Population reference values for plasma total homocysteine concentrations in US adults after the fortification of cereals with folic acid

¹ From the Department of Human Nutrition, College of Applied Health Sciences, University of Illinois at Chicago, Chicago, IL (VG), and the Department of Mathematics, San Francisco State University, San Francisco, CA (MRK)

² Reprints not available. Address correspondence to V Ganji, Department of Human Nutrition, College of Applied Health Sciences, 1919 West Taylor Street, Room 650, University of Illinois at Chicago, Chicago, IL 60612. E-mail: vganji{at}uic.edu.

ABSTRACT  
Background: Folic acid fortification has resulted in a dramatic increase in folate intake in the United States. Folate intake is inversely associated with circulating total homocysteine (tHcy). Elevated tHcy is directly associated with cardiovascular disease risk.

Objective: The aim of this study was to present the distribution of plasma concentrations of tHcy in US adults by using data from nationally representative sample surveys conducted since folic acid fortification was implemented.

Design: Data from the National Health and Nutrition Examination Surveys from 1999–2001 and 2001–2002 were used to study tHcy distribution by age, sex, and race-ethnicity in 9196 persons.

Results: Plasma concentrations of tHcy were higher in men than in women and in older persons than in younger persons. In those aged 19–30, 31–50, and 51–70 y but not in those aged >70 y, men had significantly (P < 0.0001) higher mean plasma concentrations of tHcy than did women. A race-ethnicity difference in plasma tHcy existed only in persons aged >70 y. Non-Hispanic blacks aged >70 y had significantly (P < 0.05) higher tHcy concentrations than did non-Hispanic white or Mexican American or Hispanic subjects in the same age group. Age-adjusted plasma tHcy concentrations did not differ significantly between non-Hispanic white (8.39 µmol/L), non-Hispanic black (8.92 µmol/L), and Mexican American or Hispanic (8.12 µmol/L) subjects. The rate of increase in plasma tHcy was greater in non-Hispanic blacks aged 50 y than in persons of similar age but of other races-ethnicities.

Conclusions: These plasma tHcy data reflect the effects of folic acid fortification. Sex, age, and race-ethnicity differences persist in plasma tHcy concentrations.

Key Words: Homocysteine • National Health and Nutrition Examination Survey • NHANES • heart disease • folic acid fortification • United States

INTRODUCTION  
Homocysteine is a nonessential, nonprotein-forming amino acid synthesized in a demethylation reaction from methionine. Homocysteine is either remethylated to methione or transsulfurated to cysteine. Genetic abnormalities in enzymes (1-3) and a deficiency of B vitamins involved in homocysteine metabolism (4-6) lead to elevated total homocysteine (tHcy) concentrations in blood. Evidence suggests that elevated circulating tHcy is associated with a greater risk of cardiovascular disease (CVD) (7) and decreased cognitive function (8). Approximately 10% of the population's risk of coronary artery disease is attributable to tHcy (9). Mechanisms that may explain the positive relation between tHcy and CVD risk include toxic effects on endothelial cells, increased smooth cell proliferation, LDL oxidation, and thrombus formation (10-12).

Folic acid fortification has a significant positive effect on folate status in the US population (13, 14). Because circulating tHcy concentrations are inversely associated with folate intakes, folic acid fortification of cereals and cereal products has had a significant negative effect on circulating tHcy in the United States (13, 14). Current reference data on circulating tHcy concentrations in the US population are based on studies conducted before folic acid fortification (15). Reference data on tHcy concentrations based on a representative US sample in the era since the implementation of folic acid fortification are lacking. Therefore, the objectives of this study were to report the plasma total tHcy concentration distribution and to investigate the relations between sex, age, and race-ethnicity and plasma tHcy in the US adult population by using the data from nationally representative sample surveys from 1999–2002, which were conducted after the implementation of folic acid fortification.

SUBJECTS AND METHODS  
Survey description and study sample
National Health and Nutrition Examination Surveys (NHANES) were conducted in civilian, noninstitutionalized persons by the National Center for Health Statistics of the Centers for Disease Control and Prevention. The data used in the current study were derived from the databases released for public use by the National Technical Information Service (Springfield, VA; 16, 17). We concatenated the data from NHANES 1999–2000 and NHANES 2001–2002 into one analytic database, NHANES 1999–2002 (n = 21 004). NHANES 1999–2000 and NHANES 2001–2002 were conducted as continuous annual surveys rather than as periodic surveys and were based on a complex, stratified, multistage, probability sample survey design. A detailed description of the survey methods and analytic guidelines was published previously (18).

