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.

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.

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.

Multiple imputation to evaluate the impact of an assay change in national surveys.

National health surveys, such as the National Health and Nutrition Examination Survey, are used to monitor trends of nutritional biomarkers. These surveys try to maintain the same biomarker assay over time, but there are a variety of reasons why the assay may change. In these cases, it is important to evaluate the potential impact of a change so that any observed fluctuations in concentrations over time are not confounded by changes in the assay. To this end, a subset of stored specimens previously analyzed with the old assay is retested using the new assay. These paired data are used to estimate an adjustment equation, which is then used to 'adjust' all the old assay results and convert them into 'equivalent' units of the new assay. In this paper, we present a new way of approaching this problem using modern statistical methods designed for missing data. Using simulations, we compare the proposed multiple imputation approach with the adjustment equation approach currently in use. We also compare these approaches using real National Health and Nutrition Examination Survey data for 25-hydroxyvitamin D. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

National Estimates of Serum Total 25-Hydroxyvitamin D and Metabolite Concentrations Measured by Liquid Chromatography-Tandem Mass Spectrometry in the US Population during 2007-2010.

BACKGROUND: The 2007-2010 NHANES provides the first US nationally representative serum 25-hydroxyvitamin D [25(OH)D] concentrations measured by standardized liquid chromatography-tandem mass spectrometry. OBJECTIVE: We describe patterns for total 25(OH)D and individual metabolites in persons aged ≥1 y stratified by race-ethnicity and grouped by demographic, intake, physiologic, and lifestyle variables. METHODS: We measured 25-hydroxycholecalciferol [25(OH)D3], 25-hydroxyergocalciferol [25(OH)D2], and C3-epimer of 25(OH)D3 [C3-epi-25(OH)D3] in serum samples (n = 15,652) from the 2007-2010 cross-sectional NHANES [total 25(OH)D = 25(OH)D3 + 25(OH)D2]. RESULTS: Concentrations (median, detection rate) of 25(OH)D3 (63.6 nmol/L, 100%) and C3-epi-25(OH)D3 (3.40 nmol/L, 86%) were generally detectable; 25(OH)D2 was detectable in 19% of the population. Total 25(OH)D, 25(OH)D3, and C3-epi-25(OH)D3 displayed similar demographic patterns and were strongly correlated (Spearman's r > 0.70). Concentrations of 25(OH)D2 (90th percentile) were much higher in persons aged ≥60 y (17.3 nmol/L) than in younger age groups (≤4.88 nmol/L). We noted significant race-ethnicity differences in mean total 25(OH)D [non-Hispanic blacks (NHBs), Hispanics, and non-Hispanic whites (NHWs): 46.6, 57.2, and 75.2 nmol/L, respectively] and in the prevalence of total 25(OH)D <30 nmol/L overall (24% of NHBs, 6.4% of Hispanics, and 2.3% of NHWs) as well as stratified by season (winter months: 30% of NHBs, 7.5% of Hispanics, and 3.8% of NHWs; summer months: 17% of NHBs, 4.4% of Hispanics, and 1.6% of NHWs). Persons with higher vitamin D intakes (diet, supplements, or both) and those examined during May-October had significantly higher total 25(OH)D. Significant race-ethnicity interactions in a multiple linear regression model confirmed the necessity of providing race-ethnicity-specific estimates of total 25(OH)D. CONCLUSIONS: Race-ethnicity differences in the prevalence of low total 25(OH)D remained strong even after adjustment for season to account for the NHANES design imbalance between season, latitude, and race-ethnicity. The strong correlation between C3-epi-25(OH)D3 and 25(OH)D3 may be because the epimer is a metabolite of 25(OH)D3. The presence of 25(OH)D2 mainly in older persons is likely a result of high-dose prescription vitamin D2.

Urine excretion of caffeine and select caffeine metabolites is common in the U.S. population and associated with caffeine intake.

