BACKGROUND: We compared serum vitamin C (VIC) status of the adult (≥20 y) US population in the National Health and Nutrition Examination Survey (NHANES) 2017-2018 with combined data from 2003-2004 and 2005-2006. METHODS: VIC was measured using HPLC with electrochemical detection. Mean data were stratified by age, sex, race/Hispanic origin, income, body mass index, dietary intake, supplement use, and smoking status. Prevalence of VIC deficiency (<11.4 μmol/L) was calculated. RESULTS: In NHANES 2017-2018, the mean VIC was 8 μmol/L higher in people ≥60 y compared with those 20-59 y of age, 10 μmol/L lower in men vs women, 8 μmol/L lower in low vs high income, 11 μmol/L lower in obese vs healthy weight, and 15 μmol/L lower in smokers vs nonsmokers. Differences in mean VIC across race/Hispanic origin groups ranged from 2 to 7 μmol/L. Mean VIC was 27 μmol/L higher with vitamin C-containing supplement use and positively associated (Spearman ρ = 0.33; P < 0.0001) with increasing dietary intake. The associations between mean VIC and the investigated covariates were generally consistent and the prevalence of deficiency was not significantly different between survey periods (6.8% vs 7.0%; P = 0.83). However, a few subgroups had double the risk. We found no significant survey differences in mean VIC (51.2 vs 54.0 μmol/L; P = 0.09). CONCLUSIONS: Overall VIC status of the US adult population has remained stable since last assessed in the NHANES 2005-2006 survey. Vitamin C deficiency remained high for those with low dietary intake and who smoke.

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.

Retinol-binding protein (RBP), retinol, and modified-relative-dose response (MRDR) are used to assess vitamin A status. We describe vitamin A status in Ugandan children and women using dried blood spot (DBS) RBP, serum RBP, plasma retinol, and MRDR and compare DBS-RBP, serum RBP, and plasma retinol. Blood was collected from 39 children aged 12-23 months and 28 non-pregnant mothers aged 15-49 years as a subsample from a survey in Amuria district, Uganda, in 2016. DBS RBP was assessed using a commercial enzyme immunoassay kit, serum RBP using an in-house sandwich enzyme-linked immunosorbent assay, and plasma retinol/MRDR test using high-performance liquid chromatography. We examined (a) median concentration or value (Q1, Q3); (b) R(2) between DBS-RBP, serum RBP, and plasma retinol; and (c) Bland-Altman plots. Median (Q1, Q3) for children and mothers, respectively, were as follows: DBS-RBP 1.15 µmol/L (0.97, 1.42) and 1.73 (1.52, 1.96), serum RBP 0.95 µmol/L (0.78, 1.18) and 1.47 µmol/L (1.30, 1.79), plasma retinol 0.82 µmol/L (0.67, 0.99) and 1.33 µmol/L (1.22, 1.58), and MRDR 0.025 (0.014, 0.042) and 0.014 (0.009, 0.019). DBS RBP-serum RBP R(2) was 0.09 for both children and mothers. The mean biases were -0.19 µmol/L (95% limits of agreement [LOA] 0.62, -0.99) for children and -0.01 µmol/L (95% LOA -1.11, -1.31) for mothers. DBS RBP-plasma retinol R(2) was 0.11 for children and 0.13 for mothers. Mean biases were 0.33 µmol/L (95% LOA -0.37, 1.03) for children, and 0.29 µmol/L (95% LOA -0.69, 1.27) for mothers. Serum RBP-plasma retinol R(2) was 0.75 for children and 0.55 for mothers, with mean biases of 0.13 µmol/L (95% LOA -0.23, 0.49) for children and 0.18 µmol/L (95% LOA -0.61, 0.96) for mothers. Results varied by indicator and matrix. The serum RBP-retinol R(2) for children was moderate (0.75), but poor for other comparisons. Understanding the relationships among vitamin A indicators across contexts and population groups is needed.

BACKGROUND: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 mumol/L or 20 mug/dL) that is suitable for low- and middle-income countries and uses commercially available materials. METHODS: Serum (25 muL) added to retinyl acetate was precipitated with acetonitrile (125 muL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 mug/dL) or multipoint solutions (0.52-100 mug/dL). C18 column (4.6 x 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes). RESULTS: With only 25 muL of serum, the limit of detection was 0.52 mug/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was </=7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was -0.2% (5.8%). Maximally, 180 samples were processed within 24 h. CONCLUSIONS: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.

