Dolutegravir- Versus Efavirenz-Based Treatment in Pregnancy: Impact on Red Blood Cell Folate Concentrations in Pregnant Women and Their Infants.

In IMPAACT 2010/VESTED, pregnant women were randomized to initiate dolutegravir (DTG)+emtricitabine (FTC)/tenofovir alafenamide (TAF), DTG+FTC/tenofovir disoproxil fumarate (TDF), or efavirenz (EFV)/FTC/TDF. We assessed red blood cell folate concentrations (RBC-folate) at maternal study entry and delivery, and infant birth. RBC-folate outcomes were: 1) maternal change entry to delivery (trajectory), 2) infant, 3) ratio of infant-to-maternal delivery. Generalized estimating equation models for each log(folate) outcome were fit to estimate adjusted geometric mean ratio (Adj-GMR)/GMR trajectories (Adj-GMRT) of each arm comparison in 340 mothers and 310 infants. Overall, 90% of mothers received folic acid supplements and 78% lived in Africa. At entry, median maternal age was 25 years, gestational age was 22 weeks, CD4 count was 482 cells/mm3 and log10HIV RNA was 3 copies/mL. Entry RBC-folate was similar across arms. Adj-GMRT of maternal folate was 3% higher in the DTG+FTC/TAF versus EFV/FTC/TDF arm (1.03, 95%CI 1.00, 1.06). The DTG+FTC/TAF arm had an 8% lower infant-maternal folate ratio (0.92, 95%CI 0.78, 1.09) versus EFV/FTC/TDF. Results are consistent with no clinically meaningful differences between arms for all RBC-folate outcomes and they suggest that cellular uptake of folate and folate transport to the infant do not differ in pregnant women starting DTG- vs. EFV-based ART.

CDC's Vitamin A Laboratory-External Quality Assessment Program Shows Little Change in Laboratory Performance for Multiple Nutritional Biomarkers over a 10-Year Period (2008-2017).

BACKGROUND: The Vitamin A Laboratory-External Quality Assessment (VITAL-EQA) program operated by the CDC provides analytical performance assessment to low-resource laboratories conducting serum vitamins A (VIA), D (VID), B-12 (B12), and folate (FOL), as well as ferritin (FER) and CRP measurements for public health studies. OBJECTIVES: We aimed to describe the long-term performance of VITAL-EQA participants from 2008 to 2017. METHODS: Participating laboratories received 3 blinded serum samples biannually for duplicate analysis over 3 d. We assessed results (n = 6) for relative difference (%) from the CDC target value and imprecision (% CV) and conducted descriptive statistics on the aggregate 10-year and round-by-round data. Performance criteria were based on biologic variation and deemed acceptable (optimal, desirable, or minimal performance) or unacceptable (less than minimal performance). RESULTS: Thirty-five countries reported VIA, VID, B12, FOL, FER, and CRP results from 2008-2017. The proportion of laboratories with acceptable performance ranged widely by round: VIA 48%-79% (for difference) and 65%-93% (for imprecision), VID 19%-63% and 33%-100%, B12 0%-92% and 73%-100%, FOL 33%-89% and 78%-100%, FER 69%-100% and 73%-100%, and CRP 57%-92% and 87%-100%. On aggregate, ≥60% of laboratories achieved acceptable differences for VIA, B12, FOL, FER, and CRP (only 44% for VID), and over 75% of laboratories achieved acceptable imprecision for all 6 analytes. Laboratories participating continuously in 4 rounds (2016-2017) showed generally similar performance compared to laboratories participating occasionally. CONCLUSIONS: Although we observed little change in laboratory performance over time, on aggregate, >50% of the participating laboratories achieved acceptable performance, with acceptable imprecision being achieved more often than acceptable difference. The VITAL-EQA program is a valuable tool for low-resource laboratories to observe the state of the field and track their own performance over time. However, the small number of samples per round and the constant changes in laboratory participants make it difficult to identify long-term improvements.

Comparison of Current World Health Organization Guidelines with Physiologically Based Serum Ferritin Thresholds for Iron Deficiency in Healthy Young Children and Nonpregnant Women Using Data from the Third National Health and Nutrition Examination Survey.