BACKGROUND: Caffeine is a widely consumed psychoactive stimulant and is of epidemiologic interest. Major sources of caffeine are challenging to standardize, and the use of biomarkers is proposed as an alternative means of assessing intake. OBJECTIVE: We described urine caffeine and caffeine metabolite concentrations (n = 2466) and excretion rates (n = 2261) in the US population ≥6 y by age, sex, race-ethnicity, and caffeine intake (from foods, beverages, and dietary supplements). METHODS: We measured caffeine and 14 of its metabolites in spot urine samples from the cross-sectional NHANES 2009-2010 by use of LC-tandem mass spectrometry. RESULTS: Caffeine and its metabolites were detectable in the urine of most persons, generally at concentrations ≥1 µmol/L. Median concentrations (95% CI) ranged from 0.560 (0.497, 0.620) µmol/L to 58.6 (48.6, 67.2) µmol/L; median excretion rates from 0.423 (0.385, 0.468) nmol/min to 46.0 (40.7, 50.2) nmol/min. Urine concentrations and excretion rates for 9 analytes (caffeine, theophylline, paraxanthine, 1-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 1,3,7-trimethyluric acid, and 5-acetylamino-6-amino-3-methyluracil) had moderate correlations with caffeine intake (Spearman ρ = 0.55-0.68, P < 0.0001); the remaining analytes had low correlations (ρ = 0.15-0.33, P < 0.0001). We observed larger differences in geometric mean concentrations and excretion rates between the highest vs. lowest quartiles of caffeine intake for these 9 compounds than the rest. Consistent with dietary caffeine intake, we observed that urine concentrations and excretion rates for most compounds were significantly (P < 0.05) higher in men than women, non-Hispanic whites than Hispanics and non-Hispanic blacks, and highest in persons aged 40-59 y. CONCLUSION: Excretion of caffeine and its metabolites in urine is common in the US population. According to the observed associations between spot urine concentrations or excretion rates with caffeine intake, several of these compounds show promise as potential biomarkers of caffeine intake.

Unmetabolized folic acid is detected in nearly all serum samples from US children, adolescents, and adults.

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.

Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011-2.

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.

Population Size Estimates for Men who Have Sex with Men and Persons who Inject Drugs.

Understanding geographic variation in the numbers of men who have sex with men (MSM) and persons who inject drugs (PWID) is critical to targeting and scaling up HIV prevention programs, but population size estimates are not available at generalizable sub-national levels. We analyzed 1999-2010 National Health and Nutrition Examination Survey data on persons aged 18-59 years. We estimated weighted prevalence of recent (past 12 month) male-male sex and injection drug use by urbanicity (the degree to which a geographic area is urban) and US census region and calculated population sizes. Large metro areas (population ≥1,000,000) had higher prevalence of male-male sex (central areas, 4.4% of men; fringe areas, 2.5%) compared with medium/small metro areas (1.4%) and nonmetro areas (1.1%). Injection drug use did not vary by urbanicity and neither varied by census region. Three-quarters of MSM, but only half of PWID, resided in large metro areas. Two-thirds of MSM and two-thirds of PWID resided in the South and West. Efforts to reach MSM would benefit from being focused in large metro areas, while efforts to reach PWID should be delivered more broadly. These data allow for more effective allocation of funds for prevention programs.

Robotic axial lower leg testing: repeatability and reproducibility.

