrations were potentially attributable to low vitamin concentrations, but estimation of the proportion of cases with high homocysteine concentrations that can be attributed to inadequate vitamin status is complicated by the lack of a standard definition of a high total homocysteine concentration. In the absence of a definition based on increased risk for an adverse health outcome, such as vascular disease, upper reference limits from samples of healthy persons without established risk factors for high homocysteine concentrations have been used to define a high total homocysteine concentration (10, 15-17). We previously described the distribution of total serum homocysteine concentrations in participants 12 years of age or older from the third National Health and Nutrition Examination Survey (NHANES III), a population-based sample of U.S. residents (18). These data present a unique opportunity to develop population reference ranges for serum total homocysteine concentration and to determine the extent to which elevated homocysteine concentrations are associated with low circulating vitamin concentrations in a representative sample of U.S. residents. Methods Participants The NHANES III was developed to obtain nationally representative information on the health and nutritional status of the civilian, noninstitutionalized U.S. population (19, 20). Homocysteine concentrations were measured as part of an NHANES III surplus sera project on serum samples from participants 12 years of age or older who were seen during phase II of this survey (19911994). This project is described in greater detail elsewhere (18). Homocysteine concentrations were measured at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University by using the high-performance liquid chromatography method of Araki and Sako (21). The interassay coefficient of variation for this assay was 6%. Folate and vitamin B12 were determined for phase 2 specimens at the Centers for Disease Control and Prevention central laboratory by using a Quanta Phase II radioassay kit (Bio-Rad Laboratories, Hercules, California), and analyses are described in detail in the NHANES III Laboratory Procedures Manual (22). The coefficients of variation for folate and vitamin B12 were 6% and 7%, respectively. Informed consent was obtained from all respondents. The NHANES III protocol was approved by the National Center for Health Statistics NHANES Institutional Review Board, and measurement of serum homocysteine was approved by the Human Investigations Review Committee at the New England Medical Center. We used the following search strategy, combining Medical Subject Headings (MeSH terms) and text words, to identify all population-based studies relating vitamin status to circulating homocysteine concentrations: ([homocysteine (MeSH] OR homocysteine [Text Word]) AND (vitamins [MeSH] OR vitamin [Text Word]]) AND (epidemiologic studies [MeSH] OR data collection [MeSH] OR survey [Text Word]]. This search identified 137 citations, of which 16 were reviews. We selected original studies that 1) were designed to be representative of national, regional, or local populations and 2) described the relation between circulating homocysteine concentrations and either intake or circulating concentrations of folate or vitamin B12. As of 1 March 1999, 3 articles met our criteria (8-10). Statistical Analysis We used sample weights in analyses to account for unequal probability of selection and nonresponse and to produce estimates of means and percentiles that were representative of the noninstitutionalized, civilian U.S. population. We used SUDAAN statistical software (23) to account for the complex survey design in the variance estimates. Because total homocysteine, folate, and vitamin B12 values were skewed, logarithmic transformations were applied. To show the relations between total homocysteine concentrations and vitamin concentrations, we classified participants into age- and sex-specific vitamin decile categories and estimated the geometric mean of the serum total homocysteine concentration within each decile. Analyses were adjusted for ethnicity and serum creatinine concentration. In addition, the relation between total homocysteine and folate concentrations was adjusted for vitamin B12 concentrations, and the relation between total homocysteine and vitamin B12 was adjusted for folate concentrations. We tested the associations between homocysteine and vitamins for interactions with age, sex, and ethnicity. We tested for trend of total homocysteine concentration across vitamin concentrations by using linear regression with the logarithm of the continuous vitamin concentration as the independent variable, adjusting as described above. We showed the trend by using the SYSTAT LOWESS procedure to fit smoothed curves (24) to the geometric mean total homocysteine concentrations in the vitamin decile categories (25). It has been suggested that population reference ranges for the total homocysteine concentration be established in samples of persons without established risk factors for a high homocysteine concentration (10, 15-17). For our reference sample, we included persons whom we assumed to be folate- and vitamin B12-replete (that is, their serum concentrations of both vitamins were above the 50th percentile) and had normal serum creatinine concentrations (<90 mol/L for women and<110 mol/L for men). Pregnant women were excluded. We used the 5th and the 95th percentiles from the reference sample to estimate population reference ranges. To identify the potential impact of low vitamin concentrations on high total homocysteine concentration, we needed to establish values for high total homocysteine and low vitamin concentrations. We used the sex-specific 95th percentiles in the participants 20 to 39 years of age (the reference sample) to define high total homocysteine concentrations for all age groups. We used this reference sample because homocysteine concentrations changed little with age in this group, unlike in the other age groups (18). We defined low vitamin concentrations as a folate concentration less than 11 nmol/L (26, 27) and a vitamin B12 concentration less than 185 pmol/L (28, 29). We calculated the prevalence of high total homocysteine concentration; the prevalence ratio for high total homocysteine concentration; the attributable risk percentage; and the population attributable risk percentage for persons with low concentrations of folate, vitamin B12, or both compared with persons who had adequate concentrations of both of these vitamins. The attributable risk percentage estimates the excess cases of high homocysteine concentrations among persons with low vitamin concentrations, whereas the population attributable risk percentage takes into account the prevalence of low vitamin concentrations in the population and estimates the excess of high homocysteine concentrations associated with low vitamin concentrations in the entire population. We used the design effect for total homocysteine concentration, which is the ratio of the complex sampling design variance derived by using SUDAAN software (23) to the simple random sample variance calculated by using SAS software (30), to determine the recommended minimum sample size needed to achieve stable estimates of means, proportions, and percentiles according to the National Center for Health Statistics analytic guidelines (19). On the basis of an average design effect of approximately 1.4 for our sample, means and medians derived from fewer than 42 participants, 10th and 90th percentiles derived from fewer than 112 participants, and 5th and 95th percentiles derived from fewer than 224 participants were deemed unstable. Sample size for stable estimates of the proportions varied by the magnitude of the proportion, ranging from 42 for proportions of 0.5 to 224 for proportions of 0.05 or 0.95. We indicate in the text and tables statistics that did not meet the appropriate sample size. We categorized participants into three ethnic groups: non-Hispanic white, non-Hispanic black, and Mexican American. We excluded persons from other ethnic groups (n=436) because their inclusion produced unstable estimates of mean total homocysteine concentration after adjustment for ethnicity. Our analyses are based on 8086 participants with complete data on serum total homocysteine, folate, vitamin B12, and creatinine concentrations. Results Table 1 shows selected characteristics of the sample by sex and ethnic group. On average, non-Hispanic white pa