BACKGROUND: Vitamin D is important for bone health; in 2014 it was the fifth most commonly ordered laboratory test among Medicare Part B payments. OBJECTIVES: The aim of this study was to describe vitamin D status in the US population in 2011-2014 and trends from 2003 to 2014. METHODS: We used serum 25-hydroxyvitamin D data from NHANES 2011-2014 (n = 16,180), and estimated the prevalence at risk of deficiency (<30 nmol/L) or prevalence at risk of inadequacy (30-49 nmol/L) by age, sex, race and Hispanic origin, and dietary intake of vitamin D. We also present trends between 2003 and 2014. RESULTS: In 2011-2014, the percentage aged >/=1 y at risk of vitamin D deficiency or inadequacy was 5.0% (95% CI: 4.1%, 6.2%) and 18.3% (95% CI: 16.2%, 20.6%). The prevalence of at risk of deficiency was lowest among children aged 1-5 y (0.5%; 95% CI: 0.3%, 1.1%), peaked among adults aged 20-39 y (7.6%; 95% CI: 6.0%, 9.6%), and fell to 2.9% (95% CI: 2.0%, 4.0%) among adults aged >/=60 y; the prevalence of at risk of inadequacy was similar. The prevalence of at risk of deficiency was higher among non-Hispanic black (17.5%; 95% CI: 15.2%, 20.0%) than among non-Hispanic Asian (7.6%; 95% CI: 5.9%, 9.9%), non-Hispanic white (2.1%; 95% CI: 1.5%, 2.7%), and Hispanic (5.9%; 95% CI: 4.4%, 7.8%) persons; the prevalence of at risk of inadequacy was similar. Persons with higher vitamin D dietary intake or who used supplements had lower prevalences of at risk of deficiency or inadequacy. From 2003 to 2014 there was no change in the risk of vitamin D deficiency; the risk of inadequacy declined from 21.0% (95% CI: 17.9%, 24.5%) to 17.7% (95% CI: 16.0%, 19.7%). CONCLUSION: The prevalence of at risk of vitamin D deficiency in the United States remained stable from 2003 to 2014; at risk of inadequacy declined. Differences in vitamin D status by race and Hispanic origin warrant additional investigation.

BACKGROUND: Consistent information on long-term storage stability for a broad range of nutritional biomarkers is lacking. We investigated the stability of 18 biomarkers stored at suboptimal temperatures (-20 °C and 5 °C) for up to 12 months. METHODS: Multiple vials of serum or whole blood pools (3 concentrations) were stored at -20 °C or 5 °C, removed from the -20 °C freezer after 3, 6, 9, and 12 months and from the 5 °C refrigerator after 6 and 12 months, and placed into a -70 °C freezer until analysis at study completion. Vials stored continuously at -70 °C were used as the reference condition for optimal storage. We measured 18 biomarkers: 4 iron status, 1 inflammation, 8 water-soluble vitamin, and 5 fat-soluble vitamin. For each temperature, we calculated geometric mean concentrations and average percent changes of geometric means across pools relative to the reference condition estimated from a linear mixed model. RESULTS: Most biomarkers (13 of 18) showed no difference in concentration after 12 months of storage at -20 °C. Serum ferritin (1.5%), soluble transferrin receptor (-1.7%), and folate (-10.5%) showed small to moderate significant changes at 6 months, but changes were acceptable based on biologic variability. Serum pyridoxal-5'-phosphate (-18.6% at 9 months) and vitamin C (-23% at 6 months) showed large and unacceptable changes at -20 °C. All serum fat-soluble vitamins and iron status indicators, vitamin B12, total homocysteine, and methylmalonic acid showed acceptable changes when stored at 5 °C for up to 12 months. CONCLUSIONS: Overall, we found good long-term stability for multiple nutritional biomarkers stored at suboptimal temperatures.

