LC-MS/MS for Identifying Patients with CYP24A1 Mutations Hemamalini Ketha, 1 Rajiv Kumar, 2,3,4 and Ravinder J. Singh 1* BACKGROUND: Patients have been described with loss- of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25- dihydroxyvitamin D [25(OH)D/24,25(OH) 2 D], in- creased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH) 2 D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH) 2 D (D 3 and D 2 ) and determined a 25(OH)D/24,25(OH) 2 D cutoff to identify candidates for confirmatory genetic testing. METHODS: After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline- 3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH) 2 D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS: The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH) 2 D 3 , and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH) 2 D 2 . Intra- and interassay im- precision was 4%–15% across the analytical measure- ment range of 0.1–25 ng/mL (0.2– 60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH) 2 D. 25(OH)D/24,25(OH) 2 D of 7–35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH) 2 D was significantly increased (99 – 467; P  0.001). A 25(OH)D/24,25(OH) 2 D ratio 99 identified pa- tients who were candidates for CYP24A1 genetic testing. CONCLUSIONS: Increased 25(OH)D/24,25(OH) 2 D sup- ports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/ 24,25(OH) 2 D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise un- known etiology. © 2015 American Association for Clinical Chemistry Vitamin D is essential for healthy calcium and phosphate homeostasis (1 ). Vitamin D 3 is formed in the skin by the UV light–mediated photolysis of 7-dehydrocholesterol (1, 2 ), whereas vitamin D 2 is obtained through the diet (1 ). The metabolism of both vitamin D metabolites is identical in mammals. Vitamin D is converted in the liver by 25- hydroxylase to 25-hydroxyvitamin D [25(OH)D] 5 (3, 4 ). Depending on calcium and phosphorus demands, 25(OH)D 3 is metabolized by 25(OH)D-1-hydroxylase to its bioactive form, 1,25-dihydroxyvitamin D [1,25(OH) 2 D] or by 25(OH)D-24-hydroxylase to an inactive metabolite, 24R,25-dihydroxyvitamin D [24,25(OH) 2 D] (1, 5–9 ). Vitamin D–related disorders are diagnosed by measuring serum concentrations of total (D 2 + D 3 ) 25(OH)D or 1,25(OH) 2 D (10, 11 ). Inactivating muta- tions of CYP24A1 (cytochrome P450, family 24, sub- family A, polypeptide 1) cause hypercalcemia, hypercal- ciuria, and increased 1,25(OH) 2 D concentrations (12–20 ). In studies describing LC-MS/MS assays for 24,25(OH) 2 D quantification, the relationship between 25(OH)D and 24,25(OH) 2 D has been used as a nutri- tional marker for assessment of optimum vitamin D sup- plementation (21 ). Mutations that cause reduced or complete loss of 24-hydroxylase function result in low or undetectable serum 24,25(OH) 2 D or increased 25(OH)D/24,25(OH) 2 D ratio. A ratio that could iden- tify candidates for confirmatory genetic testing has not been defined. Loss-of-function mutations in CYP24A1 have been identified as the underlying cause of hypercalcemia pre- viously considered to be idiopathic. Several groups have identified patients with similar mutations in the 24- 1 Department of Pathology, University of Michigan Health System, Ann Arbor, MI; 2 Divi- sion of Nephrology and Hypertension, 3 Department of Internal Medicine, and 4 Depart- ment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN. * Address correspondence to this author at: Hilton Bldg 7-30, Mayo Clinic, 200 2nd St, SW, Rochester, MN 55905. E-mail singh.ravinder@mayo.edu. Received June 1, 2015; accepted October 27, 2015. Previously published online at DOI: 10.1373/clinchem.2015.244459 © 2015 American Association for Clinical Chemistry 5 Nonstandard abbreviations: 25(OH)D, 25-hydroxyvitamin D; 1,25(OH) 2 D, 1,25- dihydroxyvitamin D; 24,25(OH) 2 D, 24R,25-dihydroxyvitamin D; PTAD, 4-phenyl-1,2,4,- triazoline-3,5-dione; LOD, limit of detection; LOQ, limit of quantification. Clinical Chemistry 62:1 236–242 (2016) Endocrinology and Metabolism 236 Downloaded from https://academic.oup.com/clinchem/article/62/1/236/5611793 by guest on 18 June 2022