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Nutritional Epidemiology

Trends in Serum Folate, RBC Folate, and Circulating Total Homocysteine Concentrations in the United States: Analysis of Data from National Health and Nutrition Examination Surveys, 1988–1994, 1999–2000, and 2001–2002¹

Department of Human Nutrition, College of Applied Health Sciences, University of Illinois at Chicago, Chicago, IL and ^* Department of Mathematics, San Francisco State University, San Francisco, CA

² To whom correspondence should be addressed. E-mail: vganji{at}uic.edu.

ABSTRACT

Folate intakes increased dramatically after folic acid fortification. We investigated the changes in serum folate, RBC folate, and total homocysteine (tHcy) concentrations utilizing data from National Health and Nutrition Examination Surveys (NHANES) 1988–2002. NHANES 1988–2002 were based on a stratified, multistage, probability sampling design conducted among civilian U.S. residents. The current study included 17,144, 17,213, and 11,415 measurements for serum folate, RBC folate, and tHcy, respectively. Overall, geometric mean serum folate concentrations were 149.6 and 129.8% higher in 1999–2000 and 2001–2002, respectively, than in 1988–1994 (P < 0.0001). Sex-, age-, and race-ethnicity–adjusted serum folate was significantly lower in 2001–2002 than in 1999–2000 (10.4%, P < 0.0002). The prevalence of low serum folate decreased from 18.4% in 1988–1994 to 0.8% in 1999–2000 and to 0.2% in 2001–2002 (P < 0.0001). RBC folate increased from 391 nmol/L in 1988–1994 to 618 nmol/L in 1999–2000, and to 611 nmol/L in 2001–2002. Consequently, the prevalence of low RBC folate decreased from 45.8% in 1988–1994 to 7.3% in 1999–2000 and to 7.1% in 2001–2002 (P < 0.0001). Although, RBC folate status improved after folic acid fortification in all race-ethnicities, the prevalence of low RBC folate (20.5%) continues to be high in non-Hispanic blacks. Age-, sex-, and race-ethnicity–adjusted tHcy declined from 9.5 µmol/L in 1988–1994 to 7.6 µmol/L in 1999–2000 and to 7.9 µmol/L in 2001–2002. Although folic acid fortification contributed to significant improvement in folate status, serum folate concentrations have declined recently. This may be attributable to lower folic acid intakes.

KEY WORDS: • serum folate • RBC folate • total homocysteine • folic acid fortification • NHANES

In response to the U.S. Public Health Service's recommendation that all child-bearing women consume 400 µg folic acid/d so that they are folate replete to reduce the risk of having a child with neural tube defect (NTD)³ (1), the FDA mandated that all enriched cereal grain products be fortified with folic acid at the level of 140 µg/100 g of cereal product (2). The FDA predicted that the increase in folic acid intake by the target population would be 100 µg/d, and the daily intake from all sources would remain below the recommended upper limit of 1000 µg/d by the nontarget population (2). However, the actual intake of folic acid after folic acid fortification has exceeded the predicted intake because several fortified foods contained higher amounts of folic acid than required by the FDA regulation (3,4). High intakes of folic acid raise the possibility of masking cobalamin deficiency, especially in an elderly population (5).

Folic acid fortification has markedly improved plasma folate, RBC folate, and circulating total homocysteine (tHcy) concentrations in the United States. (6–8). Previous studies that examined the relation between folic acid fortification and folate status were based on either a nonrepresentative U.S. population sample or a convenience sample (6,7). In addition, the effect of folic acid fortification on folate status in non-Hispanic blacks (NHB) and Mexican Americans/Hispanics (MA/H) has never been investigated. From a public health perspective, it is important to monitor serum and RBC folate concentrations on a continuous basis to determine whether they continue to rise or stabilize after folic acid fortification. This report presents up to date data on serum and RBC folate concentrations and prevalence estimates for low serum and RBC folate in various demographic categories using the databases from nationally representative sample surveys, National Health and Nutrition Examination Survey (NHANES) III 1988–1994, NHANES 1999–2000, and NHANES 2000–2002. Because circulating tHcy concentrations are inversely related to dietary folate intake (9), we also analyzed tHcy concentrations and prevalence estimates for high tHcy across these 3 surveys.