BACKGROUND: Reference measurement procedures (RMP) have rigorous accuracy specifications. For total 25-hydroxyvitamin D, 25(OH)D, bias </=1.7% and CV </=5% are recommended. These quality specifications are impractical for minor analytes, such as 25(OH)D2. Furthermore, documentation on RMP quality performance specifications for the individual 25(OH)D metabolites and their daily application are missing. METHODS: To assess accuracy, we used zeta-scores. Daily, 5-10 specimens (duplicate) and 3 reference materials (singleton or duplicate) were measured for 25(OH)D3 and 25(OH)D2 using JCTLM-accepted LC-MS/MS RMPs. Protocols were repeated on 3-4 occasions to generate campaign results. We used separate zeta-score acceptability criteria for daily (</=|2|) and campaign (</=|1|) evaluations. Allowable imprecision was determined experimentally. RESULTS: Across 7 campaigns, unacceptable daily zeta-scores required repeating 2 runs for 25(OH)D3 and 5 runs for 25(OH)D2. Hence, the zeta-scores of acceptable reference material results indicated high accuracy. The allowable imprecision for the RMPs was </=5% (daily) and</=3% (campaign) for 25(OH)D3 and</=7% (daily) and</=4% (campaign) for 25(OH)D2, respectively. CONCLUSIONS: Using zeta-scores and experimentally derived imprecision, we developed a straightforward approach to assess the acceptability of individual 25(OH)D reference measurements, providing also much-needed practical accuracy specifications for 25(OH)D2.

Vitamin D is an essential nutrient for bone health and may influence the risks of respiratory illness, adverse pregnancy outcomes, and chronic diseases of adulthood. Because many countries have a relatively low supply of foods rich in vitamin D and inadequate exposure to natural ultraviolet B (UVB) radiation from sunlight, an important proportion of the global population is at risk of vitamin D deficiency. There is general agreement that the minimum serum/plasma 25-hydroxyvitamin D concentration (25(OH)D) that protects against vitamin D deficiency-related bone disease is approximately 30 nmol/L; therefore, this threshold is suitable to define vitamin D deficiency in population surveys. However, efforts to assess the vitamin D status of populations in low- and middle-income countries have been hampered by limited availability of population-representative 25(OH)D data, particularly among population subgroups most vulnerable to the skeletal and potential extraskeletal consequences of low vitamin D status, namely exclusively breastfed infants, children, adolescents, pregnant and lactating women, and the elderly. In the absence of 25(OH)D data, identification of communities that would benefit from public health interventions to improve vitamin D status may require proxy indicators of the population risk of vitamin D deficiency, such as the prevalence of rickets or metrics of usual UVB exposure. If a high prevalence of vitamin D deficiency is identified (>20% prevalence of 25(OH)D < 30 nmol/L) or the risk for vitamin D deficiency is determined to be high based on proxy indicators (e.g., prevalence of rickets >1%), food fortification and/or targeted vitamin D supplementation policies can be implemented to reduce the burden of vitamin D deficiency-related conditions in vulnerable populations.

Dietary fatty acids can be both beneficial and detrimental to human health depending on the degree and type of saturation. Healthcare providers and research scientists monitor the fatty acid content of human plasma and serum as an indicator of health status and diet. In addition, both the Centers for Disease Control & Prevention (CDC) and the National Institutes of Health - Office of Dietary Supplements are interested in circulating fatty acids (FAs) because they may be predictive of coronary heart disease. The National Institute of Standards and Technology (NIST) provides a wide variety of reference materials (RMs) and Standard Reference Materials(R) (SRM(R)s) including blood, serum, plasma, and urine with values assigned for analytes of clinical interest. NIST SRM 2378 Fatty Acids in Frozen Human Serum was introduced in 2015 to help validate methods used for the analysis of FAs in serum, and consists of three different pools of serum acquired from (1) healthy donors who had taken fish oil dietary supplements (at least 1000 mg per day) for at least one month (level 1 material), (2) healthy donors who had taken flaxseed oil dietary supplements (at least 1000 mg per day) for at least one month (level 2 material), and (3) healthy donors eating "normal" diets who had not taken dietary supplements containing fish or plant oils (level 3 material). The use of dietary supplements by donors provided SRMs with natural endogenous ranges of FAs at concentrations observed in human populations. Results from analyses using two methods at NIST, including one involving a novel microwave-assisted acid hydrolysis procedure, and one at the CDC are presented here. These results and their respective uncertainties were combined to yield certified values with expanded uncertainties for 12 FAs and reference values with expanded uncertainties for an additional 18 FAs.

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Health's Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.