Demographic, socioeconomic, dietary, and health-related data were collected in participants' homes as part of the household interview. Persons participating in the household interview were asked to undergo a health examination in a Mobile Examination Center (MEC). NHANES 1999–2000 was conducted between March 1999 and December 2000 in 9965 persons (all were interviewed at home; 9282 of these 9965 were examined in a MEC); NHANES 2001–2002 was conducted between January 2001 and December 2002 on 11 039 persons (all were interviewed at home; 10 477 of these 11 039 were examined in a MEC). In NHANES 1999–2002, young children, older persons, non-Hispanic blacks, and Mexican Americans were oversampled. We excluded persons aged <19 y (n = 10 150) and persons with missing values for the plasma tHcy concentrations (n = 1401). Because of the small sample size, we also excluded persons of other races or ethnicities (n = 257). After these exclusions, the final analytic sample consisted of 9196 adults (4360 men and 4836 women).

Measurements
Blood was collected in a MEC by venipuncture according to standard protocols. Subjects fasted for varied lengths of time. Approximately 43.5% of the subjects (n = 3998) fasted for <10 h, and 56.4% (n = 5188) fasted for 10 h. Data on the duration of fasting were missing for 10 subjects. Duration of fasting had no appreciable effect on circulating tHcy concentrations. The mean plasma tHcy concentration was 8.59 µmol/L in subjects who fasted <10 h and 8.26 µmol/L in subjects who fasted 10 h. For tHcy measurement, blood was collected into tubes containing EDTA and immediately centrifuged at 2900 rpm for 10 min at 4–8 °C (V6500; Hamilton Bell, Montvale, NJ) to yield plasma. Plasma tHcy concentrations were measured at the laboratories at the Centers of Disease Control and Prevention by using the fluorescence polarization immunoassay (Abbott Laboratories, Abbott Park, IL). Detailed methods for blood collection and laboratory procedures were described elsewhere (19).

Statistical analysis
We used SUDAAN for WINDOWS statistical software (version 9.0; Research Triangle Institute, Research Triangle Park, NC), which takes complex survey design into consideration. Sample weights, masked variance units, and stratification variables were incorporated to account for differential probabilities of selection and noncoverage and nonresponse bias. We also used SAS for WINDOWS software (version 9.1; SAS Institute Inc, Cary, NC) in conjunction with SUDAAN to manage and analyze the data files.

We analyzed 2-way interactions between sex and age, sex and race-ethnicity, and age and race-ethnicity and 3-way interactions between sex, age, and race-ethnicity. Interactions between sex and age (P < 0.0183) and age and race-ethnicity (P < 0.0261) were significant, but the sex x race-ethnicity interaction (P < 0.1483) and the 3-way sex x age x race-ethnicity interaction (P < 0.2214) were not significant. On the basis of these interactions, we analyzed the tHcy data by age x sex and age x race-ethnicity. Because plasma tHcy concentrations were asymmetric in nature, geometric means were calculated for comparisons of age and sex and of age and race-ethnicity. Geometric means ± SEs were determined by using the Taylor series linearization method. Differences between men and women aged 19–30, 31–50, 51–70, and >70 y were determined by using a t test. Differences between races-ethnicities in subjects aged 19–30, 31–50, 51–70, and >70 y were determined by using Tukey-Kramer multiple comparison test. The Tukey-Kramer test was also used to determine the differences between age groups within sex and race-ethnicity categories after the hypothesis was tested by using analysis of covariance. Age-adjusted least-squares mean ± SE plasma tHcy concentrations in men and women and in non-Hispanic white, non-Hispanic black, and Mexican American or Hispanic subjects were measured by using analysis of covariance after the data were log transformed.

In addition, the rate of increase in plasma tHcy concentrations in men and women and in non-Hispanic white, non-Hispanic black, and Mexican American or Hispanic subjects by age was plotted for those aged 19–24, 25–29, 30–34, 35–39, 40–44, 45–49, 50–54, 55–59, 60–64, 65–69, and 70 y by using a cubic spline smoothing. In all analyses, P < 0.05 was considered significant.

RESULTS  
The study sample consisted of 4548 (49.5%) non-Hispanic white (2186 men and 2362 women), 1808 (19.7%) non-Hispanic black (849 men and 959 women), and 2840 (30.9%) Mexican American or Hispanic (1325 men and 1515 women) subjects. Distribution of plasma tHcy concentrations by sex and age in non-Hispanic white, non-Hispanic black, and Mexican American or Hispanic subjects are shown in Table 1. Arithmetic means and percentiles of plasma tHcy concentrations tended to be higher in men than in women and in older persons than in younger persons.