PURPOSE: The purpose of this study was to determine the test-retest reliability and the repeatability over multiple days of a robotic testing device when used to measure laxity of the lower leg during a simulated dial test. METHODS: Ten healthy subjects were evaluated using an instrumented robotic lower leg testing system over 4 days. Three testing cycles were performed each day. Each leg was rotated into external and then internal rotation by servomotors until a torque threshold of 5.65 N m was reached. Load-deformation curves were generated from torque and rotation data. Both average-measure and single-measure intraclass correlation coefficients (ICC) were compared across the curves. ICC scores were also compared for features of the curves including: maximum external rotation at -5.65 N m of torque, maximum internal rotation at 5.65 N m of torque, rotation at torque 0, compliance (slope of load-deformation curve) at torque 0, endpoint compliance in external rotation, endpoint compliance in internal rotation, and play at torque 0. Play at torque 0 was defined as the width of the hysteresis curve at torque 0. RESULTS: Average-measure ICC scores and test-retest scores were >0.95 along the entire load-deformation curve except around zero torque. ICC scores at maximum internal and external rotation ranged from 0.87 to 0.99 across the left and right knees. ICC scores for the other features of the curves ranged from 0.61 to 0.98. The standard error of the mean ranged from 0.0497 to 1.1712. CONCLUSIONS: The robotic testing device in this study proved to be reliable for testing a subject multiple times both within the same day and over multiple days. These findings suggest that the device can provide a level of reliability in rotational testing that allows for clinical use of test results. Objective laxity data can improve consistency and accuracy in diagnosing knee injuries and may enable more effective treatment.

Prevalence of HIV, sexually transmitted infections, and viral hepatitis by Urbanicity, among men who have sex with men, injection drug users, and heterosexuals in the United States.

BACKGROUND: Men who have sex with men (MSM), injection drug users (IDUs), and certain subgroups of heterosexuals are disproportionately affected by the syndemics of HIV, other sexually transmitted infections, and viral hepatitis. Although understanding the burden of these infections in these populations by urbanicity (the degree to which a geographic area is urban) is critical to targeting prevention programs, few studies have done so. METHODS: We analyzed nationally representative 1999 to 2010 data from the National Health and Nutrition Examination Survey on persons aged 18 to 59 years. We estimated the weighted prevalence of HIV, herpes simplex virus type 2 (HSV-2), human papillomavirus, chlamydia, hepatitis B, and hepatitis C, stratified by urbanicity level, for the overall sample, MSM, IDUs, and heterosexuals. Geographic areas with population at least million are classified into large central and large fringe metropolitan counties. RESULTS: Overall, large central metropolitan areas had a higher prevalence of HIV, HSV-2, and hepatitis B. HIV prevalence among MSM was elevated in large central and large fringe metro areas (14.5% and 16.9%, respectively). Among heterosexuals, large central metropolitan areas had elevated prevalence of HSV-2, chlamydia, and hepatitis B. Human papillomavirus and hepatitis C prevalence did not vary significantly by urbanicity for any population, including IDUs. CONCLUSIONS: Infections with higher prevalence in urban areas merit a geographically focused approach to screening and prevention programs, whereas those with uniform prevalence across levels of urbanicity would benefit from a generalized prevention approach. These nationally representative, population-based data allow for more effective planning for prevention programs.

The loss of 5-methyltetrahydrofolate in human serum under suboptimal preanalytical conditions can only partially be recovered by an oxidation product.

BACKGROUND: Maintaining folate stability during sample handling is important, yet challenging. OBJECTIVE: We investigated the effects of suboptimal preanalytical conditions on serum folate stability. METHODS: By using an HPLC-tandem MS method we measured folates [5-methyltetrahydrofolate (5-methylTHF), folic acid, MeFox (5-methylTHF oxidation product, pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF), and other minor folate forms at or below the limit of detection] in human serum exposed to suboptimal conditions. RESULTS: Whole blood samples (n = 21) stored at 32°C for ≤ 3 d (Expt. 1: delayed processing) showed significant decreases in serum total folate (tFOL; sum of folate forms: 11-32%, 5.5-15.9 nmol/L) and 5-methylTHF (36-62%, 14.5-25.1 nmol/L) and a significant increase in MeFox (346-415%, 7.17-8.63 nmol/L). Serum samples (n = 21) stored at 11°C for 7-14 d (Expt. 2: delayed freezing) also showed significant decreases in tFOL (4.6-10.4%, 2.3-5.1 nmol/L) and 5-methylTHF (8.4-29%, 3.4-11.6 nmol/L) and significant increases in MeFox (88-320%, 1.82-6.62 nmol/L). The molar loss in 5-methylTHF exceeded the gain in MeFox in these 2 experiments. When we exposed 3 serum pools (tFOL: 16.7-58.3 nmol/L) for 24 h to an elevated temperature of 37°C (Expt. 3), the significant decrease in 5-methylTHF (33% on average) was compensated for by an equimolar gain in MeFox. Repeated freeze/thaw cycles (≤ 3 cycles) of serum [closed (Expt. 4) and open (Expt. 5) vials] showed generally stable folates with small (<1 nmol/L) changes. Long-term (≤ 12 mo) exposure of 3 serum pools (tFOL: 17.5-63.7 nmol/L) to a suboptimal (-20°C) freezing temperature (Expt. 6) showed significant decreases in tFOL (5% on average) already after 3 mo. The molar loss in 5-methylTHF exceeded the gain in MeFox. Folic acid generally showed good stability. CONCLUSIONS: To avoid folate losses, unprocessed whole blood should be protected from elevated temperatures and serum should not be refrigerated for >2 d or for a long term stored at -20°C.