OBJECTIVE: To evaluate the role of vitamin D intake and serum levels on conception of clinical pregnancy and live birth. DESIGN: Prospective cohort study. SETTING: Academic medical centers. PATIENT(S): Healthy, nulliparous women, age 18-39 years, and their male partners. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy and live birth were compared between those who did or did not meet the vitamin D estimated average requirement (EAR) intake (10 mug/d) and with serum 25-hydroxyvitamin D (25(OH)D) considered at risk for inadequacy or deficiency (<50 nmol/L) or sufficient (≥50 nmol/L). RESULT(S): Among 132 women, 37.1% did not meet the vitamin D EAR and 13.9% had serum levels at risk for inadequacy or deficiency. Clinical pregnancies were significantly higher among women who met the vitamin D EAR (67.5% vs. 49.0%) and with sufficient serum 25(OH)D (64.3% vs. 38.9%) compared with those who did not. Live births were higher among those who met the vitamin D EAR (59.0% vs. 40.0%). The adjusted odds ratio (AOR) of conceiving a clinical pregnancy was significantly higher among those who met the EAR (AOR = 2.26; 95% confidence interval [CI], 1.05-4.86) and had sufficient serum 25(OH)D (AOR = 3.37; 95% CI, 1.06-10.70). The associations were not significant after controlling for selected nutrients and dietary quality. CONCLUSION(S): Women with vitamin D intake below EAR and serum 25(OH)D levels at risk for inadequacy or deficiency may be less likely to conceive and might benefit from increased vitamin D intake to achieve adequacy. CLINICAL TRIAL REGISTRATION NUMBER: NCT00642590.

BACKGROUND: In field studies, hemoglobin (Hb) is often measured using a battery-operated, portable HemoCue(R) hemoglobinometer. METHODS: We compared the performance of 2 HemoCue(R) models (Hb-201+ and Hb-301) and investigated effects of preanalytical factors on Hb results by simulating unfavorable field conditions. RESULTS: The Hb-301 produced 2.6% higher results compared to the Hb-201+. Hb had to be measured within 1min of filling the Hb-301 cuvette to avoid artificially elevated concentrations (1.3% per min). The Hb-301 cuvettes withstood elevated temperature (37 degrees C) and humidity (72%) for 3weeks, while the Hb-201+ cuvettes degraded within 10min under those conditions. Both cuvette types withstood elevated temperature for 3weeks. Properly-collected venous and capillary blood produced comparable results. Pooled capillary blood produced comparable results to the second and third but not the fourth drop of blood (3.3% lower). Blood could be stored for ≤4days at 10-30 degrees C before Hb-201+ measurement, but only for 1day at 10-23 degrees C before Hb-301 measurement (≤1% change in Hb). CONCLUSIONS: Higher Hb results obtained with the Hb-301 may influence the interpretation of anemia prevalence in health surveys. While the Hb-301 performed better in high humidity conditions, the Hb-201+ provided more user flexibility regarding delayed Hb reading.

For the past 45 y, the National Center for Health Statistics at the CDC has carried out nutrition surveillance of the US population by collecting anthropometric, dietary intake, and nutritional biomarker data, the latter being the focus of this publication. The earliest biomarker testing assessed iron and vitamin A status. With time, a broad spectrum of water- and fat-soluble vitamins was added and biomarkers for other types of nutrients (e.g., fatty acids) and bioactive dietary compounds (e.g., phytoestrogens) were included in NHANES. The cross-sectional survey is flexible in design, and biomarkers may be measured for a short period of time or rotated in and out of surveys depending on scientific needs. Maintaining high-quality laboratory measurements over extended periods of time such that trends in status can be reliably assessed is a major goal of the testing laboratories. Physicians, health scientists, and policy makers rely on the NHANES reference data to compare the nutritional status of population groups, to assess the impact of various interventions, and to explore associations between nutritional status and health promotion or disease prevention. Focusing on the continuous NHANES, which started in 1999, this review uses a "lessons learned" approach to present a series of challenges that are relevant to researchers measuring biomarkers in NHANES and beyond. Some of those challenges are the use of multiple related biomarkers instead of a single biomarker for a specific nutrient (e.g., folate, vitamin B-12, iron), adhering to special needs for specimen collection and handling to ensure optimum specimen quality (e.g., vitamin C, folate, homocysteine, iodine, polyunsaturated fatty acids), the retrospective use of long-term quality-control data to correct for assay shifts (e.g., vitamin D, vitamin B-12), and the proper planning for and interpretation of crossover studies to adjust for systematic method changes (e.g., folate, vitamin D, ferritin).

