Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)₂D₃ in Affected Patients

CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D₃:24,25-(OH)₂D₃ ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D₃ by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)₂D₃ remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)₂D₃ peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)₂D₃ as a contaminant of the 24,25-(OH)₂D₃ peak. In normals, the concentration of 24,25-(OH)₂D₃ greatly exceeds 25,26-(OH)₂D₃; however, 25,26-(OH)₂D₃ becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)₂D₃ levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)₂D₃ levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)₂D₃ from 25,26-(OH)₂D₃ produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)₂D₃ in IIH patients appears to be multifactorial.