Standard Reference Material (SRM) 2378 fatty acids in frozen human serum. Certification of a clinical SRM based on endogenous supplementation of polyunsaturated fatty acids.

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® (SRM®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.

Development of a Standard Reference Material for metabolomics research.

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.

Vitamin C Status of US Adults Assessed as Part of the National Health and Nutrition Examination Survey Remained Unchanged between 2003-2006 and 2017-2018.

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.

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

Interlaboratory analytical comparison of fatty acid concentrations in serum or plasma.

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.

Serum concentrations of an aflatoxin-albumin adduct in the National Health and Nutrition Examination Survey (NHANES) 1999-2000.

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 a study 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 (≥0.02µg/l) of aflatoxin B1-lysine. Of those with detectable levels, the geometric mean (95% confidence interval) was 0.038 (0.024-0.060) µg/l (equivalent to 0.842 (0.530-1.34) pg/mg albumin). The highest value was 0.2µg/l (4.43pg/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.

Analysis of aflatoxin B1-lysine adduct in serum using isotope-dilution liquid chromatography/tandem mass spectrometry.

A method for quantitative analysis of aflatoxin B1-lysine adduct (B1-Lys) in serum by liquid chromatography using tandem mass spectrometry (LC/MS/MS) is presented. The protein in a 250-microL sample was digested in the presence of a stable-isotope internal standard during a 4-h incubation at 37 degrees C with Pronasetrade mark. B1-Lys and the internal standard were extracted using mixed-mode solid-phase extraction cartridges and eluted with 2% formic acid in methanol. Following evaporation and reconstitution, extracts were injected onto a Luna C-18(2) column and eluted with a step gradient of acetonitrile and 0.06% formic acid. The B1-Lys and the internal standard were detected in a positive ionization selective reaction monitoring mode with a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer. Calibration curves were linear for concentrations from 0.05-8.0 ng/mL. The method was validated with aflatoxin B1 dosed rat serum diluted to anticipated high and low concentrations. Total imprecision determined from 30 measurements over 15 days was 5.6% and 9.1%, respectively. Recoveries of 78.8 +/- 6.4% for B1-Lys and 85.4 +/- 12.4% for the internal standard were based on the full extraction and reconstitution processes. The method can be used to quantitate B1-Lys at the 0.5 pg/mg albumin level and is suitable for routine analysis.