BACKGROUND: There are a large number of clinical studies focusing on the measurement of individual fatty acids in serum or plasma; however, few studies have focused on the interlaboratory comparisons of these measurements. The National Institutes of Standards and Technology (NIST), in collaboration with the National Institutes of Health Office of Dietary Supplements (NIH-ODS) and the Centers for Disease Control and Prevention (CDC), has initiated a quality assurance program for assessing and improving the comparability of individual fatty acid measurements in serum and plasma. METHODS: This is a performance-based study so participants are encouraged to use their laboratory's methods for the quantification of the individual fatty acids that they typically measure in the unknown serum or plasma samples along with a control material. The control materials used to date are SRM 1950 Metabolites in Human Plasma and SRM 2378 Fatty Acids in Frozen Human Serum. RESULTS: To date, two studies of the Fatty Acid Quality Assurance Program (FAQAP) have been completed with 11 and 14 participants, respectively. The agreement among the laboratories for individual fatty acids was within 20% for 70% of the data submitted. Laboratories were also requested to run triplicate analyses for each unknown sample. The precision of the individual laboratory data was generally good, with relative standard deviations <20%. CONCLUSIONS: The results from the first two exercises indicate the need for additional assessment of the comparability among laboratories doing these measurements. Future studies will be conducted with the goals of increasing the number of participating laboratories, increasing awareness of the need to use control materials, and improving the comparability among laboratories.

BACKGROUND: Temporal trends in the US population's vitamin D status have been uncertain because of nonstandardized serum 25-hydroxyvitamin D [25(OH)D] measurements. OBJECTIVE: To accurately assess vitamin D status trends among those aged ≥12 y, we used data from the cross-sectional NHANESs. DESIGN: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 25(OH)D (sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3), calibrated to standard reference materials, was used to predict LC-MS/MS-equivalent concentrations from radioimmunoassay data (1988-2006 surveys; n = 38,700) and to measure LC-MS/MS concentrations (2007-2010 surveys; n = 12,446). Weighted arithmetic means and the prevalence of 25(OH)D above or below cutoff concentrations were calculated to evaluate long-term trends. RESULTS: Overall, mean predicted 25(OH)D showed no time trend from 1988 to 2006, but during 2007-2010 the mean measured 25(OH)D was 5-6 nmol/L higher. Those groups who showed the largest 25(OH)D increases (7-11 nmol/L) were older, female, non-Hispanic white, and vitamin D supplement users. During 1988-2010, the proportions of persons with 25(OH)D <40 nmol/L were 14-18% (overall), 46-60% (non-Hispanic blacks), 21-28% (Mexican Americans), and 6-10% (non-Hispanic whites). CONCLUSIONS: An accurate method for measuring 25(OH)D showed stable mean concentrations in the US population (1988-2006) and recent modest increases (2007-2010). Although it is unclear to what extent supplement usage compared with different laboratory methods explain the increases in 25(OH)D, the use of higher vitamin D supplement dosages coincided with the increase. Marked race-ethnic differences in 25(OH)D concentrations were apparent. These data provide the first standardized information about temporal trends in the vitamin D status of the US population.

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)D2was detectable in 19% of the population. Total 25(OH)D, 25(OH)D3, and C3-epi-25(OH)D3displayed similar demographic patterns and were strongly correlated (Spearman'sr> 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)D3and 25(OH)D3may be because the epimer is a metabolite of 25(OH)D3.The presence of 25(OH)D2mainly in older persons is likely a result of high-dose prescription vitamin D2.