BACKGROUND: Current WHO serum ferritin (SF) thresholds for iron deficiency (ID) in children (<12 µg/L) and women (<15 µg/L) are derived from expert opinion based on radiometric assays in use decades ago. Using a contemporary immunoturbidimetry assay, higher thresholds (children, <20 µg/L; women, <25 µg/L) were identified from physiologically based analyses. OBJECTIVE: We examined relationships of SF measured using an immunoradiometric assay from the era of expert opinion with 2 independently measured indicators of ID, hemoglobin (Hb) and erythrocyte zinc protoporphyrin (eZnPP), using data from the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994). The SF at which circulating Hb begins to decrease and eZnPP begins to increase provides a physiological basis for identifying the onset of iron-deficient erythropoiesis. METHODS: We analyzed NHANES III cross-sectional data from 2616 apparently healthy children, aged 12-59 mo, and 4639 apparently healthy nonpregnant women, aged 15-49 y. We used restricted cubic spline regression models to determine SF thresholds for ID. RESULTS: SF thresholds identified by Hb and eZnPP did not differ significantly in children, 21.2 µg/L (95% confidence interval: 18.5, 26.5) and 18.7 µg/L (17.9, 19.7), and, in women, were similar although significantly different, 24.8 µg/L (23.4, 26.9) and 22.5 µg/L (21.7, 23.3). CONCLUSIONS: These NHANES results suggest that physiologically based SF thresholds are higher than the thresholds from expert opinion established during the same era. SF thresholds found using physiological indicators detect the onset of iron-deficient erythropoiesis, whereas the WHO thresholds identify a later, more severe stage of ID.

Maternal Periconceptional Folic Acid Supplementation and DNA Methylation Patterns in Adolescent Offspring.

BACKGROUND: Folate, including the folic acid form, is a key component of the one-carbon metabolic pathway used for DNA methylation. Changes in DNA methylation patterns during critical development periods are associated with disease outcomes and are associated with changes in nutritional status in pregnancy. The long-term impact of periconceptional folic acid supplementation on DNA methylation patterns is unknown. OBJECTIVES: To determine the long-term impact of periconceptional folic acid supplementation on DNA methylation patterns, we examined the association of the recommended dosage (400 µg/d) and time period (periconceptional before pregnancy through first trimester) of folic acid supplementation with the DNA methylation patterns in the offspring at age 14-17 y compared with offspring with no supplementation. METHODS: Two geographic sites in China from the 1993-1995 Community Intervention Program of folic acid supplementation were selected for the follow-up study. DNA methylation at 402,730 CpG sites was assessed using saliva samples from 89 mothers and 179 adolescents (89 male). The mean age at saliva collection was 40 y among mothers (range: 35-54 y) and 15 y among adolescents (range: 14-17 y). Epigenome-wide analyses were conducted to assess the interactions of periconceptional folic acid exposure, the 5,10-methylenetetrahydrofolate reductase (MTHFR)-C677T genotype, and epigenome-wide DNA methylation controlling for offspring sex, geographic region, and background cell composition in the saliva. RESULTS: In the primary outcome, no significant differences were observed in epigenome-wide methylation patterns between adolescents exposed and those non-exposed to maternal periconceptional folic acid supplementation after adjustment for potential confounders [false discovery rate (FDR) P values < 0.05]. The MTHFR-C677T genotype did not modify this lack of association (FDR P values < 0.05). CONCLUSIONS: Overall, there were no differences in DNA methylation between adolescents who were exposed during the critical developmental window and those not exposed to the recommended periconceptional/first-trimester dosage of folic acid.

Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays.

OBJECTIVES: The clinical use of soluble transferrin receptor (sTfR) as an iron status indicator is hindered by a lack of assay standardization and common reference ranges and decision thresholds. In 2009, the WHO and National Institute for Biological Standards and Controls (NIBSC) released a sTfR reference material (RM), 07/202, for assay standardization; however, a comprehensive, formal commutability study was not conducted. METHODS: This study evaluated the commutability of WHO 07/202 sTfR RM and human serum pools and the impacts of their use as common calibrators. Commutability was assessed for six different measurement procedures (MPs). Serum pools were prepared according to updated CLSI C37-A procedures (C37) or non-C37 procedures. The study design and analyses were based on Parts 2 and 3 of the 2018 IFCC Commutability in Metrological Traceability Working Group's Recommendations for Commutability Assessment. WHO 07/202 and serum pools were used for instrument/assay and mathematical recalibration, respectively, to determine if their use decreases inter-assay measurement variability for clinical samples. RESULTS: The WHO 07/202 RM dilutions were commutable for all 6 MPs assessed and, when used for instrument calibration, decreased inter-assay variability from 208 to 55.7 %. Non-C37 and C37 serum pools were commutable for all 6 MPs assessed and decreased inter-assay variability from 208 to 13.8 % and 4.6 %, respectively, when used for mathematical recalibration. CONCLUSIONS: All materials evaluated, when used as common calibrators, substantially decreased inter-assay sTfR measurement variability. MP calibration to non-C37 and C37 serum pools may reduce the sTfR IMPBR to a greater extent than WHO 07/202 RM.

