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Background: Folate cutoffs for risk of deficiency compared with possible deficiency were originally derived differently (experimental compared with epidemiologic data), and their interpretations are different. The matching of cutoffs derived from one assay with population-based data derived from another assay requires caution., Objective: We assessed the extent of folate-status misinterpretation with the use of inappropriate cutoffs., Design: In the cross-sectional NHANES, serum and red blood cell (RBC) folate were first measured with the use of a radioprotein-binding assay (RPBA) (1988-2006) and, afterwards, with the use of a microbiologic assay (2007-2010). We compared prevalence estimates for assay-matched cutoffs (e.g., with the use of an RPBA cutoff with RPBA data) and assay-mismatched cutoffs (e.g., with the use of microbiologic assay cutoff with RPBA data) for risk of deficiency on the basis of megaloblastic anemia as a hematologic indicator in persons ≥4 y of age (e.g., serum folate concentration <7 nmol/L and RBC folate concentration <305 nmol/L derived with the use of a microbiologic assay), possible deficiency on the basis of rising homocysteine as a metabolic indicator in persons ≥4 y of age (e.g., serum folate concentration <10 nmol/L and RBC folate concentration <340 nmol/L derived with the use of an RPBA), and insufficiency on the basis of elevated risk of neural tube defects in women 12-49 y old (e.g., RBC folate concentration <906 nmol/L derived with the use of a microbiologic assay)., Results: Pre-folic acid fortification (1988-1994), risks of deficiency for assay-matched compared with assay-mismatched cutoffs were 5.6% compared with 16% (serum folate), respectively, and 7.4% compared with 28% (RBC folate), respectively; risks declined postfortification (1999-2006) to <1% compared with <1% (serum folate), respectively, and to <1% compared with 2.5% (RBC folate), respectively. Prefortification (1988-1994), risks of possible deficiency for assay-matched compared with assay-mismatched cutoffs were 35% compared with 56% (serum folate), respectively, and 37% compared with 84% (RBC folate), respectively; risks declined postfortification (1999-2006) to 1.9% compared with 7.0% (serum folate), respectively, and to 4.8% compared with 53% (RBC folate), respectively. Postfortification (2007-2010), risks of insufficiency were 3% (assay matched) compared with 39% (assay mismatched), respectively., Conclusions: The application of assay-mismatched cutoffs leads to a misinterpretation of folate status. This confusion likely applies to clinical assays because no comparability data are available, to our knowledge., (© 2016 American Society for Nutrition.)

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC-MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011-2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0·4) but significant (P< 0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

Background: Serum total folate consists mainly of 5-methyltetrahydrofolate (5-methylTHF). Unmetabolized folic acid (UMFA) may occur in persons consuming folic acid-fortified foods or supplements., Objectives: We describe serum 5-methylTHF and UMFA concentrations in the US population ≥1 y of age by demographic variables and fasting time, stratified by folic acid-containing dietary supplement use. We also evaluate factors associated with UMFA concentrations >1 nmol/L., Methods: Serum samples from the cross-sectional NHANES 2007-2008 were measured for 5-methylTHF (n = 2734) and UMFA (n = 2707) by HPLC-tandem mass spectrometry., Results: In supplement users compared with nonusers, we found significantly higher geometric mean concentrations of 5-methylTHF (48.4 and 30.7 nmol/L, respectively) and UMFA (1.54 and 0.794 nmol/L, respectively). UMFA concentrations were detectable (>0.3 nmol/L) in >95% of supplement users and nonusers, regardless of demographic or fasting characteristics; concentrations differed significantly by age and fasting time, but not by sex and race-ethnicity, both in supplement users and nonusers. The prevalence of UMFA concentrations >1 nmol/L was 33.2% overall and 21.0% in fasting (≥8 h) adults (≥20 y of age). Using multiple logistic regression analysis, UMFA concentrations >1 nmol/L were associated with being older, non-Hispanic black, nonfasting (<8 h), having smaller body surface area, higher total folic acid intake (diet and supplements), and higher red blood cell folate concentrations. In fasting adults, a decrease in the mean daily alcohol consumption was also associated with increased odds of UMFA concentrations >1 nmol/L., Conclusions: UMFA detection was nearly ubiquitous, and concentrations >1 nmol/L were largely but not entirely explained by fasting status and by total folic acid intake from diet and supplements. These new UMFA data in US persons ≥1 y of age provide much-needed information on this vitamer in a fortified population with relatively high use of dietary supplements., (© 2015 American Society for Nutrition.)

The NHANES measured serum and RBC folate concentrations by using a radioassay during prefortification (1988-1994) and postfortification (1999-2006) periods followed by the use of a microbiologic assay (MBA) from 2007-2010. The MBA produces higher concentrations than does the radioassay and is considered to be more accurate. To allow for accurate long-term trending (1988-2010), we evaluated different regression models (linear, piecewise linear, and fractional polynomial) to assay-adjust the radioassay results to be comparable to the MBA results. The data used to derive the regression models originated from 2 crossover studies in which the 2 assays were applied to a set of 325 serum and 171 whole-blood samples. Fractional polynomial regression of logarithmically transformed data provided the best fit for serum folate. Linear regression of logarithmically transformed whole-blood data provided an equally good fit compared with the other models and was the simplest to apply for RBC folate. Prefortification serum and RBC folate geometric mean concentrations increased after adjustment from 13.0 to 16.7 nmol/L and from 403 to 747 nmol/L, respectively. Postfortification serum folate concentrations increased from ~30 to ~43 nmol/L, and RBC folate concentrations increased from ~600 to ~1100 nmol/L after adjustment, with some variation across survey cycles. The presented regression equations allow the estimation of more accurate prevalence estimates and long-term trends in blood folate concentrations in the U.S. population by using results that are equivalent to the MBA. This information will be useful to public health officials in the United States who are dealing with folic acid fortification issues.