The inaccuracy of routine serum 25-hydroxyvitamin D measurements hampers the interpretation of data in patient care and public health research. We developed and validated a candidate reference measurement procedure (RMP) for highly accurate quantitation of two clinically important 25-hydroxyvitamin D metabolites in serum, 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3]. The two compounds of interest together with spiked deuterium-labeled internal standards [d 3-25(OH)D2 and d 6-25(OH)D3] were extracted from serum via liquid-liquid extraction. The featured isotope-dilution LC-MS/MS method used reversed-phase chromatography and atmospheric pressure chemical ionization in positive ion mode. A pentafluorophenylpropyl-packed UHPLC column together with isocratic elution allowed for complete baseline resolution of 25(OH)D2 and 25(OH)D3 from their structural C-3 isomers within 12 min. We evaluated method trueness, precision, potential interferences, matrix effects, limits of quantitation, and measurement uncertainty. Calibration materials were, or were traceable to, NIST Standard Reference Materials 2972. Within-day and total imprecision (CV) averaged 1.9 and 2.0 % for 25(OH)D3, respectively, and 2.4 and 3.5 % for 25(OH)D2, respectively. Mean trueness was 100.3 % for 25(OH)D3 and 25(OH)D2. The limits of quantitation/limits of detection were 4.61/1.38 nmol/L for 25(OH)D3 and 1.46/0.13 nmol/L for 25(OH)D2. When we compared our RMP results to an established RMP using 40 serum samples, we found a nonsignificant mean bias of 0.2 % for total 25(OH)D. This candidate RMP for 25(OH)D metabolites meets predefined method performance specifications (≤5 % total CV and ≤1.7 % bias) and provides sufficient sample throughput to meet the needs of the Centers for Disease Control and Prevention Vitamin D Standardization Certification Program. Graphical abstract Bias assessment using NIST standard reference materials. Legend CDC mean mass fractions (ng/g) +/- U 95 (6 replicates per mean). NIST-certified mass fractions (ng/g) +/- U 95 from the Certificates of Analysis.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

BACKGROUND: Preanalytical conditions encountered during collection, processing, and storage of biospecimens may influence laboratory results. The National Children's Study (NCS) is a planned prospective cohort study of 100,000 families to examine the influence of a wide variety of exposures on child health. In developing biospecimen collection, processing, and storage procedures for the NCS, we identified several analytes of different biochemical categories for which it was unclear to what extent deviations from NCS procedures could influence measurement results. METHODS: A pilot study was performed to examine effects of preanalytic sample handling conditions (delays in centrifugation, freezing delays, delays in separation from cells, additive delay, and tube type) on concentrations of eight different analytes. A total of 2825 measurements were made to assess 15 unique combinations of analyte and handling conditions in blood collected from 151 women of childbearing age (≥20 individuals per handling condition). RESULTS: The majority of analytes were stable under the conditions evaluated. However, levels of plasma interleukin-6 and serum insulin were decreased in response to sample centrifugation delays of up to 5.5 h post-collection (p<0.0001). In addition, delays in freezing centrifuged plasma samples (comparing 24, 48 and 72 h to immediate freezing) resulted in increased levels of adrenocorticotropic hormone (p=0.0014). CONCLUSION: Determining stability of proposed analytes in response to preanalytical conditions and handling helps to ensure high-quality specimens for study now and in the future. The results inform development of procedures, plans for measurement of analytes, and interpretation of laboratory results.

CONTEXT: A reverse J-shaped association between serum 25-hydroxyvitamin D (25[OH]D) concentration and all-cause mortality was suggested in a 9-year follow-up (1991-2000) analysis of the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994). OBJECTIVE: Our objective was to repeat the analyses with 6 years additional follow-up to evaluate whether the association persists through 15 years of follow-up. PARTICIPANTS: The study included 15 099 participants aged ≥20 years with 3784 deaths. MAIN OUTCOME MEASURE: Relative risk (RR) of death from all causes was adjusted for age, sex, race/ethnicity, and season using 2 Poisson regression approaches: traditional categorical and cubic splines. Results were given for 9 25(OH)D levels: <20, 20 to 29, 30 to 39, 40 to 49, 50 to 59, 60 to 74, 75 to 99 (reference), 100 to 119, and ≥120 nmol/L. RESULTS: The reverse J-shaped association became stronger with longer follow-up and was not affected by excluding deaths within the first 3 years of follow-up. Similar results were found from both statistical approaches for levels <20 through 119 nmol/L. Adjusted RR (95% confidence interval [CI]) estimates for all levels <60 nmol/L were significantly >1 compared with the reference group. The nadir of risk was 81 nmol/L (95% CI, 73-90 nmol/L). For 25(OH)D ≥120 nmol/L, results (RR, 95% CI) were slightly different using traditional categorical (1.5, 1.02-2.3) and cubic splines approaches (1.2, 0.9-1.4). The association appeared in men, women, adults ages 20 to 64 years, and non-Hispanic whites but was weaker in older adults. The study was too small to evaluate the association in non-Hispanic black and Mexican-American adults. CONCLUSIONS: A reverse J-shaped association between serum 25(OH)D and all-cause mortality appears to be real. It is uncertain whether the association is causal.

