1β,25-Dihydroxyvitamin D3: A new vitamin D metabolite in human serum

Background: The measurement of 1 alpha,25(OH)(2)D-3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere. Materials and methods: During optimization of our in-house LC-MSMS method for serum 1 alpha,25(OH)(2)D-3 a previously co-eluting isobaric interference was separated. The isobar was identified as 1 beta,25(OH)(2)D-3 by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D3 analogs). 1 beta,25(OH)(2)D-3 showed specific cluster formation (water), not present in 1 alpha,25(OH)(2)D-3.1 beta,25(OH)(2)D-3 was measured in serum of apparently healthy human volunteers (n = 20), patients with high serum 25-hydroxyvitamin D [25(OH)D] concentrations (>50 ng/mL) (n=33 among which 4 with very high levels (>150 ng/mL)) and patients with kidney failure (n = 68; 39 stage 1-3, 29 stage 4-5). Pearson's r was calculated for correlations and Mann-Whitney statistic to compare group medians. Results: Median serum 1 beta,25(OH)(2)D-3 was 11 pg/mL in apparently healthy volunteers and increased to 20 pg/mL for serum 25(OH)D concentrations above 80 ng/mL (n = 22) (p < 0.0001). 1 beta,25(OH)(2)D-3 concentrations were significantly correlated to serum 25(OH)D concentrations (r = 0.85) for the combined results from healthy volunteers and patient sera (n = 53) (p < 0.0001). For patients with kidney failure, median serum 1 beta,25(OH)(2)D-3 was 7 pg/mL and not different from the median level in healthy volunteers (p = 0.06). The median concentration did not vary with different stages. Conclusions: We present evidence for the widespread presence of 1 beta,25(OH)(2)D-3, a new vitamin D metabolite, in human serum. The level increases with rising serum 25(OH)D concentrations and is particularly high in patients with very high 25(OH)D levels. We previously demonstrated that 1 beta,25(OH)(2)D-3 is a poor genomic agonist but a potent non-genomic antagonist of 1 alpha,25(OH)(2)D-3. The clinical implications of the presence of this analog therefore require further exploration. (C) 2017 Elsevier Ltd. All rights reserved.