The NHANES has monitored folate status of the U.S. population from prefortification (1988-1994) to postfortification (1999-2010) by measuring serum and RBC folate concentrations. The Bio-Rad radioassay (BR) was used from 1988 to 2006, and the microbiologic assay (MBA) was used from 2007 to 2010. The MBA produces higher concentrations than the BR and is considered to be more accurate. Thus, to bridge assay differences and to examine folate trends over time, we adjusted the BR results to be comparable to the MBA results. Postfortification, assay-adjusted serum and RBC folate concentrations were 2.5 times and 1.5 times prefortification concentrations, respectively, and showed a significant linear trend (P < 0.001) to slightly lower concentrations during 1999-2010. The postfortification prevalence of low serum (<10 nmol/L) or RBC (<340 nmol/L) folate concentrations was ≤ 1%, regardless of demographic subgroup, compared with 24% for serum folate and 3.5% for RBC folate prefortification, with substantial variation among demographic subgroups. The central 95% reference intervals for serum and RBC folate varied by demographic subgroup during both pre- and postfortification periods. Age and dietary supplement use had the greatest effects on prevalence estimates of low folate concentrations during the prefortification period. In summary, the MBA-equivalent blood folate concentrations in the U.S. population showed first a sharp increase from pre- to postfortification, then showed a slight decrease (17% for serum and 12% for RBC folate) during the 12-y postfortification period. The MBA-equivalent pre- and postfortification reference concentrations will inform countries that plan folic acid fortification or that need to evaluate its impact.

A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.

Three laboratories participated with their laboratory-specific microbiologic growth assays (MA) in the NHANES 2007-2008 to assess whether the distributions of serum (n = 2645) and RBC folate (n = 2613) for the same one-third sample of participants were comparable among laboratories. Laboratory (L) 2 produced the highest and L1 the lowest serum and RBC folate geometric means (nmol/L) in the NHANES sample (serum: L1, 39.5; L2, 59.2; L3, 47.7; and RBC: L1, 1120; L2, 1380; L3, 1380). Each laboratory produced different reference intervals for the central 95% of the population. Pearson correlation coefficients were highest between L3 and L1 (serum, r = 0.95; RBC, r = 0.92) and lowest between L2 and L1 (serum, r = 0.81; RBC, r = 0.65). Notable procedural differences among the laboratories were the Lactobacillus rhamnosus microorganism (L1 and L3: chloramphenicol resistant, L2: wild type) and the calibrator [L1: [6S]5-methyltetrahydrofolate (5-methylTHF), L2: [6R,S] 5-formyltetrahydrofolate ([6R,S] 5-formylTHF), L3: folic acid (FA)]. Compared with 5-methylTHF as calibrator, the folate results were 22-32% higher with FA as calibrator and 8% higher with 5-formylTHF as calibrator, regardless of the matrix (n = 30 serum, n = 28 RBC). The use of different calibrators explained most of the differences in results between L3 and L1 but not between L2 and L1. The use of the wild-type L. rhamnosus by L2 appeared to be the main reason for the differences in results between L2 and the other 2 laboratories. These findings indicate how assay variations influence MA folate results and how those variations can affect population data. To ensure data comparability, better assay harmonization is needed.

Data from the National Health and Nutrition Examination Surveys. Very large increases in blood folate levels of the U.S. population occurred between 1988-1994 and 1999-2000. Small fluctuations in blood folate levels occurred over the time period 1999-2006. The median red blood cell (RBC) folate level of the U.S. population 4 years of age and older was 266 ng/mL in 2005-2006. The median serum folate level of the U.S. population 4 years of age and older was 12.2 ng/mL in 2005-2006. In 2005-2006, the prevalence of low RBC folate (less than 140 ng/mL) among U.S. women of childbearing age (15-45 years) was 4.5%. In 2005-2006, the prevalence of low serum folate (less than 3 ng/mL) among U.S. women of childbearing age was 0.5%. Folate is an essential vitamin for good health. Women of childbearing age are among the population subgroups that have been shown previously to have low blood folate levels. Low blood folate levels are associated with an increased risk of neural tube birth defects. Beginning in 1998, the Food and Drug Administration (FDA) required the addition of folic acid (a form of folate) to all enriched breads, cereals, flours, corn meal, pasta products, rice, and other cereal grain products sold in the United States. Blood folate data from the National Health and Nutrition Examination Surveys (NHANES) have documented improvements in the folate status of the U.S. population after folate fortification was implemented. Red blood cell (RBC) folate measures long-term folate intake and low levels are associated with adverse health effects. Serum folate reflects recent folate intake and low levels are an early indicator of inadequate folate status. Pre- and postfortification blood folate levels of the U.S. population 4 years of age and older and prevalence of low blood folate among women of childbearing age (15-45 years) are reported., (All material appearing in this report is in the public domain and may be reproduced or copied without permission; citation as to source, however, is appreciated.)

A roundtable to discuss the measurement of vitamin B- 12 (cobalamin) status hiomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-l2-related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-I 2 status used in past NHANES—serum vitamin B-12, methylnialonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B- 12—related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-l2 (serum vitamin B-12 or hoand one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-l2 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B- 2 and MMA. [ABSTRACT FROM AUTHOR]