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

Abstract

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.