Development of an improved standard reference material for folate vitamers in human serum.

The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material® (SRM®) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-µg folic acid supplement 1 h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates.

The VitMin Lab Sandwich-ELISA Assays for Iron and Inflammation Markers Compared Well with Clinical Analyzer Reference-Type Assays in Subsamples of the Nepal National Micronutrient Status Survey.

BACKGROUND: The low cost and small specimen volume of the VitMin Lab ELISA assays for serum ferritin (Fer), soluble transferrin receptor (sTfR), C-reactive protein (CRP), and α-1-acid glycoprotein (AGP) have allowed their application to micronutrient surveys conducted in low-resource countries for ∼2 decades. OBJECTIVES: We conducted a comparison between the ELISA and reference-type assays used in the US NHANES. METHODS: Using the Roche clinical analyzer as a reference, we measured random subsets of the 2016 Nepal National Micronutrient Status Survey (200 serum samples from children aged 6-59 mo; 100 serum samples from nonpregnant women) for Fer, sTfR, CRP, and AGP. We compared the combined data sets with the ELISA survey results using descriptive analyses. RESULTS: The Lin's concordance coefficients between the 2 assays were ≥0.89 except for sTfR (Lin's ρ = 0.58). The median relative difference to the reference was as follows: Fer, -8.5%; sTfR, 71.2%; CRP, -19.5%; and AGP, -8.2%. The percentage of VitMin samples agreeing within ±30% of the reference was as follows: Fer, 88.5%; sTfR, 1.70%; CRP, 74.9%; and AGP, 92.9%. The prevalence of abnormal results was comparable between the 2 assays for Fer, CRP, and AGP, and for sTfR after adjusting to the Roche assay. Continued biannual performance (2007-2019) of the VitMin assays in CDC's external quality assessment program (6 samples/y) demonstrated generally acceptable performance. CONCLUSIONS: Using samples from the Nepal survey, the VitMin ELISA assays produced mostly comparable results to the Roche reference-type assays for Fer, CRP, and AGP. The lack of sTfR assay standardization to a common reference material explains the large systematic difference observed for sTfR, which could be corrected by an adjustment equation pending further validation. This snapshot comparison together with the long-term external quality assessment links the survey data generated by the VitMin Lab to the Roche assays used in NHANES.

Iron Deficiency in the United States: Limitations in Guidelines, Data, and Monitoring of Disparities.

Iron deficiency and the more severe sequela, iron deficiency anemia, are public health problems associated with morbidity and mortality, particularly among pregnant women and younger children. The 1998 Centers for Disease Control and Prevention recommendations for prevention and control of iron deficiency in the United States is old and does not reflect recent evidence but is a foundational reference for many federal, clinical, and program guidelines. Surveillance data for iron deficiency are sparse at all levels, with critical gaps for pregnant women and younger children. Anemia, iron deficiency, and iron deficiency anemia are often conflated but should not be. Clinical guidelines for anemia, iron deficiency, and iron deficiency anemia give inconsistent recommendations, causing nonsystematic assessment of iron deficiency. Screening for iron deficiency typically relies on identifying anemia, despite anemia's low sensitivity for iron deficiency. In the National Health and Nutrition Examination Survey, more than 70% of iron deficiency is missed among pregnant women and children by relying on hemoglobin for iron deficiency screening. To improve assessment and diagnosis and strengthen surveillance, better and more complete data and updated foundational guidance on iron deficiency and anemia are needed that consider new evidence for measuring and interpreting laboratory results. (Am J Public Health. 2022;112(S8):S826-S835. https://doi.org/10.2105/AJPH.2022.306998).

Urinary nitrate and sodium in a high-risk area for upper gastrointestinal cancers: Golestan Cohort Study☆.

