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

The measurement of 1α,25(OH)₂D₃ in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere.

During optimization of our in-house LC-MSMS method for serum 1α,25(OH)₂D₃ a previously co-eluting isobaric interference was separated. The isobar was identified as 1β,25(OH)₂D₃ by comparing retention time and fragmentation spectra to standards (other isobaric dihydroxylated vitamin D₃ analogs). 1β,25(OH)₂D₃ showed specific cluster formation (water), not present in 1α,25(OH)₂D₃. 1β,25(OH)₂D₃ was measured in serum of apparently healthy human volunteers (n=20), patients with high serum 25-hydroxyvitamin D [25(OH)D] concentrations (>50ng/mL) (n=33 among which 4 with very high levels (>150ng/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.

Median serum 1β,25(OH)₂D₃ was 11pg/mL in apparently healthy volunteers and increased to 20pg/mL for serum 25(OH)D concentrations above 80ng/mL (n=22) (p<0.0001). 1β,25(OH)₂D₃ 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 1β,25(OH)₂D₃ was 7pg/mL and not different from the median level in healthy volunteers (p=0.06). The median concentration did not vary with different stages.

We present evidence for the widespread presence of 1β,25(OH)₂D₃, 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 1β,25(OH)₂D₃ is a poor genomic agonist but a potent non-genomic antagonist of 1α,25(OH)₂D_3. The clinical implications of the presence of this analog therefore require further exploration.