Glenville Jones and Martin Kaufmann, Queen’s University Kingston, Ontario, Canada.
Abstract Text: Improvements in liquid chromatography-tandem mass spectrometry (LC-MS/MS) have triggered a revolution in small molecule clinical chemistry, including vitamin D. The additional sensitivity/selectivity provided by these new LC-MS/MS techniques makes it now feasible to measure at least the 8 metabolites: vitamin D, 25-OH-D, 3-epi-25-OH-D3, 24,25-(OH)2D3, 25,26-(OH)2D3, 1,25-(OH)2D3, 1,24,25-(OH)3D3 and 25-OH-D3-26,23-lactone simultaneously by use of liquid-liquid-extraction and immunoextraction steps. But the principal question is: Does assay of these metabolites have clinical utility? Working with clinical collaborators around the world, we would answer yes, they do for specific metabolites in particular conditions as follows:
1,25-(OH)2D3
Chronic Kidney Disease- Numerous studies have documented a fall in serum 25-OH-D & 1,25-(OH)2D with the decline in renal function but studies of the full vitamin D metabolome over the stages of CKD 1-5 have revealed that the same phenomenon applies to 24,25-(OH)2D3 as well. Both human and pre-clinical CKD show changes in the levels of 24,25-(OH)2D3 and 1,24,25-(OH)3D3 with changes in GFR (Turner et al Scientific Reports, 2022). The clinical significance of these changes remains to be elucidated.
Low Birthweight Infants/ Neonatal Conditions-
Recent studies (Romero-Lopez et al, VDW Cork 2024; Jain et al VDW Montreal 2025) reveal big increases in 1,25-(OH)2D3 during the neonatal period, presumably to correct stresses on calcium homeostasis. Changes in 3-epimerization and 24-hydroxylation also occur during this time period.
24,25-(OH)2D3
Infantile hypercalcemia, type 1 caused by defects in CYP24A1- The utility of measuring 24-hydroxylated forms, in particular, the 25-OH-D3:24,25-(OH)2D3 ratio has been established as a useful screening tool. Ratios are elevated from 5-25 in normals to >80 in affected IH individuals (e.g. Capellani et al Euro J Endocrinol, 2021).
Other hypercalcemias- Included in these are patients with Williams Syndrome, Hypervitaminosis D and those with excessive VDR-mediated gene expression (Kaufmann et al J Bone Mineral Res, 2021) who show normal 24,25-(OH)2D3 but elevated 25-OH-D3-26,23,lactone.
1,24,25-(OH)3D3
Clinical Trials of Vitamin D Supplementation. Reanalysis of the Calgary bone study (Burt et al, JAMA 2019), which used doses up to 10,000 IU of vitamin D3/day and showed falls in BMD, has revealed rises in several metabolites in addition to 25-OH-D3 e.g. 24,25-(OH)2D3 & 1,24,25-(OH)3D3 but not 1,25-(OH)2D3, suggesting that they could be responsible. (Burt et al JBMR 2023).
We conclude that study of a wider array of vitamin D metabolites from the vitamin D metabolome is providing clinical researchers with valuable additional information that improves diagnosis & understanding of diseases involving dysfunctional calcium and phosphate metabolism.