Performance evaluation of four 25-hydroxyvitamin D assays to measure 25-hydroxyvitamin D2.

OBJECTIVES: The ability of current immunoassays to accurately measure equimolar amounts of 25(OH)D2 and 25(OH)D3 has been recently questioned. This study determined serum 25(OH)D2, 25(OH)D3 and total serum 25(OH)D concentrations in healthy vitamin D2-supplemented subjects by isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS); and, evaluated the ability of the Siemens, DiaSorin, Roche, and Abbott Vitamin D Total assays to monitor total serum 25(OH)D concentrations compared to an ID-LC-MS/MS method traceable to the National Institute of Standards and Technology (NIST), and that has achieved certification from the Centers for Disease Control and Prevention (CDC) Vitamin D Standardization Certification Program (VDSCP). DESIGN AND METHODS: Twenty (20) healthy adults, with no history of prior vitamin D supplementation were administered oral vitamin D2 (2400IU/day for 6months). Serum samples (140) from baseline and monthly blood draws were tested. RESULTS: After one month, the mean serum 25(OH)D2 concentrations rose from 0.8 to 43.6nmol/L, whereas 25(OH)D3 concentrations declined from 84.0 to 63.4nmol/L; total serum 25(OH)D concentrations rose from 86.6 to 107.0nmol/L. The overall mean bias to ID-LC-MS/MS was -7.1% for the Siemens ADVIA Centaur assay, -15.3% for the DiaSorin LIAISON assay; -8.4% for the Roche ELECSYS assay and -16.3% for the Abbott ARCHITECT assay. Correlation coefficients (r) were 0.94, 0.79, 0.74, and 0.73; the mean bias for baseline [25(OH) D3-containing] versus six-month [25(OH)D2- and 25(OH)D3-containing] samples was -13.4% and -5.7%; -3.5% and 20.3%, 9.6% and -12.1%, and 0.2% and -17.8%, respectively. CONCLUSIONS: The bias results obtained for the Siemens ADVIA Centaur assay and Roche ELECSYS assay were slightly lower than those for the DiaSorin LIAISON assay and the Abbott ARCHITECT assay, but all 25(OH)D assays demonstrated acceptable performance.

The 3 epimer of 25-hydroxycholecalciferol is present in the circulation of the majority of adults in a nationally representative sample and has endogenous origins.

Fundamental knowledge gaps in relation to the 3 epimer of 25-hydroxycholecalciferol [3-epi-25(OH)D3] limit our understanding of its relevance for vitamin D nutrition and health. The aims of this study were to characterize the 3-epi-25(OH)D3 concentrations in a nationally representative sample of adults and explore its determinants. We also used data from a recent randomized controlled trial (RCT) of supplemental cholecalciferol (vitamin D3) conducted in winter in older adults to directly test the impact of changes in vitamin D status on serum 3-epi-25(OH)D3 concentrations. Serum 25-hydroxycholecalciferol [25(OH)D3] and 3-epi-25(OH)D3 concentrations (via LC-tandem mass spectrometry) from our vitamin D3 RCT in adults (aged ≥50 y) and data on dietary, lifestyle, and biochemical characteristics of participants of the recent National Adult Nutrition Survey in Ireland (aged 18-84 y; n = 1122) were used in the present work. In the subsample of participants who had serum 3-epi-25(OH)D3 concentrations greater than the limit of quantification (n = 1082; 96.4%), the mean, 10th, 50th (median), and 90th percentile concentrations were 2.50, 1.05, 2.18, and 4.30 nmol/L, respectively, whereas the maximum 3-epi-25(OH)D3 concentration was 15.0 nmol/L. A regression model [explaining 29.9% of the variability in serum 3-epi-25(OH)D3] showed that age >50 y, vitamin D supplement use, dietary vitamin D, meat intake, season of blood sampling, and sun exposure habits were significant positive determinants, whereas increasing waist circumference and serum 25-hydroxyergocalciferol concentration were significant negative determinants. The RCT data showed that mean serum 25(OH)D3 and 3-epi-25(OH)D3 concentrations increased (49.3% and 42.1%, respectively) and decreased (-28.0% and -29.1%, respectively) significantly (P < 0.0001) with vitamin D3 (20 µg/d) and placebo supplementation, respectively, over 15 wk of winter. In conclusion, we provide data on serum 3-epi-25(OH)D3 in a nationally representative sample of adults. Our combined observational and RCT data might suggest that both dietary supply and dermal synthesis of vitamin D3 contribute to serum 3-epi-25(OH)D3 concentration.

Case report: Three patients with substantial serum levels of 3-epi-25(OH)D including one with 3-epi-25(OH)D2 while on high-dose ergocalciferol.

CONTEXT: We report the presence of substantial concentrations of 3-epi-25(OH)D3 in two patients and a third patient with 3-epi-25(OH)D2. PATIENTS: The first patient, a 66-year-old female receiving cholecalciferol 4000 IU daily was originally found to have 53 ng/mL of 25(OH)D3 and almost an equal amount of 3-epi-25(OH)D3. Subsequently, the patient had four additional samples, each of which has similar levels of both 25(OH)D3 and 3-epi-25(OH)D3. The second patient, a 7-year-old male receiving cholecalciferol 1000 IU daily, had a 25(OH)D3 concentration of 37 ng/mL and 3-epi-25(OH)D3 of approximately 10 ng/mL. The third and most intriguing patient, a 55-year-old female was receiving ergocalciferol 50,000 IU twice weekly for approximately 3 months, at which time her serum 25(OH)D2 was 64 ng/mL and her 3-epi-25(OH)D2 was approximately 32 ng/mL. This patient's physician changed her vitamin D therapy to cholecalciferol 1000 IU daily, discontinuing ergocalciferol, and a second specimen was collected 5 months later. Analysis of this last specimen found both 25(OH)D3 and 25(OH)D2 at concentrations of 12 and 24 ng/mL respectively, plus corresponding 3-epimer peaks for both 25(OH)D3 and 25(OH)D2 observed chromatographically. CONCLUSION: The presence of a substantial concentration of 3-epi-25(OH)D in these three patients documents that one cannot assume 3-epi is a trivial metabolite of 25(OH)D for all patients. In addition, the appearance of 3-epi-25(OH)D3 when the last patient changed her vitamin D supplementation from ergocalciferol to cholecalciferol demonstrates that the 3-epimer is probably an endogenous metabolite of 25(OH)D in humans.