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25-Hydroxyvitamin D Can Interfere With a Common Assay for 1,25-Dihydroxyvitamin D in Vitamin D Intoxication
Colin P Hawkes1, Sarah Schnellbacher1, Ravinder J Singh1
1Division of Endocrinology and Diabetes (C.P.H., S.S., M.A.L.), The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; National Children's Research Centre (C.P.H.), Dublin, Ireland; Department of Laboratory Medicine and Pathology (R.J.S.), The Mayo Clinic, Rochester, Minnesota 55905; and Department of Pediatrics (M.A.L.), Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
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)2D 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)2D 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.
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