MSACL 2025 Abstract

Distribution of Serum and Urinary 24,25(OH)₂D₃ and VMR and Their Association with PTH in an Adult Health Checkup Cohort

Nau Ishimine (1), Shixing Wu (1, 2), Taichi Shimazaki (3), Koji Takahashi (3), Masaki Takiwaki (3), Kentaro Abe (3)
(1) Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan, (2) Department of Medical Sciences, Graduate School of Medicine, Science and Technology, Shinshu University, Matsumoto, Japan,(3) ME Business Operations, JEOL Ltd., Tokyo, Japan.

Nau Ishimine, PhD (Presenter) Shinshu University Hospital

Relevant Financial Disclosures (within past 24 months, reported on Jul 07, 2025) Other Potential Conflicts JEOL (Japan Electron Optics Laboratory) / Research collaboration and reagent support / Ongoing Conflict mitigation NOT REQUIRED.

BACKGROUND:
Serum 25-hydroxyvitamin D (25[OH]D) is the standard marker of vitamin D status; however, it may not fully reflect functional vitamin D sufficiency. In particular, serum 25(OH)D shows a weak inverse correlation with parathyroid hormone (PTH) levels, suggesting that additional vitamin D metabolites are necessary to better reflect their biological effects. Two markers, 24,25-dihydroxyvitamin D₃ (24,25[OH]₂D₃) and the ratio of 24,25(OH)₂D₃ to 25(OH)D (vitamin D metabolite ratio, VMR), have emerged as potential improved indicators of vitamin D status. Non-invasive methods, such as urinary metabolite profiling, offer clinical advantages; however, they have not been thoroughly investigated. Herein, we aimed to investigate vitamin D metabolite distributions in both serum and urine in a large adult cohort and to examine their correlations with PTH to evaluate the utility of urine-based vitamin D markers compared with traditional serum measures.

METHODS:
We included 506 adults undergoing annual health checkups. Paired blood and spot urine samples were collected. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), concentrations of 25(OH)D₃, 25(OH)D2, and 24,25(OH)₂D₃ were measured in both serum and urine. Urine samples were pre-treated with β-glucuronidase to hydrolyze glucuronide conjugates. All samples were derivatized with DAP-PA (JeoQuantTM VD kit, JEOL, Japan) to enhance electrospray ionization. The derivatized metabolites were quantified by LC-MS/MS using isotope-labeled internal standards. Serum PTH was measured by immunoassays. Serum vitamin D status was classified as deficient (< 20 ng/mL, <50 nmol/L), insufficient (20–30 ng/mL, 50-75 nmol/L), or sufficient (> 30 ng/mL, > 75 nmol/L), based on thresholds widely used in clinical and epidemiological studies, including those of the Endocrine Society and Japanese guidelines. Furthermore, cut-offs were based on those commonly used in clinical and epidemiological studies. VMR was calculated using the following formula: VMR = [24,25(OH)₂D₃/25(OH)D] × 100. Correlations between vitamin D metabolites and VMR with PTH were analyzed.

RESULTS:
A total of 506 individuals (156 males, 350 females; median age 41 years) were included. Vitamin D deficiencies were identified in 39.5% of participants (serum 25[OH]D < 20 ng/mL), and 42.1% were insufficient (20–30 ng/mL). Thus, 81.6% of participants had suboptimal vitamin D status. The median serum 25(OH)D was 22.33 ng/mL and the median serum VMR was approximately 4.5%. Notably, 33.6% of participants had serum VMR < 4%, a cutoff proposed for low vitamin D metabolite profiles.
Differences by sex were found in serum 25(OH)D₃, 24,25(OH)₂D₃, and VMR. Urinary 24,25(OH)₂D₃ was the predominant metabolite, often exceeding urinary 25(OH)D₃ in concentration. Correlation analysis showed that urinary 25(OH)D and 24,25(OH)₂D₃ levels were significantly correlated with their corresponding serum metabolites (ρ > 0.6, p < 0.001). Urinary 24,25(OH)₂D₃ showed significantly stronger correlation with serum 25(OH)D than other urinary metabolites (p < 0.001, Steiger’s test). Both serum and urinary vitamin D metabolites showed significant inverse correlations with serum PTH. Notably, urinary 24,25(OH)₂D₃ and VMR demonstrated stronger correlations with PTH than their corresponding serum metabolites. Quartile analysis further confirmed that lower urinary metabolite levels were associated with higher PTH concentrations (p < 0.001).

CONCLUSIONS:
This study found a high prevalence of vitamin D insufficiency in this adult cohort. Moreover, low 24,25(OH)₂D₃ levels and low VMR were characteristic findings of vitamin D deficiency. We demonstrated that urinary vitamin D metabolites, particularly 24,25(OH)₂D₃ and urinary VMR, are more strongly correlated with PTH than serum 25(OH)D, suggesting that these non-invasive markers better reflect functional vitamin D status. These findings support the use of urinary vitamin D metabolite profiling as a practical and biologically meaningful tool. Its potential application in over-the-counter tests, such as self-health checkups, warrants further exploration.