BACKGROUND: In developing countries, deficiencies in essential micronutrients are common, particularly in pregnant women. Although, biochemical indicators of diet and nutrition are useful to assess nutritional status, few studies have examined such indicators throughout pregnancy in women in developing countries. METHODS: The primary objective of this study was to assess the nutritional status of 78 Peruvian women throughout pregnancy for 16 different nutritional indicators including fat-soluble vitamins and carotenoids, iron-status indicators, and selenium. Venous blood samples from which serum was prepared were collected during trimesters one (n = 78), two (n = 65), three (n = 62), and at term via the umbilical cord (n = 52). Questionnaires were completed to determine the demographic characteristics of subjects. Linear mixed effects models were used to study the associations between each maternal indicator and the demographic characteristics. RESULTS: None of the women were vitamin A and E deficient at any stage of pregnancy and only 1/62 women (1.6%) was selenium deficient during the third trimester. However, 6.4%, 44% and 64% of women had ferritin levels indicative of iron deficiency during the first, second and third trimester, respectively. Statistically significant changes (p ≤ 0.05) throughout pregnancy were noted for 15/16 nutritional indicators for this Peruvian cohort, with little-to-no association with demographic characteristics. Three carotenoids (beta-carotene, beta-cryptoxanthin and trans-lycopene) were significantly associated with education status, while trans-lycopene was associated with age and beta-cryptoxanthin with SES (p < 0.05). Concentrations of retinol, tocopherol, beta-cryptoxanthin, lutein + zeaxanthin and selenium were lower in cord serum compared with maternal serum (p < 0.05). Conversely, levels of iron status indicators (ferritin, transferrin saturation and iron) were higher in cord serum (p < 0.05). CONCLUSION: The increasing prevalence of iron deficiency throughout pregnancy in these Peruvian women was expected. It was surprising though not to find deficiencies in other nutrients. The results highlight the importance of continual monitoring of women throughout pregnancy for iron deficiency which could be caused by increasing fetal needs and/or inadequate iron intake as pregnancy progresses.

BACKGROUND: During 1998, weather conditions in the United States favored the growth of Aspergillus species leading to widespread contamination of Midwestern and Southern corn with hepatotoxic and hepatocarcinogenic aflatoxins. We designed to provide the first national prevalence estimate of aflatoxin exposure using the National Health and Nutrition Examination Survey (NHANES), a representative cross-sectional survey of the noninstitutionalized civilian population of the US. METHODS: Isotope dilution liquid chromatography-tandem mass spectrometry was used to quantitate serum concentrations of aflatoxin B1-lysine in a one-third random subset of participants from NHANES 1999-2000. RESULTS: About 1% of the U.S. population had detectable levels (≥0.02 mcg/l) of aflatoxin B1-lysine. Of those with detectable levels, the geometric mean (95% confidence interval) was 0.038 (0.024-0.060) mcg/l (equivalent to 0.842 (0.530-1.34) pg/mg albumin). The highest value was 0.2 mcg/l (4.43 pg/mg albumin). Based on liver function biomarkers, there was no evidence of increased liver dysfunction in these persons. CONCLUSIONS: During a time when exposure to aflatoxins in food products might have been expected to be increased, we identified few exposed persons. Although none of the subgroup analyses provided reliable estimates due to high relative standard errors, they suggested that additional targeted surveillance may be warranted.

The CDC's National Report on Biochemical Indicators of Diet and Nutrition in the U.S. Population (Nutrition Report) is a serial publication that provides ongoing assessment of the population's nutritional status. The Nutrition Report presents data on blood and urine biomarker concentrations (selected water- and fat-soluble vitamins and nutrients, trace elements, dietary bioactive compounds) from a representative sample of the population participating in the NHANES. The Second Nutrition Report (released in 2012) contains reference information (means and percentiles) for 58 biomarkers measured during all or part of 2003-2006, stratified by age, sex, and race-ethnicity. Where available, we presented cutoff-based prevalence data during 2003-2006 and data on changes in biomarker concentrations or prevalence since 1999. Blood vitamin concentrations were generally higher in older (≥60 y) than in younger (20-39 y) adults and lower in Mexican Americans and non-Hispanic blacks than in non-Hispanic whites. Nearly 80% of Americans (aged ≥6 y) were not at risk of deficiencies in any of the 7 vitamins studied (vitamins A, B-6, B-12, C, D, and E and folate). Deficiency rates varied by age, sex, and race-ethnicity. Approximately 90% of women (aged 12-49 y) were not at risk of iron deficiency, but only 68% were not at risk of deficiencies in iron and all 7 vitamins. Young women (20-39 y) had median urine iodine concentrations bordering on insufficiency. First-time data are presented on plasma concentrations of 24 saturated and mono- and polyunsaturated fatty acids. Tabulation and graphical presentation of NHANES data in the Second Nutrition Report benefits those organizations involved in developing and evaluating nutrition policy.