View this table:
TABLE 1. Distribution of plasma total homocysteine concentrations in US adults by race-ethnicity, sex, and age in the era since implementation of folic acid fortification: data from the National Health and Nutrition Examination Survey, 1999–2002¹

 
Geometric mean plasma tHcy concentrations by age and sex are shown in Table 2. Age-adjusted mean plasma tHcy concentrations were significantly (P < 0.0001) higher in men than in women. In the groups aged 19–30, 31–50, and 51–70 y, the men had significantly (P < 0.0001) higher mean plasma tHcy concentrations than did the women. Whereas, in the group aged >70 y, no significant difference was found in plasma tHcy concentrations between men and women (P = 0.2109). Older men and women (ie, aged >70 y) had significantly (P < 0.05) higher plasma tHcy than did younger men and women (ie, aged 19–30 y).

View this table:
TABLE 2. Plasma total homocysteine concentrations in US adults by age and sex in the era since implementation of folic acid fortification: data from the National Health and Nutrition Examination Survey, 1999–2002

 
Geometric mean plasma tHcy concentrations by age and race-ethnicity are shown in Table 3. Age-adjusted mean plasma tHcy concentrations did not differ significantly by race-ethnicity between non-Hispanic white, non-Hispanic black, and Mexican American or Hispanic subjects. In addition, in the groups aged 19–30, 31–50, and 51–70 y, no significant differences were found in geometric mean plasma tHcy concentrations between races-ethnicities. However, in the group aged >70 y, non-Hispanic blacks had significantly (P < 0.05) higher tHcy than did non-Hispanic white and Mexican American or Hispanic subjects. In all races-ethnicities, geometric mean plasma tHcy concentrations were significantly higher in older persons than in younger persons. For example, among the non-Hispanic white subjects, those aged >70 y had 53.0% higher tHcy concentrations than did those aged 19–30 y group (P < 0.0001). Among the non-Hispanic black subjects, those aged >70 y had 73.9% higher tHcy concentrations than did those aged 19–30 y (P < 0.0001). Among the Mexican American or Hispanic subjects, those aged >70 y had 66.1% higher tHcy concentrations than did those aged 19–30 y (P < 0.0001).

View this table:
TABLE 3. Plasma total homocysteine concentrations in US adults by age and race-ethnicity in the era since implementation of folic acid fortification: data from the National Health and Nutrition Examination Survey, 1999–2002¹

 
The rates of increase in plasma tHcy concentrations in men and women and in non-Hispanic white, non-Hispanic black, and Mexican American or Hispanic subjects by age are shown in Figure 1. The rate of increase in plasma tHcy was significantly greater in women than in men and in non-Hispanic blacks than in other races-ethnicities after the age of 45–49 y.

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FIGURE 1.. Smoothed geometric  

DISCUSSION  
In this study, we report the first reference data on plasma tHcy concentrations in the era since the implementation of folic acid fortification by using the data from a nationally representative sample survey of US residents. Mandatory folic acid fortification of processed cereals and cereal products was begun in January 1998 in response to the Public Health Service recommendation that all women who are capable of becoming pregnant should receive 400 µg folic acid/d to reduce the risk of having a child with neural tube defects (20). Because folate nutritional status is inversely related to tHcy concentrations (21, 22), it was anticipated that folic acid fortification would reduce the circulating tHcy in the US population. In fact, we recently reported (14) that circulating tHcy concentrations in the period since the implementation of folic acid fortification were 7.6 µmol/L in 1999–2000 and 7.9 µmol/L in 2001–2002, as compared with 9.5 µmol/L in the period before the implementation (1998–1994). These concentrations translate into tHcy concentrations 21.3% lower in 1999–2000 and 18.0% lower in 2001–2002 than in 1988–1994 (14). Using data from the Framingham Study of middle-aged and older adults, Jacques et al (13) reported that folic acid fortification reduced mean plasma tHcy concentrations from 10.1 to 9.4 µmol/L, a decrease of 7.5% from the concentrations from the period before the implementation of folic acid fortification. This decrease was observed within the first 3 mo of mandatory folic acid fortification (ie, by the end of March 1998). The discrepancy between these studies can be explained partly by the difference in duration of exposure to folic acid by the study population. However, it is difficult to assess the exact decrease in tHcy concentrations because of differences in study sample, survey methods, and measurement of tHcy concentrations between studies conducted before and after folic acid fortification (14, 23). Specifically, when comparing the tHcy data between NHANES III (conducted in 1988–1994) and NHANES 1999+, caution should be exercised because of differences in tHcy measurement (23).