BACKGROUND: The epidemiological evidence regarding the carcinogenicity of nitrate and sodium in drinking water is limited, partly because measuring the exposure at the individual level is complex. Most studies have used nitrate in water supplies as a proxy for individual exposure, but dietary intakes and other factors may contribute to the exposure. The present study investigates the factors associated with urinary nitrate and sodium in a high-risk area for esophageal and gastric cancers. METHODS: For this cross-sectional study, we used data and samples collected in 2004-2008 during the enrollment phase of the Golestan Cohort Study from a random sample of 349 participants (300 individuals from 24 rural villages and 49 from the city of Gonbad), stratified by average water nitrate in their district, the source of drinking water, and the usual dietary intake of nitrate and sodium. Nitrate, sodium, and creatinine were measured in a spot urine sample collected at the time of interview. We used the provincial cancer registry data to calculate the cumulative incidence rates of esophageal and gastric cancers for each location through June 1, 2020, and used weighted partial Pearson correlation to compare the incidence rates with median urinary nitrate and sodium in each village or the city. RESULTS: Among 349 participants (mean age±SD: 50.7 ± 8.6 years), about half (n = 170) used groundwater for drinking, and the use of groundwater was significantly more common in high-elevation locations (75.8%). The geometric mean of the creatinine-corrected urinary nitrate concentration was 68.3 mg/g cr (95%CI: 64.6,72.3), and the corresponding geometric mean for urinary sodium was 150.0 mmoL/g cr (95%CI: 139.6,161.1). After adjusting for confounders, urinary nitrate was associated with being a woman, drinking groundwater, and living in high-elevation locations, but not with estimated dietary intake. Urinary sodium concentration was significantly associated with monthly precipitation at the time of sampling but not with elevation or drinking water source. There were significant positive correlations between both median urinary nitrate and sodium in each location and esophageal cancer incidence rates adjusted for sex and age (r = 0.65 and r = 0.58, respectively, p < 0.01), but not with gastric cancer incidence. CONCLUSION: In a rural population at high risk for esophageal and gastric cancers, nitrate excretion was associated with living at a higher elevation and using groundwater for drinking. The associations between nitrate and sodium excretion with esophageal cancer incidence warrant future investigation.

Folate Forms in RBC and Whole-Blood Lysates Appear Stable When Stored Frozen for 2 Years.

BACKGROUND: The use of RBC lysate (RBC-Lys) eliminates the need for serum folate and hematocrit (Hct) measurement to calculate RBC folate. Information on the long-term frozen storage stability of RBC-Lys is missing. OBJECTIVES: We aimed to assess the comparability of RBC folate forms in whole-blood lysate (WB-Lys) and RBC-Lys and the folate stability in both matrices. METHODS: We prepared conventional WB-Lys (1:11 dilution with 1% ascorbic acid) and RBC-Lys (1:11 dilution of washed and saline-diluted RBCs with 1% ascorbic acid) from EDTA blood (n = 60 adult donors) and stored lysates at -70°C until analysis at baseline (1 wk), 3, 6, 12, and 24 mo. Before analysis by HPLC-tandem MS, we incubated the WB-Lys (4 h at 37°C) and treated the RBC-Lys with human recombinant γ-glutamyl hydrolase for folate polyglutamate deconjugation. We analyzed RBC-Lys samples for hemoglobin (Hb) (same aliquot) to normalize for the preanalytical dilution; Hb-folate was converted to RBC folate for each folate form using the mean corpuscular Hb concentration. We analyzed Hct as well as folate forms in matching serum samples for traditional RBC folate calculation. We conducted descriptive data analyses (correlation, Bland-Altman plot, Deming regression). RESULTS: At baseline, results for RBC folate forms derived from WB-Lys compared with RBC-Lys samples showed excellent correlation (Pearson r ≥ 0.97). Mean ± SD concentrations compared well for total folate (WB-Lys: 886 ± 255 compared with RBC-Lys: 899 ± 271 nmol/L), 5-methyltetrahydrofolate (WB-Lys: 831 ± 258 compared with RBC-Lys: 843 ± 276 nmol/L), and nonmethyl folate (WB-Lys: 53.3 ± 74.4 compared with RBC-Lys: 52.9 ± 70.7 nmol/L), but were 17% higher in RBC-Lys for pyrazino-s-triazine derivative of 4α-hydroxy-5-CH3-H4folate (MeFox) (WB-Lys: 147 ± 44.1 compared with RBC-Lys: 172 ± 53.5 nmol/L). Frozen storage of WB-Lys and RBC-Lys samples for ≤24 mo showed ≤5%, ≤5%, ≤13%, and ≤11% change in total folate, 5-methyltetrahydrofolate, nonmethyl folate, and MeFox, respectively. CONCLUSIONS: Erythrocyte folate forms appear to be stable in RBC-Lys samples stored frozen at -70°C for ≤2 y. The relatively small changes in folate concentrations over time were comparable between RBC-Lys and conventionally prepared WB-Lys samples.

