25-hydroxyvitamin D can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication

Context: Vitamin D intoxication is characterized by elevated serum 25-hydroxyvitamin D (25(OH)D) and suppressed serum 1,25-dihydroxvitamin D (1,25(OH)₂D). We evaluated two adolescents with hypercalcemia due to vitamin D intoxication; both had elevated serum 1,25(OH)₂D by Diasorin RIA, but normal serum 1,25(OH)₂D concentrations by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Objective: This study aimed to determine the effect of 25(OH)D₂ and 25(OH)D₃ on 1,25(OH)₂D concentration determined using RIA and LC-MS/MS. Methods: Pools of normal serum and an artificial serum matrix were prepared and aliquots were spiked with >99% pure 25(OH)D₂ or 25(OH)D₃ (50-700 ng/mL). Samples were maintained at 4°C or heated to 56°C, and the concentrations of vitamin D metabolites were measured by LC-MS/MS and Diasorin RIA. Results: Median 1,25(OH)2 D increased by 114% with RIA and 21% with LC-MS/MS with addition of 100 ng/mL 25(OH)D₃, and 349% (RIA) and 117% (LC-MS/MS) with 700 ng/mL of 25(OH)D₃. Each 1-ng/mL increase in 25(OH)D₃ increased 1,25(OH)₂D by 0.231 pg/mL (RIA) and 0.121 pg/mL (LC-MS/MS). Spiking with 25(OH)D₂ led to similar changes. Heat inactivation of serum, and using an artificial serum matrix, were associated with similar effects of 25(OH)D on 1,25(OH)₂D assays. Conclusions: Vitamin D intoxication with high serum levels of 25(OH)D₂ or 25(OH)D₃ can be associated with elevated levels of 1,25(OH)₂D due to interference in a commonly used RIA. A similar but attenuated effect also occurs when 1,25(OH)2 D is measured using LC-MS/MS but does not seem to be clinically significant. The basis for this effect on the LC-MS/MS assay is presently uncertain.