Establishing Population Values for Chlorine Exposure in the United States (2015-2016) Using 2 Chlorine Biomarkers, 3-Chlorotyrosine and 3,5-Dichlorotyrosine.

BACKGROUND: In the United States, 12 million short tons of chlorine are manufactured and transported each year. Due to the volume of this volatile chemical, large- and small-scale chemical exposures occur frequently. To diagnose and treat potentially exposed individuals, reference range values for confirmatory biomarkers are required to differentiate between normal and abnormal exposure levels. METHODS: Serum surplus samples (n = 1780) from the National Health and Nutrition Examination Survey (NHANES) 2015-2016 were measured for 2 chlorine biomarkers, 3-chlorotyrosine (Cl-Tyr) and 3,5-dichlorotyrosine (Cl2-Tyr), by liquid chromatography coupled to a triple quadrupole mass spectrometer. We evaluated demographic factors associated with elevated biomarker levels. RESULTS: Participant samples were analyzed for the chlorine biomarkers Cl-Tyr and Cl2-Tyr. In the unweighted analysis of these samples, 1349 (75.8%) were under the limit of detection (< LOD) of 2.50 ng/mL for Cl-Tyr and 1773 (99.6%) were < LOD for Cl2-Tyr. Samples within the method reportable range were 2.50 to 35.6 ng/mL for Cl-Tyr and 2.69 to 11.2 ng/mL for Cl2-Tyr. Since only 7 of the 1780 participants had detectable Cl2-Tyr, statistical analysis was limited to Cl-Tyr. Of the demographic characteristics examined, age, body mass index (BMI), estimated glomerular filtration rate (eGFR), and sex exhibited statistically significant differences in the weighted prevalence of detectable Cl-Tyr. CONCLUSIONS: This is the first reported set of Cl-Tyr and Cl2-Tyr population values for the United States. This population range coupled with NHANES demographic information could help healthcare professionals distinguish between normal and abnormal chlorine biomarker levels in an emergency. With this information, an inference could be made when determining acute chlorine exposure in individuals.

Race-ethnicity is a strong correlate of circulating fat-soluble nutrient concentrations in a representative sample of the U.S. population.