Supplemental Vitamin D Increased Serum Total 25-Hydroxyvitamin D in the US Adult Population During 2007-2014.

BACKGROUND: Data from the 2007-2010 NHANES suggested that vitamin D supplements contributed to increased serum concentrations of 25-hydroxyvitamin D [25(OH)D] in the US population. OBJECTIVES: We sought to determine whether 25(OH)D continued to increase during NHANES 2011-2014 and whether associations of 25(OH)D with preselected covariates differed across time periods. METHODS: For this study, 25(OH)D was measured in adults (≥20 y) using LC-MS/MS. Descriptive and regression analyses were stratified by survey period to investigate the effects of age, race-Hispanic origin, sex, season, BMI, dietary vitamin D, and vitamin D-containing supplements. A multiple linear regression model was used to assess 25(OH)D changes between two 4-y survey periods, namely 2007-2010 and 2011-2014. RESULTS: We observed several significant concomitant increases between 2007-2010 and 2011-2014: unadjusted mean 25(OH)D increased by 2.7 nmol/L (95% CI: 0, 5.4 nmol/L; P = 0.048), the percentage of persons taking any vitamin D-containing supplements increased 2.9% (95% CI: 0.03, 5.5%; P = 0.0314), and the percentage of persons taking high-dose (≥1000 IU/d) vitamin D-containing supplements increased 8.6% (95% CI: 6.9, 9.9%; P < 0.0001). With covariate adjustment, the increase in 25(OH)D from 2007-2010 to 2011-2014 was no longer statistically significant [1.4 nmol/L (95% CI: -3.0, 0.23 nmol/L; P = 0.09)]. After adjustments, several large differences in 25(OH)D remained, namely non-Hispanic blacks had 25(OH)D 22 nmol/L lower than that of non-Hispanic whites, and users of vitamin D-containing supplements ≥1000 IU/d had 25(OH)D 31 nmol/L higher than that of nonusers. CONCLUSIONS: After adjusting for vitamin D supplement dose, the overall adjusted increase in 25(OH)D was no longer statistically significant, suggesting that changes in US adults' 25(OH)D concentrations between NHANES periods 2007-2010 and 2011-2014 may primarily be associated with changes in vitamin D supplementation.

Demographic, Physiologic, and Lifestyle Characteristics Observed with Serum Total Folate Differ Among Folate Forms: Cross-Sectional Data from Fasting Samples in the NHANES 2011-2016.

BACKGROUND: Serum folate forms were measured in the US population during recent NHANES to assess folate status. OBJECTIVE: We describe post-folic acid-fortification concentrations of serum folate forms in the fasting US population ≥1 y from the NHANES 2011-2016. METHODS: We measured 5 biologically active folates and 1 oxidation product (MeFox) of 5-methyltetrahydrofolate (5-methyl-THF). We calculated geometric means of 5-methyl-THF, unmetabolized folic acid (UMFA), nonmethyl folate (sum of tetrahydrofolate, 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate), total folate (sum of above biomarkers), and MeFox by demographic, physiologic, and lifestyle variables; estimated the magnitude of variables on biomarker concentrations after covariate adjustment; and determined the prevalence of UMFA >2 nmol/L. RESULTS: After demographic adjustment, age, sex, and race-Hispanic origin were significantly associated with most folate forms. MeFox increased with age, while 5-methyl-THF, UMFA, and nonmethyl folate displayed U-shaped age patterns. Compared with non-Hispanic whites, non-Hispanic blacks had 23% lower predicted 5-methyl-THF but comparable UMFA; non-Hispanic Asians had comparable 5-methyl-THF but 28% lower UMFA; Hispanics, non-Hispanic Asians, and non-Hispanic blacks had ∼20% lower MeFox. After additional physiologic and lifestyle adjustment, predicted UMFA and MeFox concentrations were 43% and 112% higher, respectively, in adults with chronic kidney disease and 17% and 15% lower, respectively, in adults consuming daily 1-<2 alcoholic beverages; 5-methyl-THF concentrations were 20% lower in adult smokers. The prevalence of UMFA >2 nmol/L was highest in persons aged ≥70 y (9.01%) and lowest in those aged 12-19 y (1.14%). During 2011-2014, the prevalence was 10.6% in users and 2.22% in nonusers of folic acid-containing supplements. CONCLUSIONS: In fasting persons ≥1 y, the demographic, physiologic, and lifestyle characteristics observed with serum total folate differed among folate forms, suggesting biological and/or genetic influences on folate metabolism. High UMFA was mostly observed in supplement users and older persons.