SUBJECTS AND METHODS

    Surveys description and study sample. The National Center for Health Statistics, part of the CDC conducts NHANES among civilian, noninstitutionalized residents of the United States. The data used in this study were derived from the databases released for public use by the National Technical Information Service, Springfield, VA. The design of NHANES was a complex, stratified, multistage probability sample survey. Demographic, socioeconomic, dietary, and health-related data were collected in the participants' homes as part of the household interview. All persons interviewed in the household were asked to complete the physician-administered health examination component in the Mobile Examination Centers (MEC). Participants who were unable to come to the MEC received home examination. In the NHANES, young children, older persons, NHB, and MA/H were oversampled. In this study, we used data from 3 surveys, NHANES III 1988–1994 (10–13), NHANES 1999–2000 (14) and NHANES 2001–2002 (15). The detailed description of the survey methodologies and analytical guidelines were reported elsewhere (16,17).

NHANES III was conducted in 2 phases from 1988 to 1994. The first phase of the survey was conducted in 1988–1991 at 44 locations; the second phase of the survey was conducted in 1991–1994 at 45 locations. NHANES III included 39,695 subjects 2 mo old. Of those, 33,994 were interviewed in their homes, 30,818 were examined in a MEC, and 493 were examined at home. NHANES 1999–2000 and 2001–2002 were conducted as continuous, annual surveys rather than periodic surveys. NHANES 1999–2000 was conducted between March 1999 and December 2000 on 9965 individuals (all were home interviewed; 9282 were examined in a MEC); NHANES 2001–2002 was conducted between January 2001 and December 2002 among 11,039 individuals (all were home interviewed; 10,477 were examined in a MEC). We excluded pregnant women, individuals who had fasted <10 h before phlebotomy, and persons with missing values for serum folate, RBC folate, and tHcy concentrations.

    Measurements. Blood was collected by venipuncture at the MEC according to standard protocols. In the NHANES III, blood samples were held at room temperature for 30–60 min and then the serum was separated by centrifugation (1115 x g for 15 min). For the RBC folate assay, whole blood was collected into tubes containing EDTA as an anticoagulant. The specimens were frozen at or below –20°C and transported on dry ice to the CDC for laboratory analysis. Serum and RBC folate concentrations were assayed using the Quantaphase II folate radioassay kit (Bio-Rad Laboratories) during phase II of NHANES III, NHANES 1999–2000, and NHANES 2001–2002. Surplus serum samples were frozen at –70°C for 8 mo to 3 y. Serum tHcy concentrations were measured in these surplus serum samples at the USDA Human Nutrition Research Center on Aging. Repeated freezing and thawing of serum samples had no effect on the concentration of serum tHcy (18). Serum tHcy concentrations were measured using HPLC (19).

In NHANES 1999–2000 and 2001–2002, for tHcy measurement, blood was collected into the EDTA tubes and immediately centrifuged (2900 x g for 10 min) to yield plasma. Plasma tHcy concentrations were measured at the CDC laboratories using the fluorescence polarization immunoassay (Abbott Laboratories). The methodology employed for the analysis of serum and RBC folate concentrations in phase I of NHANES III was Quantaphase I folate radioassay kit (Bio-Rad Laboratories). Serum folate and RBC folate data from phase I of NHANES III were corrected to correspond to the data from phase II of the NHANES III (corrected value = –0.1411 + 0.6849 x uncorrected value). A detailed description of specimen processing and laboratory methods was given elsewhere (20,21).

Data analysis was based on 17,144 measurements for serum folate, 17,213 measurements for RBC folate, and 11,415 measurements for circulating tHcy concentrations across 3 surveys. The poverty income ratio (PIR) was used to define the socioeconomic status. PIR is the ratio of a family's income to their appropriate threshold income (22). Income with a PIR value of <1.0 is considered to be below the poverty level. We categorized PIR values into <1.0, 1.0 to <2.5, 2.5 to <4.0, and 4.0.