Sociodemographic and lifestyle factors exert important influences on nutritional status; however, information on their association with biomarkers of fat-soluble nutrients is limited, particularly in a representative sample of adults. Serum or plasma concentrations of vitamin A, vitamin E, carotenes, xanthophylls, 25-hydroxyvitamin D [25(OH)D], SFAs, MUFAs, PUFAs, and total fatty acids (tFAs) were measured in adults (aged ≥ 20 y) during all or part of NHANES 2003-2006. Simple and multiple linear regression models were used to assess 5 sociodemographic variables (age, sex, race-ethnicity, education, and income) and 5 lifestyle behaviors (smoking, alcohol consumption, BMI, physical activity, and supplement use) and their relation to biomarker concentrations. Adjustment for total serum cholesterol and lipid-altering drug use was added to the full regression model. Adjustment for latitude and season was added to the full model for 25(OH)D. Based on simple linear regression, race-ethnicity, BMI, and supplement use were significantly related to all fat-soluble biomarkers. Sociodemographic variables as a group explained 5-17% of biomarker variability, whereas together, sociodemographic and lifestyle variables explained 22-23% [25(OH)D, vitamin E, xanthophylls], 17% (vitamin A), 15% (MUFAs), 10-11% (SFAs, carotenes, tFAs), and 6% (PUFAs) of biomarker variability. Although lipid adjustment explained additional variability for all biomarkers except for 25(OH)D, it appeared to be largely independent of sociodemographic and lifestyle variables. After adjusting for sociodemographic, lifestyle, and lipid-related variables, major differences in biomarkers were associated with race-ethnicity (from -44 to 57%), smoking (up to -25%), supplement use (up to 21%), and BMI (up to -15%). Latitude and season attenuated some race-ethnicity differences. Of the sociodemographic and lifestyle variables examined, with or without lipid adjustment, most fat-soluble nutrient biomarkers were significantly associated with race-ethnicity.

Regression modeling plan for 29 biochemical indicators of diet and nutrition measured in NHANES 2003-2006.

The collection of articles in this supplement issue provides insight into the association of various covariates with concentrations of biochemical indicators of diet and nutrition (biomarkers), beyond age, race, and sex, using linear regression. We studied 10 specific sociodemographic and lifestyle covariates in combination with 29 biomarkers from NHANES 2003-2006 for persons aged ≥ 20 y. The covariates were organized into 2 sets or "chunks": sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle (dietary supplement use, smoking, alcohol consumption, BMI, and physical activity) and fit in hierarchical fashion by using each category or set of related variables to determine how covariates, jointly, are related to biomarker concentrations. In contrast to many regression modeling applications, all variables were retained in a full regression model regardless of significance to preserve the interpretation of the statistical properties of ß coefficients, P values, and CIs and to keep the interpretation consistent across a set of biomarkers. The variables were preselected before data analysis, and the data analysis plan was designed at the outset to minimize the reporting of false-positive findings by limiting the amount of preliminary hypothesis testing. Although we generally found that demographic differences seen in biomarkers were over- or underestimated when ignoring other key covariates, the demographic differences generally remained significant after adjusting for sociodemographic and lifestyle variables. These articles are intended to provide a foundation to researchers to help them generate hypotheses for future studies or data analyses and/or develop predictive regression models using the wealth of NHANES data.

Sociodemographic and lifestyle variables are compound- and class-specific correlates of urine phytoestrogen concentrations in the U.S. population.

Isoflavones and lignans are plant-derived dietary compounds generally believed to be beneficial to human health. We investigated the extent to which sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle variables (smoking, alcohol consumption, BMI, physical activity, and dietary supplement use) were correlates of spot urine concentration for daidzein, genistein, O-desmethylangolensin (DMA), equol, enterodiol, and enterolactone in the U.S. population aged ≥ 20 y (NHANES 2003-2006). We performed correlation analyses with continuous variables and calculated stratified unadjusted geometric means for each sociodemographic and lifestyle variable. We used bivariate significance testing and covariate adjustment by use of multiple regression models to identify influential variables and used ß coefficients to estimate relative effects. Urine creatinine was also included in our analyses because of its use in correcting for variable dilution in spot urine samples. We observed many significant (P < 0.05) associations with the sociodemographic and lifestyle variables that withstood covariate adjustment. Smoking was a significant correlate of urine DMA and enterolactone, with concentrations at least 25% lower in smokers vs. nonsmokers. Consumers of 1 daily alcoholic drink vs. none were estimated to have 18-21% lower urine equol and DMA concentrations. A 25% increase in BMI was associated with a 21% lower urine enterolactone concentration, and increasing physical activity was associated with a >6% higher urine enterolactone concentration. Dietary supplement use was not significantly associated with any of the urine phytoestrogens. Overall, we found that relationships between sociodemographic and lifestyle variables and urine phytoestrogen concentration were highly compound and class specific.