Harmonizing the Calibrator and Microorganism Used in the Folate Microbiological Assay Increases the Comparability of Serum and Whole-Blood Folate Results in a CDC Round-Robin Study.

Background: Harmonizing critical reagents for the folate microbiological assay (MBA) may improve among-laboratory comparability. Objective: We assessed the comparability of the MBA for serum folate (S-FOL) and whole-blood folate (WB-FOL) in an international comparison study. Methods: Eight laboratories obtained a kit containing CDC microorganism inoculum (chloramphenicol-resistant Lactobacillus rhamnosus), CDC calibrator (5-methyltetrahydrofolate), and 23 serum and WB hemolysate samples each. Laboratories analyzed the samples in single measurement over 2 d using 4 conditions: in-house microorganism and in-house calibrator (IH-MO & IH-CAL), in-house microorganism and CDC calibrator (IH-MO & CDC-CAL), CDC microorganism and in-house calibrator (CDC-MO & IH-CAL), and CDC microorganism and CDC calibrator (CDC-MO & CDC-CAL). We calculated geometric mean concentrations for each laboratory and condition and compared data to the CDC MBA (target). Results: The among-laboratory arithmetic mean S-FOL concentrations for the 4 conditions were 30.2, 28.1, 30.0, and 29.9 (group 1, 5-methyltetrahydrofolate IH-CAL) compared with 35.3, 33.3, 33.6, and 30.7 nmol/L (group 2, folic acid IH-CAL), respectively; and 428, 405, 398, and 393 (group 1) compared with 469, 423, 477, and 418 nmol/L (group 2), respectively, for WB-FOL. Differences to the CDC MBA target values were smaller for group 1 (range across conditions; S-FOL: 9.9-21%; WB-FOL: 9.0-18%) compared with group 2 laboratories (S-FOL: 13-30%; WB-FOL: 16-32%) and smaller when CDC reagents were used compared with in-house reagents (S-FOL: 12% compared with 22%; WB-FOL: 13% compared with 25%). A linear mixed model estimated a small microorganism effect (S-FOL: 2.3%; WB-FOL: 2.3%) and a larger mean calibrator effect; folic acid compared with 5-methyltetrahydrofolate calibrator produced 12% higher S-FOL and 15% higher WB-FOL results. When laboratories used CDC reagents, the estimated among-laboratory variability was ∼10% for S-FOL and WB-FOL. Conclusion: Harmonizing the calibrator and microorganism for the folate MBA has the potential to improve the among-laboratory comparability in future surveys.

Biomarkers of Nutrition for Development (BOND)-Iron Review.

This is the fifth in the series of reviews developed as part of the Biomarkers of Nutrition for Development (BOND) program. The BOND Iron Expert Panel (I-EP) reviewed the extant knowledge regarding iron biology, public health implications, and the relative usefulness of currently available biomarkers of iron status from deficiency to overload. Approaches to assessing intake, including bioavailability, are also covered. The report also covers technical and laboratory considerations for the use of available biomarkers of iron status, and concludes with a description of research priorities along with a brief discussion of new biomarkers with potential for use across the spectrum of activities related to the study of iron in human health.The I-EP concluded that current iron biomarkers are reliable for accurately assessing many aspects of iron nutrition. However, a clear distinction is made between the relative strengths of biomarkers to assess hematological consequences of iron deficiency versus other putative functional outcomes, particularly the relationship between maternal and fetal iron status during pregnancy, birth outcomes, and infant cognitive, motor and emotional development. The I-EP also highlighted the importance of considering the confounding effects of inflammation and infection on the interpretation of iron biomarker results, as well as the impact of life stage. Finally, alternative approaches to the evaluation of the risk for nutritional iron overload at the population level are presented, because the currently designated upper limits for the biomarker generally employed (serum ferritin) may not differentiate between true iron overload and the effects of subclinical inflammation.

Estimated 24-Hour Urinary Sodium and Potassium Excretion in US Adults.