    Statistical analyses. SUDAAN statistical software (SUDAAN for Windows, version 8.0.2, Research Triangle Institute) was used to account for complex survey design. Sample weights, primary sampling units, and stratification variables were considered in the data analysis so that the differential probabilities of selection and adjustments for noncoverage and nonresponse bias were taken into account. We also used SAS (SAS for Windows, version 8.0, SAS Institute) in conjunction with SUDAAN to manage and analyze data files.

Due to the asymmetric nature of the data, geometric mean serum folate, RBC folate, and tHcy concentrations were determined according to sex, race-ethnicity [non-Hispanic White (NHW), NHB, MA/H, and Others], age (<18 y, 19–30 y, 31–50 y, 51–70, and 70 y), and PIR (<1.0, 1.0 to <2.5, 2.5 to <4.0, and 4.0). Prevalences (%) of at risk for low serum folate (<6.8 nmol/L) (6), low RBC folate (<362.6 nmol/L) (8), and high tHcy concentrations (>13 µmol/L) (6) were determined. Standard errors were determined using the Taylor series linearization method. The differences in serum folate, RBC folate, and tHcy and the prevalence of at risk for low serum folate and RBC folate and high tHcy between 1988 and 1994, 1999 and 2000, and 2001 and 2002 were determined with Bonferroni adjustment after testing the hypothesis with t test for independent samples. Additionally, we determined sex-, age-, and race-ethnicity–adjusted mean serum folate, RBC folate, and tHcy concentrations across the 3 NHANES with analysis of covariance (ANCOVA). Differences in sex-, age-, and race-ethnicity–adjusted mean serum folate, RBC folate, and tHcy concentrations among the 3 NHANES were analyzed with Bonferroni adjustment after testing the hypothesis with ANCOVA. Data are presented as means ± SE. In all other analyses, = 0.05 was considered significant.

RESULTS

Fasting serum folate, RBC folate, and circulating tHcy concentrations by sex, race-ethnicity, age, and PIR before and after folic acid fortification are presented in Table 1. We presented serum folate, RBC folate, and tHcy concentrations as geometric means ± SE because the distributions of these blood analytes were skewed to the right. Prevalence estimates for low serum and RBC folates and high tHcy from national surveys conducted before and after folic acid fortification in the U.S. population are presented in Table 2. Additionally, sex-, age-, and race-ethnicity–adjusted changes in serum folate, RBC folate, and circulating tHcy concentrations are presented in Figure 1.

View larger version (15K): [in this window] [in a new window]   FIGURE 1  Sex-, age-, and race-ethnicity–adjusted serum folate (A), RBC folate (B), and circulating tHcy concentrations (C) in the United States, 1988–2002. Error bars indicate SE. Means with different letters differ, P < 0.016 (Bonferroni adjustment for multiple comparisons). Due to methodological differences in tHcy concentration measurement, statistical comparisons between NHANES 1988–1994 and NHANESs 1999+ for tHcy should be interpreted with caution.

Overall, geometric mean serum folate concentrations were 149.6% higher in 1999–2000 and 129.8% higher in 2001–2002 than in 1988–1994. A notable observation in this study was a decrease in serum folate concentrations from 1999–2000 to 2001–2002. Although the decline in serum folate concentration was observed in all demographic groups of the U.S. population, a significant decline occurred in men (7.3%, P = 0.0012), in women (8.5%, P = 0.0028), in NHW (9.7%, P = 0.0037), in those 18 y old (7.3%, P = 0.0081), in those 31–50 y old (7.6%, P = 0.0021), in those 51–70 y old (10.8%, P = 0.0146), and in those with PIR < 1.0 (12.5%, P = 0.0063) and 4.0 (9.9%, P = 0.0068). When the data were adjusted for sex, age, and race-ethnicity, there was a reduction of 10.4% in serum folate concentrations from 1999–2000 to 2001–2002 (P < 0.0002). Between 1999–2000 and 2001–2002, plasma tHcy increased, but the differences were significant only in women (6.3%, P = 0.0014), in NHW (5.6%, P = 0.0117), in those 18 y old (8.3%, P = 0.0015), and in those >70 y old (11.7%, P = 0.0076).