Dietary supplement use and smoking are important correlates of biomarkers of water-soluble vitamin status after adjusting for sociodemographic and lifestyle variables in a representative sample of U.S. adults.

Biochemical indicators of water-soluble vitamin (WSV) status were measured in a nationally representative sample of the U.S. population in NHANES 2003-2006. To examine whether demographic differentials in nutritional status were related to and confounded by certain variables, we assessed the association of sociodemographic (age, sex, race-ethnicity, education, income) and lifestyle (dietary supplement use, smoking, alcohol consumption, BMI, physical activity) variables with biomarkers of WSV status in adults (aged ≥ 20 y): serum and RBC folate, serum pyridoxal-5'-phosphate (PLP), serum 4-pyridoxic acid, serum total cobalamin (vitamin B-12), plasma total homocysteine (tHcy), plasma methylmalonic acid (MMA), and serum ascorbic acid. Age (except for PLP) and smoking (except for MMA) were generally the strongest significant correlates of these biomarkers (|r| ≤ 0.43) and together with supplement use explained more of the variability compared with the other covariates in bivariate analysis. In multiple regression models, sociodemographic and lifestyle variables together explained from 7 (vitamin B-12) to 29% (tHcy) of the biomarker variability. We observed significant associations for most biomarkers (≥ 6 of 8) with age, sex, race-ethnicity, supplement use, smoking, and BMI and for some biomarkers with PIR (5 of 8), education (1 of 8), alcohol consumption (4 of 8), and physical activity (5 of 8). We noted large estimated percentage changes in biomarker concentrations between race-ethnic groups (from -24 to 20%), between supplement users and nonusers (from -12 to 104%), and between smokers and nonsmokers (from -28 to 8%). In summary, age, sex, and race-ethnic differentials in biomarker concentrations remained significant after adjusting for sociodemographic and lifestyle variables. Supplement use and smoking were important correlates of biomarkers of WSV status.

Among 10 sociodemographic and lifestyle variables, smoking is strongly associated with biomarkers of acrylamide exposure in a representative sample of the U.S. Population.

Hemoglobin adducts of acrylamide (HbAA) and glycidamide (HbGA) have been measured as biomarkers of acrylamide exposure and metabolism in a nationally representative sample of the U.S. population in the NHANES 2003-2004. We assessed the association of sociodemographic (age, sex, race-ethnicity, education, and income) and lifestyle (smoking, alcohol consumption, BMI, physical activity, and dietary supplement use) variables with these biomarkers in U.S. adults (aged ≥ 20 y). We used bivariate and multiple regression models and assessed the magnitude of an estimated change in biomarker concentration with change in a covariable for 2 biomarkers of acrylamide exposure. Smoking was strongly and significantly correlated with HbAA and HbGA concentrations (rs = 0.51 and 0.42, respectively), with biomarker concentrations being 126 and 101% higher in smokers compared with nonsmokers after adjusting for sociodemographic and lifestyle covariates. Age was moderately and significantly correlated with both biomarkers (rs = -0.21 and -0.22, respectively). BMI (rs = -0.11) and alcohol consumption (rs = 0.13) were weakly yet significantly correlated with HbAA concentrations only. The estimated percentage change in biomarker concentration was ≤ 20% for all variables other than smoking after adjusting for sociodemographic and lifestyle covariates. Using multiple regression models, the sociodemographic variables explained 9 and 7% whereas the sociodemographic and lifestyle variables together explained 46 and 25% of the variability in HbAA and HbGA, respectively, showing the importance of considering and adequately controlling for these variables in future studies. Our findings will be useful in the design and analysis of future studies that assess and evaluate exposure to acrylamide and its metabolism to glycidamide.