Decoding liver injury: Vitamin D3 metabolite fingerprints in cholestatic liver disease diagnosis

Conventional indicators for diagnosing liver injury in cholestatic liver diseases (CLD) include a combination of biochemical, imaging, and clinical assessments, which are critically limited by non-specificity, delayed detection windows, and poor correlation with cholestasis-specific mechanisms, underscoring the need for novel biomarker-driven strategies. Emerging biomarkers (e.g., vitamin D metabolites) are being explored to improve early diagnosis and specificity in liver injury assessment.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for simultaneous quantification of 25-hydroxyvitamin D3 [25-(OH)-D₃], 1,25-dihydroxyvitamin D3 [1,25-(OH)₂-D₃], 24,25-dihydroxyvitamin D3 [24,25-(OH)₂-D₃], and the vitamin D metabolite ratio (VMR), providing a robust framework for assessing both systemic vitamin D deficiency and cholestatic liver injury severity. To interrogate vitamin D3 metabolism dynamics in CLD progression, statistical analyses-one-way ANOVA, correlation analysis, receiver operating characteristic (ROC) curves, and heatmaps-were applied to evaluate associations between metabolic profiles and liver injury severity.

Stratified by the Child-Pugh scoring system, serum levels of 25-(OH)-D₃, 1,25-(OH)₂-D₃, 24,25-(OH)₂-D₃, and VMR exhibited a progressive decline across cohorts-healthy controls (HC), moderate liver injury (MLI), and severe liver injury (SLI)-correlating inversely with advancing disease severity. Notably, 24,25-(OH)₂-D₃ demonstrated superior diagnostic accuracy for CLD, achieving an area under the curve (AUC) of 0.994. Robust correlations between 25-(OH)-D₃ and 24,25-(OH)₂-D₃ were observed in all cohorts (HC, MLI, SLI), though the regression slopes progressively attenuated in MLI and SLI cohorts, indicating preserved but diminished metabolic flux from 25-(OH)-D₃ to 24,25-(OH)₂-D₃ as liver injury worsened. This attenuation aligns with progressive functional impairment in vitamin D3 metabolism. Clinically, post-treatment profiling following one week of hospitalization revealed that 25-(OH)-D₃ exhibited superior sensitivity in tracking therapeutic responses compared to conventional cholestatic liver disease (CLD) indicators, underscoring their utility as dynamic biomarkers.

The metabolites, particularly 24,25-(OH)₂-D₃, demonstrated excellent diagnostic accuracy for liver injury in CLD, while 25-(OH)-D₃ outperformed conventional indicators in monitoring therapeutic response during cholestasis management.