Overall, geometric mean RBC folate concentrations were 58.2% higher in 1999–2000 and 56.5% higher in 2001–20002 than in 1988–1994. tHcy concentrations were 19.5% lower in 1999–2000 and 16.1% lower in 2001–2002 compared with 1988–1994. When the data were adjusted for sex, age, and race-ethnicity, similar trends were present for RBC folate and tHcy (P < 0.0167) (Fig. 1).

Among all race-ethnicities, the increase in RBC folate concentrations ranged from 50.8 to 66.5% from 1988–1994 to 1999–2002. The prevalence of low RBC folate in the NHB population was highest in both the pre- (69.6%) and post-folic acid fortification periods (17.9% in 1999–2000 and 20.5% in 2001–2002). Between 1988–1994 and 1999–2000, and 1988–1994 and 2001–2002, the decline in the prevalence of low RBC folate was greatest in the NHB population. The prevalence of high tHcy concentration was highest in those >70 y old in both the pre- and post-folic acid fortification periods (31.8, 16.8, and 20.9% in 1988–1994, 1999–2000, and 2001–2002, respectively).

Between 1988–1994 and 1999–2000, persons > 70 y old had the largest increase in serum folate (26.1 nmol/L). Across the age groups, the increase in RBC folate from 1988–1994 to 1999–2000 ranged from 54 to 61%. The decrease in the prevalence of low RBC folate in those <18 y old was greatest, a 90.4% decrease in 1999–2000 from 1988–1994. During this period, this population experienced greatest decline in tHcy concentrations (2.1 µmol/L).

DISCUSSION

In this report, we present up to date data on serum folate, RBC folate, and circulating tHcy concentrations, and prevalence estimates of low serum and RBC folates and high tHcy in the United States. We also examined the temporal association between folic acid fortification and folate status in various demographic groups of the U.S. population using the data from nationally representative sample surveys, 1988–2002. Because the NHANES III 1988–1994 was conducted before the implementation of folic acid fortification, and the 1999–2000 and 2001–2002 surveys were conducted after folic acid fortification, we were able to assess the effect of folic acid fortification on folate status in a nationally representative sample. After folic acid fortification of cereal products, folic acid intake increased by >200 µg/d (23). This increase was approximately twice as large as originally estimated (4). Thus, the differences we observed in serum folate, RBC folate, and tHcy concentrations between 1988–1994 and 1999–2002 are more likely due to increased folic acid intake from fortified foods. Increased use of vitamin supplements may have also contributed to the improved folate status.

What is surprising is the significant overall decline we observed in serum folate concentrations between 1999–2000 and 2001–2002 in both men and women, although the decline was modest (7.3% in men and 8.5% in women). This decline remained significant (10.4%) after adjusting the analysis for sex, age, and race-ethnicity. After folic acid fortification, measurement of actual folate content in folic acid–fortified foods showed that these products contained higher amounts of folic acid than required by law (3,4). Recently, Johnston and Tamura (24) reported that the folate content in fortified foods declined significantly after 2001. They reported that the mean folate content of all breads was 154 ± 76 (n = 29), 113 ± 43 (n = 31), and 109 ± 49 (n = 32) µg/100 g fresh weight in 2001, 2002, and 2003, respectively. The folate content in breads in 2001 is similar to the folate content of breads before 2000 (3,24). It remains to be seen whether the folate content in other fortified cereal and cereal products has also decreased. The decrease in folate content of breads coincides with the decline in serum folate concentrations in our study. However, the exact reasons for the recent decline in serum folate concentrations warrant further investigation. Whether the recent decline we observed in serum folate concentrations would continue in the future also remains to be seen. If this trend continues, the effect of folic acid fortification may not be as high as in the early years of folic acid fortification.

Folic acid fortification had a dramatic effect on serum and RBC folate in all demographic groups, suggesting that folic acid fortification efforts have reached all segments of the U.S. population. Consequently, the prevalences of low serum and RBC folate, and high tHcy concentrations have decreased substantially in 1999–2002 compared with 1988–1994. Although the overall prevalence of low serum folate and RBC folate decreased to just below 1% and 7%, respectively, in 1999–2002 from 1988–1994, low RBC folate continues to be a problem in some populations. A substantial NHB population is at risk for low RBC folate, although the mean prevalence of low RBC folate in this population declined by 71% from 1988–1994 to 2001–2002. Similar to our observation, Perry et al. (25) found that serum and RBC folate concentrations were significantly lower in African Americans than in Caucasians. In addition, they excreted less urinary folate, suggesting that African Americans have a higher folate requirements rather than lower folate intakes (25).

