This study reports 1,24,25(OH)₃D₃ levels in humans across a spectrum of measured GFR. To address the limitations of a cross-sectional study, we evaluated the same parameters longitudinally in the circulation of rats with the induction and progression of CKD. 1,25(OH)₂D₃ catabolism reflected by the 1,25-VMR increased whereas 25(OH)D₃ catabolism (25-VMR) decreased as mGFR declined, a finding that we also observed longitudinally in rats.

The progressive reduction in the 25-VMR with decreasing mGFR in humans, and with the induction and progression of CKD in rats, supports the work of others¹² and indicates stagnation of 25(OH)D₃ catabolism_.. The biological significance of 24-hydroxylated metabolites in the circulation is unclear; however, there is evidence that 1,24,25(OH)₃D₃ is biologically active and 24,25(OH)₂D₃ facilitates fracture repair^13,14,15,16. Global reduction of 24-hydroxylation in CKD has been proposed to explain low levels of 24,25(OH)₂D₃, however, this theory requires careful further evaluation given that the 1,25-VMR does not trend similarly to the 25-VMR. The kidneys are the main site of origin of 1,25(OH)₂D₃ in the circulation, yet increased or unchanged expression of CYP24A1 in kidney tissue has been reported^17,18,19,20. While those finding are not consistent with the reduction in the 25-VMR, they are consistent with the 1,25-VMR.

Participants taking cholecalciferol had higher levels of 1,24,25(OH)₃D₃. One possibility is that cholecalciferol may have increased 1,25(OH)₂D₃ production, and subsequent catabolism, despite low kidney function. If this hypothesis were true, this could support the use of cholecalciferol in patients with kidney disease. Alternatively, it could also suggest the presence of pathways that convert 25(OH)D₃ to 1,24,25(OH)₃D₃ independently of a 1,25(OH)₂D₃ intermediary as suggested by Martineau et al.¹⁶ In a study of participants with moderate to severe CKD, cholecalciferol supplementation increased levels of 25(OH)D₃ substantially, but the change in 24,25(OH)₂D₃ was more than proportional to the increase in 25(OH)D suggesting that supplementation increased delivery of 25(OH)D to CYP24A1 and/or increased CYP24A1 activity²¹. Levels of 1,25(OH)₂D₃ did not change. In a study involving participants of the Multi-Ethnic Study of Atherosclerosis, lower 25(OH)D and a lower 25-VMR was associated with a greater treatment response to cholecalciferol supplementation, as assessed by a change in PTH²². Similar to the previous study, there was no change in 1,25(OH)₂D₃ in the cholecalciferol supplemented participants.

Study strengths include the LC–MS/MS assessment of vitamin D metabolites which allows for accurate assessment of these structurally very similar metabolites, a limitation of other methods^4,23 as well as the measurement of GFR, as opposed to estimation based on endogenous markers. The cross-sectional design of the human study limits interpretation, however the longitudinal rat data demonstrated the identical evolution of changes in vitamin D metabolites with progressing CKD. The participants were predominantly white limiting generalizability. Future studies should consider acute challenges of different metabolites to assess specificity and kinetics of transformation. We acknowledge that variability in metabolite-to-parent ratios may not solely reflect true enzymatic activity. For example, Hsu et al. demonstrated higher 25(OH)D clearance in Black individuals, despite lower a 25-VMR²⁴. Further, given 1α-hydroxylated metabolites circulate at ~ 1000× lower concentration than their non-1α-hydroxylated counterparts, differences unrelated to 24-hydroxylation, for example intra-individual variation or assay variability, may meaningfully alter the 1,25-VMR, without altering other metabolites circulating at a higher-levels, and thus the circadian rhythm and the metabolism of 1,24,25(OH)₃D₃ is an important area of future research before use as a potential diagnostic tool. The development of a vitamin D profile that includes VMRs and evaluates the response over time to supplementation may better define adequacy in this population and guide future therapy as suggested by Melamed et al.²⁵.

In summary, the circulating 24-hydroxylated 1,25(OH)₂D₃ ratio does not decrease like the 25-VMR as kidney function declines. The clinical implications of the divergence between catabolism of 25(OH)D₃ versus 1,25(OH)₂D₃ requires further study. Understanding vitamin D catabolism and the potential that 24-hydroxylation products may have biological activity may help inform treatment strategies in the future.