C3-Epimer Inclusion Influences Vitamin D Deficiency Classification Near Diagnostic Thresholds in Early Childhood

Background: Vitamin D deficiency in early childhood is commonly defined using fixed 25(OH)D thresholds, most often <20 ng/mL. However, inclusion of the C3-epimer (3-epi-25(OH)D₃) in total 25(OH)D measurements may influence classification, particularly in children with borderline concentrations. Methods: In this cross-sectional study of 128 children aged 0-36 months, vitamin D metabolites were quantified using LC-MS/MS in dried blood spot (DBS) samples. Vitamin D deficiency was defined as <20 ng/mL. Total 25(OH)D was recalculated after subtraction of the C3-epimer to assess changes in deficiency classification. Agreement was evaluated using Cohen's κ, and systematic differences were tested with McNemar's test. Diagnostic performance parameters were calculated using epimer-resolved 25(OH)D as the reference standard. Results: Mean 25(OH)D₃ concentration was 20.3 ± 6.2 ng/mL, and 57% of children were classified as deficient. After epimer subtraction, deficiency classification changed in 22 of 128 children (17.2%). Agreement between classifications was substantial (κ = 0.67), but McNemar's test demonstrated a significant systematic shift (p < 0.001). Sensitivity of total 25(OH)D including the epimer for detecting deficiency was 70.3% (95% CI: 59.0-80.0%), with specificity of 100% (95% CI: 94.3-100%). Reclassification was strongly concentrated among children with borderline 25(OH)D₃ concentrations (18-22 ng/mL), where 54.5% were reclassified compared with 4.2% outside this range. Reclassification was not associated with age. Conclusions: In young children, inclusion of the C3-epimer in total 25(OH)D measurement leads to potentially clinically relevant misclassification of vitamin D deficiency, particularly near diagnostic thresholds. Epimer-resolved assessment may improve diagnostic precision in cases with borderline vitamin D concentrations.