Importance: In 2010, the Institute of Medicine (now the National Academy of Medicine) recommended collecting 24-hour urine to estimate US sodium intake because previous studies indicated 90% of sodium consumed was excreted in urine. Objective: To estimate mean population sodium intake and describe urinary potassium excretion among US adults. Design, Setting, and Participants: In a nationally representative cross-sectional survey of the US noninstitutionalized population, 827 of 1103 (75%) randomly selected, nonpregnant participants aged 20 to 69 years in the examination component of the National Health and Nutrition Examination Survey (NHANES) collected at least one 24-hour urine specimen in 2014. The overall survey response rate for the 24-hour urine collection was approximately 50% (75% [24-hour urine component response rate] × 66% [examination component response rate]). Exposures: 24-hour collection of urine. Main Outcomes and Measures: Mean 24-hour urinary sodium and potassium excretion. Weighted national estimates of demographic and health characteristics and mean electrolyte excretion accounting for the complex survey design, selection probabilities, and nonresponse. Results: The study sample (n = 827) represented a population of whom 48.8% were men; 63.7% were non-Hispanic white, 15.8% Hispanic, 11.9% non-Hispanic black, and 5.6% non-Hispanic Asian; 43.5% had hypertension (according to 2017 hypertension guidelines); and 10.0% reported a diagnosis of diabetes. Overall mean 24-hour urinary sodium excretion was 3608 mg (95% CI, 3414-3803). The overall median was 3320 mg (interquartile range, 2308-4524). In secondary analyses by sex, mean sodium excretion was 4205 mg (95% CI, 3959-4452) in men (n = 421) and 3039 mg (95% CI, 2844-3234) in women (n = 406). By age group, mean sodium excretion was 3699 mg (95% CI, 3449-3949) in adults aged 20 to 44 years (n = 432) and 3507 mg (95% CI, 3266-3748) in adults aged 45 to 69 years (n = 395). Overall mean 24-hour urinary potassium excretion was 2155 mg (95% CI, 2030-2280); by sex, 2399 mg (95% CI, 2253-2545) in men and 1922 mg (95% CI, 1757-2086) in women; and by age, 1986 mg (95% CI, 1878-2094) in adults aged 20 to 44 years and 2343 mg (95% CI, 2151-2534) in adults aged 45 to 69 years. Conclusions and Relevance: In cross-sectional data from a 2014 sample of US adults, estimated mean sodium intake was 3608 mg per day. The findings provide a benchmark for future studies.

The Validity of Predictive Equations to Estimate 24-Hour Sodium Excretion: The MESA and CARDIA Urinary Sodium Study.

We examined the population distribution of urinary sodium concentrations and the validity of existing equations predicting 24-hour sodium excretion from a single spot urine sample among older adults with and without hypertension. In 2013, 24-hour urine collections were obtained from 554 participants in the Multi-Ethnic Study of Atherosclerosis and the Coronary Artery Risk Development in Young Adults study, who were aged 45-79 years and of whom 56% were female, 58% were African American, and 54% had hypertension, in Chicago, Illinois. One-third provided a second 24-hour collection. Four timed (overnight, morning, afternoon, and evening) spot urine specimens and the 24-hour collection were analyzed for sodium and creatinine concentrations. Mean 24-hour sodium excretion was 3,926 (standard deviation (SD), 1,623) mg for white men, 2,480 (SD, 1,079) mg for white women, 3,454 (SD, 1,651) mg for African-American men, and 3,397 (SD, 1,641) mg for African-American women, and did not differ significantly by hypertensive status. Mean bias (difference) in predicting 24-hour sodium excretion from the timed spot urine specimens ranged from -182 (95% confidence interval: -285, -79) to 1,090 (95% confidence interval: 966, 1,213) mg/day overall. Although the Tanaka equation using the evening specimen produced the least bias overall, no single equation worked well across subgroups of sex and race/ethnicity. A single spot urine sample is not a valid indicator of individual sodium intake. New equations are needed to accurately estimate 24-hour sodium excretion for older adults.

Two International Round-Robin Studies Showed Good Comparability of 5-Methyltetrahydrofolate but Poor Comparability of Folic Acid Measured in Serum by Different High-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods.

