The 2018 roadmap for global control programs for thiamine deficiency disorders calls out the lack of biochemical population data on thiamine status (1). This report states that “urgent public health responses are warranted in high-risk regions, considering the contribution of thiamine deficiency to infant mortality and research suggesting that even subclinical thiamine deficiency in childhood may have lifelong neurodevelopmental consequences.” This situation poses a bit of a chicken and egg dilemma, as investigators need suitable analytical methods to produce high-quality biomarker data and policymakers need comparable and interpretable biomarker data to develop policies and evaluate their impact. Furthermore, analytical methods need to be paired with practical preanalytical conditions that allow for the collection and generation of valid biological specimens. For years, the field of thiamine research has been hampered due to the labile physiochemical properties of thiamine compounds and the lack of simple, yet reliable, analytical methods.

BACKGROUND: Elevated plasma methylmalonic acid (MMA) and/or total homocysteine (tHcy), as well as low serum vitamin B12 and/or holotranscobalamin (holoTC) are indicative of vitamin B12 deficiency. Combined indicators (cB12), which pool some or all 4 markers into an index, may be a more reliable diagnostic tool to overcome inconclusive diagnoses with individual markers. OBJECTIVES: We aimed to describe different cB12 score combinations and estimate the prevalence of low or transitional vitamin B12 status compared with individual markers. DESIGN: Using cross-sectional data for B12, MMA, and tHcy in persons ≥20 y participating in NHANES 1999-2004 (n = 12,335), we examined raw and covariate-adjusted regression models to assess determinants of 3cB12 (all 3 markers) and combinations of 2cB12 (2 markers). RESULTS: 3cB12 was significantly associated with B12 (Spearman r = 0.75), MMA (r = -0.70), and tHcy (r = -0.59). The 3cB12 reference interval (2.5th to 97.5th percentile) was -0.538 to 1.60. In covariate-adjusted models, we found no association of 3cB12 with age; adult females and users of B12 supplements had higher, while adults with advanced chronic kidney disease had lower 3cB12 levels regardless of race-Hispanic origin group (self-reported). Only 2.7% of adults had low or transitional vitamin B12 status using the proposed cB12 cutoff of ≤-0.5, while the prevalence of low (or low-normal) status depended on the selected individual marker and its cutoff: 2.2% and 13% for B12 < 148 and 148-222 pmol/L, respectively; 6.0% for MMA exceeding an age-specific cutoff (250-320 nmol/L); and 8.4% for tHcy > 13 µmol/L. The reference intervals for B12, MMA, and tHcy overlapped from the low (<-2.5) to the transitional (-2.5 to -0.5) and to the adequate (>-0.5) cB12 categories. CONCLUSIONS: Vitamin B12 deficiency may be overestimated among US adults when individual, conventional markers are used. When only 2 markers are available, the combination of B12 and MMA provides results comparable to 3cB12.

BACKGROUND: Serum vitamin B-12 is measured to evaluate vitamin B-12 status. Serum methylmalonic acid (MMA) is a specific functional indicator of vitamin B-12 status; however, concentrations increase with impaired renal function. OBJECTIVE: The aim of this study was to describe the distribution of serum vitamin B-12 and MMA in US adults, and estimate age-specific reference intervals for serum MMA in a healthy subpopulation with replete vitamin B-12 status and normal renal function. METHODS: We examined cross-sectional data for serum vitamin B-12 and MMA in adults participating in the NHANES from 2011 to 2014. Vitamin B-12 was measured by electrochemiluminescence assay and MMA by isotope-dilution liquid chromatography-tandem mass spectrometry. RESULTS: In both bivariate and multivariate analyses, age, race/Hispanic origin, and vitamin B-12 supplement use were generally significantly associated with serum vitamin B-12 and MMA concentrations. Serum MMA concentrations increased with age, particularly in persons aged >/=70 y. Non-Hispanic white persons had lower vitamin B-12 and higher MMA concentrations than non-Hispanic black persons. Shorter fasting times and impaired renal function were significantly associated with higher serum MMA concentrations, but not with serum vitamin B-12 concentrations after controlling for covariates. The central 95% reference intervals for serum vitamin B-12 and MMA concentrations were widest for persons aged >/=70 y compared with younger age groups. Compared with the overall population, the central 95% reference intervals for serum MMA concentrations were considerably narrower for a vitamin B-12-replete subpopulation with normal renal function, but still age-dependent. Serum vitamin B-12 showed little, whereas serum MMA showed notable, increases with impaired renal function. CONCLUSIONS: The higher serum MMA concentrations throughout the entire distribution in older persons (especially persons aged >/=70 y) who are vitamin B-12-replete and have normal renal function indicate the need for age-specific MMA reference intervals to better interpret vitamin B-12 status in epidemiologic research.

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.

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.

Methylmalonic acid (MMA), a functional indicator of vitamin B12 insufficiency, was measured in the US population in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2004 using a GC/MS procedure that required 275 muL of sample and had a low throughput (36 samples/run). Our objective was to introduce a more efficient yet highly accurate LC-MS/MS method for NHANES 2011-2014. We adapted the sample preparation with some modifications from a published isotope-dilution LC-MS/MS procedure. The procedure utilized liquid-liquid extraction and generation of MMA dibutyl ester. Reversed-phase chromatography with isocratic elution allowed baseline resolution of MMA from its naturally occurring structural isomer succinic acid within 4.5 min. Our new method afforded an increased throughput (≤160 samples/run) and measured serum MMA with high sensitivity (LOD = 22.1 nmol/L) in only 75 muL of sample. Mean (+/-SD) recovery of MMA spiked into serum (2 d, 4 levels, 2 replicates each) was 94 % +/- 5.5 %. Total imprecision (41 d, 2 replicates each) for three serum quality control pools was 4.9 %-7.9 % (97.1-548 nmol/L). The LC-MS/MS method showed excellent correlation (n = 326, r = 0.99) and no bias (Deming regression, Bland-Altman analysis) compared to the previous GC/MS method. Both methods produced virtually identical mean (+/-SD) MMA concentrations [LC-MS/MS: 18.47 +/- 0.71 ng/mL (n = 17), GC/MS: 18.18 +/- 0.67 ng/mL (n = 11)] on a future plasma reference material compared with a GC/MS method procedure from the National Institute of Standards and Technology [18.41 +/- 0.70 ng/mL (n = 15)]. No adjustment will be necessary to compare previous (1999-2004) to future (2011-2014) NHANES MMA data.