In the era since the implementation of folic acid fortification, using the data from a nationally representative sample survey of US population, we report that plasma tHcy concentrations were significantly higher in men than in women and in older persons than in younger persons. These observations are similar to the observations made by several investigators in studies conducted before folic acid fortification (15, 21, 22). In the NHANES III population, we found an 21.1% higher tHcy in men (n = 2965) than in women (n = 3580) (21); in the Framingham Offspring Cohort, Jacques et al (22) reported an 11% higher tHcy in men (n = 920) than in women (n = 1040); and in the Hordland Homocysteine Study, Nygard et al (1) reported an 12–19% higher tHcy in men than in women. The differences in tHcy concentrations between men and women can be explained by the higher creatinine, lower folate and vitamin B-12 status, higher incidence of smoking, and higher alcohol consumption in men than in women (21). In addition, body size and estrogen status may contribute to the differences in plasma tHcy between men and women (24, 25).

Age is a significant predictor of plasma tHcy concentrations in the era since the implementation of folic acid fortification. Regardless of sex, the age-related rate of increase in plasma tHcy is higher in those aged 50 y than in those aged 19–50 y. The difference in plasma tHcy concentrations between sexes was more pronounced in younger persons than in those >70 y old. In subjects aged >70 y, the tHcy concentrations do not differ significantly between the sexes. The age-related increase in tHcy appears to be more rapid in women than in men. High concentrations of plasma tHcy in persons aged >70 y cannot be attributed to poor folate status. In fact, in the era since folic acid fortification, serum folate and RBC folate concentrations are highest in those aged >70 y (14). Elevated circulating tHcy concentrations in older persons may be due to reduced activity of cystathionine ß-synthase (26), a vitamin B-6–dependent enzyme that is essential to the transsulfuration of homocysteine to cystathionine. In addition, age-related increases in circulating tHcy concentrations may be related to the compromised renal function (27) and greater cobalamin deficiency that result from malabsorption of cobalamin by the aging gut (28).

In this large population study, race-ethnicity differences in plasma tHcy concentrations were not as evident except in those aged >70 y. In this age group, non-Hispanic blacks had significantly higher plasma tHcy than did non-Hispanic white and Mexican American or Hispanic subjects. Higher plasma tHcy concentrations in a population of older non-Hispanic blacks than in that of older non-Hispanic whites can be attributed to low folate status and greater incidence of renal failure in the former group (29). In addition, the differences in lifestyle factors between the races or ethnicities may account for some of the variation in tHcy concentrations (30).

Only a few studies have focused on race-ethnicity differences in tHcy concentrations. Earlier we reported significantly higher tHcy in non-Hispanic white than in non-Hispanic black and Mexican American or Hispanic subjects in the NHANES III population (21). In a study from South Africa, Ubbink et al (31) reported that plasma tHcy concentrations were significantly lower in black men (Venda tribe) than in white men. Ubbink et al (32) suggested that low tHcy concentrations in black subjects was due to their more efficient homocysteine metabolism than in white subjects, despite low vitamin B-6 status in blacks. Ubbink et al concluded that enzyme polymorphism may explain the efficient homocysteine metabolism in blacks (32). In fact, point mutations (677CT) in N⁵,N¹⁰–methylenetetrahydrofolate reductase that converts N⁵,N¹⁰–methylenetetrahydrofolate to 5-methyltetra-hydrofolate (methyl donor for remethylation of homocysteine to methionine) are more common in whites than in blacks (33). The 677CT mutation results in reduced activity of methylenetetrahydrofolate reductase (34) which in turn results in higher circulating tHcy concentrations in persons with low dietary folate intakes (35). However, after folate fortification, folate intake have dramatically increased in the US population (14, 36, 37), as evidenced by the significant increase in serum and red blood cell folate concentrations (13, 14, 38). Thus, the enzyme polymorphism in methylenetetrahydrofolate reductase may not be a factor in predicting circulating tHcy concentrations in the era since the implementation of folic acid fortification.

In conclusion, population reference data on plasma tHcy presented in this study reflect the scenario of the era since the implementation of folic acid fortification. Plasma tHcy concentrations in US adults vary by sex, age, and race-ethnicity in this era. It is not conclusively known whether elevated circulating tHcy leads to the development of CVD or whether it is a marker for CVD. If the causal association between circulating tHcy and the risk of CVD is proven, folic acid fortification may positively influence the CVD risk. Furthermore, predictors of plasma tHcy concentrations in the era of folic acid fortification should be established.

ACKNOWLEDGMENTS  
VG contributed to the study design and writing of the manuscript. MK contributed to data management and data analysis. Both authors contributed to the interpretation of the results. Neither of the authors had any personal or financial conflict of interest.

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