Although serum folate concentrations declined slightly in 2001–2002 from 1999–2000, the prevalence of low folate became less frequent in 2001–2002 than in 1999–2000. This indicates that the modest decline in serum folate concentrations in 2001–2002 from 1999–2000 may have occurred predominantly in people with high folate intake levels rather than in those with lower intake levels. This trend is not undesirable given the unresolved concerns about the safety of long-term excess folic acid intake.

The increase in plasma tHcy concentrations in 2001–2002 from 1999–2000 is not surprising considering that there was a decline in serum folate during the same period. An inverse association between dietary folic acid or serum folate and tHcy concentrations has been well documented (26,27). The increase in tHcy in 2001–2002 from 1999–2000 was not always matched by a decline in serum folate concentrations, suggesting that slight and variable folate changes were not solely responsible for the variability in the tHcy concentrations.

It is important to note that methodological changes have occurred in tHcy concentration measurements between NHANES III and NHANES 1999–2002. Pfeiffer et al. (28) determined the potential differences in tHcy measurement between these 2 surveys. They estimated that tHcy concentrations were overestimated by 10% in NHANES III due to the use of serum rather than plasma. There was a 6% method bias between NHANES III and NHANES 1999+. Although these 2 effects with respect to comparison of tHcy concentrations between NHANES III and NHANES 1999+ might cancel each other out, caution should be used in interpreting the tHcy data between NHANES III and NHANES 1999–2002 (28).

Folic acid fortification had a significant effect on the risk for NTD. Based on the national birth certificate data, Honein et al. (29) reported that the prevalence of NTD decreased by 19% in the United States when pre-folic acid fortification data were compared with the post-folic acid fortification data. In Ontario, Canada, the NTD incidence decreased by 47% from 1995 to 1999 (30). The decline in the prevalence of NTD was attributed to folic acid fortification although other factors may have contributed. Another potential benefit of folic acid fortification is decreased risk for heart diseases (31). Between 1988–1994 and 1999–2002, we observed a substantial decrease in age-, sex-, and race-ethnicity–adjusted tHcy concentrations due to increased intake of folic acid from folic acid fortification. A reduction of this proportion cannot be attributed to methodological differences in tHcy measurement between NHANES 1988–1994 and NHANES 1999–2002. Whether a reduction of this magnitude in tHcy concentrations would lower mortality and morbidity from cardiovascular diseases remains unknown.

This study has several strengths. Because the results in this study were based on nationally representative sample surveys, these findings can be applied to the general U.S. population. In this study, we included only subjects who had fasted 10 h because folate concentration measured in a fasted state is a better indicator of folate status. In conclusion, serum folate concentrations have declined significantly in recent years in the United States. This is likely due to decreased folate intakes associated with decreased folic acid added to fortified cereals. Based on the improved folate status, folic acid fortification efforts have reached all segments of the U.S. population. Low RBC folate concentrations in NHB continue to be a problem despite a significant improvement in the overall folate nutritional status. Because few data exist on the safety of high intake of folic acid, it is important to monitor the serum and RBC folate concentrations on a regular basis, especially in younger and older populations.

FOOTNOTES

¹ Presented in part at Experimental Biology 05, April 2005, San Diego, CA [Ganji V, Kafai MR. Effect of folic acid fortification on serum folate, red blood cell folate, and total homocysteine concentrations in the US (abstract). FASEB J. 2005;19:A1619].

³ Abbreviations used: ANCOVA, analysis of covariance; MA/H, Mexican American/Hispanic, MEC, Mobile Examination Centers, NHANES, National Health and Nutrition Examination Survey; NHB, non-Hispanic black, NHW, non-Hispanic white, NTD, neural tube defect; PIR, poverty income ratio; tHcy, total homocysteine.

Manuscript received 17 May 2005. Initial review completed 23 June 2005. Revision accepted 12 October 2005.

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