23,25-Dihydroxyvitamin D₃ is liberated as a major vitamin D₃ metabolite in human urine after treatment with β-glucuronidase: Quantitative comparison with 24,25-dihydroxyvitamin D₃ by LC/MS/MS

The complete understanding of the excretion of surplus 25-hydroxyvitamin D₃ [25(OH)D₃] in humans remains to be accomplished. In our previous study, 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] 24-glucuronide was identified as a major urinary vitamin D₃ metabolite, while the glucuronide of 23,25-dihydroxyvitamin D₃ [23,25(OH)₂D₃] is another metabolite of interest but has not been sufficiently evaluated. Although the quantitative analysis of 24,25(OH)₂D₃ liberated in urine by the treatment with β-glucuronidase (GUS) has been conducted, no information was provided about the amount of the glucuronidated 23,25(OH)₂D₃ in the urine. In this study, we first developed and validated a liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS)-based method for the simultaneous quantification of 23,25(OH)₂D₃ and 24,25(OH)₂D₃ liberated in urine by GUS. The analysis of the urine samples revealed that the amount of 23,25(OH)₂D₃ was almost as much as that of 24,25(OH)₂D₃, in contrast to the fact that the plasma concentration of 23,25(OH)₂D₃ was much lower than that of 24,25(OH)₂D₃. These results strongly suggested that 23,25(OH)₂D₃ is more susceptible to glucuronidation and more promptly excreted into urine than 24,25(OH)₂D₃. Furthermore, the amount ratios of 23,25(OH)₂D₃ to 24,25(OH)₂D₃ in the urine samples did not markedly vary during the day (morning/evening) and even by a week-long vitamin D₃ supplementation (1000 IU/body/day). We concluded that the C-23 hydroxylation plays a crucial role in the urinary excretion of surplus 25(OH)D₃.