Background: Serum folate methods produce different results. The comparability of HPLC-mass spectrometry (MS)/MS methods is not well documented.Objective: We conducted an international "round-robin" investigation to assess the comparability, precision, and accuracy of serum folate HPLC-MS/MS methods.Methods: The CDC laboratory, 7 laboratories with independently developed methods (group 1), and 6 laboratories with an adapted CDC method (group 2) analyzed folate forms in 6 serum pools and 6 calibrators from the CDC (duplicate analysis over 2 d) and in two 3-level reference materials (duplicate analysis).Results: All laboratories measured 5-methyltetrahydrofolate (5-methylTHF) and folic acid; some measured additional folate forms. The geometric mean (range) concentrations (nanomoles per liter) for 5-methylTHF in the 6 serum pools were 18.3 nmol/L (CDC), 13.8-28.9 nmol/L (group 1), and 16.8-18.6 nmol/L (group 2); for folic acid the concentrations were 3.42 nmol/L (CDC), 1.09-4.74 nmol/L (group 1), and 1.74-2.90 nmol/L (group 2). The median imprecision (CV) for 5-methylTHF was 4.1% (CDC), 4.6-11% (group 1), and 1.7-6.0% (group 2); for folic acid it was 6.9% (CDC), 4.9-20% (group 1), and 3.9-23% (group 2). The mean ± SD (range) recovery of 5-methylTHF spiked into serum was 98% ± 27% (59-138%) for group 1 and 98% ± 10% (82-111%) for group 2; for folic acid it was 93% ± 29% (67-198%) for group 1 and 81% ± 16% (64-102%) for group 2. The mean relative bias for 5-methylTHF compared with the reference material certificate value was 12% (CDC), -24% to 30% (group 1), and -0.6% to 16% (group 2); for folic acid it was 73% (CDC), -47% to 578% (group 1), and -3.3% to 67% (group 2).Conclusions: For 5-methylTHF, group 2 laboratories demonstrated better agreement and precision, less variable spiking recovery, and less bias by using a reference material. Laboratory performance for folic acid was highly variable and needs improvement. Certified reference materials for serum folate forms and total folate are needed to improve method accuracy.

A Daily Dose of 5 mg Folic Acid for 90 Days Is Associated with Increased Serum Unmetabolized Folic Acid and Reduced Natural Killer Cell Cytotoxicity in Healthy Brazilian Adults.

Background: The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown.Objective: The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR), interferon γ (IFNG), tumor necrosis factor α (TNFA), and interleukin 8 (IL8) genes; and concentrations of serum inflammatory markers.Methods: This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m2) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells.Results: Serum folate concentrations increased by ∼5-fold after the intervention (P < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline (P < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number (P < 0.001) and cytotoxicity (P = 0.003) of NK cells after 45 and 90 d. Compared with baseline, DHFR mRNA expression was higher at 90 d (P = 0.006) and IL8 and TNFA mRNA expressions were higher at 45 and 90 d (P = 0.001 for both).Conclusion: This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.

Association between Serum Unmetabolized Folic Acid Concentrations and Folic Acid from Fortified Foods.

OBJECTIVE: To investigate the association between serum unmetabolized folic acid (UMFA) concentrations and folic acid from fortified foods and nutrients known as dietary methyl-group donors (folate, methionine, choline, betaine and vitamins B2, B6 and B12) in participants exposed to mandatory fortification of wheat and maize flours with folic acid. METHODS: Cross-sectional study carried out with 144 healthy Brazilian participants, both sexes, supplement nonusers. Serum folate, UMFA, vitamin B12 and total plasma homocysteine (tHcy) were biochemically measured. Dietary intake was assessed by 2 non-consecutive 24-hour dietary recalls (24-HRs) and deattenuated energy-adjusted nutrient data were used for statistical analysis. RESULTS: Ninety eight (68.1%) participants were women. Median (interquartile range) age was 35.5 (28.0-52.0) years. Elevated serum folate concentrations (>45 nmol/L) were found in 17 (11.8%), while folate deficiency (<7 nmol/L) in 10 (6.9%) participants. No one had vitamin B12 deficiency (<148 pmol/L). An elevated serum UMFA concentration was defined as > 1 nmol/L (90th percentile). UMFA concentrations were positively correlated with folic acid intake and negatively correlated to choline, methionine and vitamin B6 intakes. Participants in the lowest quartile of UMFA concentrations had lower dietary intake of total folate (DFEs) and folic acid, and higher dietary intake of methionine, choline and vitamin B6 than participants in the highest quartile of UMFA. Folic acid intake (OR [95% CI] = 1.02 [1.01-1.04)] and being a male (OR [95% CI] = 0.40 [0.19-0.87) were associated with increased and reduced odds for UMFA concentrations > 0.55 nmol/L (median values), respectively. CONCLUSION: UMFA concentrations were directly influenced by folic acid intake from fortified foods in a healthy convenience sample of adult Brazilians exposed to mandatory flour fortification with folic acid.