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.

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 beta 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.

The physiologic status of an individual may influence biomarkers of nutritional status. To help researchers with planning studies and interpreting data, we assessed the associations between common physiologic variables (fasting, inflammation, renal function, and pregnancy) and 29 biomarkers of diet and nutrition measured in blood or urine in a representative sample of the adult U.S. population (aged ≥20 y; pregnancy variable and iron indicators limited to women aged 20-49 y) participating in NHANES 2003-2006. We compared simple linear regression (model 1) with multiple linear regression [model 2, controlling for age, sex, race-ethnicity, smoking, supplement use, and the physiologic factors (and urine creatinine for urine biomarkers)] and report significant findings from model 2. Not being fasted was positively associated with most water-soluble vitamins (WSVs) and related metabolites (RMs). Some WSV, fat-soluble vitamin (FSV) and micronutrient (MN), and phytoestrogen concentrations were lower in the presence of inflammation (C-reactive protein ≥5 mg/L), whereas fatty acids and most iron indicators were higher. Most WSVs and RMs were higher when renal function was impaired [estimated glomerular filtration rate <60 mL/(min 1.73 m2)]. Most WSV, FSV and MN, and fatty acid concentrations were higher in pregnant compared with nonpregnant women, but vitamins A and B-12 and most iron indicators were lower. The estimated changes in biomarker concentrations with different physiologic status were mostly small to moderate (≤25%) and generally similar between models; renal function, however, showed several large differences for WSV and RM concentrations. This descriptive analysis of associations between physiologic variables and a large number of nutritional biomarkers showed that controlling for demographic variables, smoking, and supplement use generally did not change the interpretation of bivariate results. The analysis serves as a useful basis for more complex future research.

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.

The National Health and Nutrition Examination Survey (NHANES) provides the most comprehensive assessment of the health and nutrition status of the US population. Up-to-date reference intervals on biomarkers and dietary intake inform the scientific and public health policy communities on current status and trends over time.The main purpose of dietary assessment methods such as the food-frequency questionnaire, food record (or diary), and 24-hr dietary recall is to estimate intake of nutrients and, together with supplement usage information, describe total intake of various foods or nutrients. As with all self-reporting methods, these tools are challenging to use and interpret. Yet, they are needed to establish dietary reference intake recommendations and to evaluate what proportion of the population meets these recommendations. While biomarkers are generally expensive and, to some degree, invasive, there is no question as to their ability to assess nutrition status. In some cases biomarkers can also be used to assess intake or function, although rarely can one biomarker fulfill all these purposes. For example, serum folate is a good indicator of folate intake, red blood cell (RBC) folate is a good status indicator, and plasma total homocysteine is a good functional indicator of one-carbon metabolism. Using folate and vitamin D - two vitamins that are currently hotly debated in the public health arena - as two case studies, we discuss the complexities of using biomarkers and total intake information to assess nutrition status. These two examples also show how biomarkers and intake provide different information and how both are needed to evaluate and set public health policy. We also provide guidance on general requirements for using nutrition biomarkers and food and supplement intake information in longitudinal, population-based surveys.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health's Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.

BACKGROUND: An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D(2) (25OHD(2)), 25-hydroxyvitamin D(3) (25OHD(3)) and the C-3 epimer of 25OHD(3) (epi-25OHD(3)) in human serum. METHODS: Tri- and hexa-deuterated internal standards were added to serum (100mul) to monitor recovery. Liquid-liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8-100nmol/L 25OHD(2,) 12-150nmol/L 25OHD(3), and 4-50nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1x100mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4ml/min, run time was 14min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2)m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3. RESULTS: Recovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20nmol/l. Bias averaged <5%. Detection limits were <5nmol/l. Median (nmol/l) 25OHD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (<LOD, 56.0, <LOD) and 35 pregnant women (<LOD, 87.6, 3.70). CONCLUSIONS: This method is highly accurate, precise and specific.