LC-MS/MS for Identifying Patients with CYP24A1 Mutations. (6th January 2020)

• Record Type: Journal Article
• Authors: Ketha, Hemamalini; Kumar, Rajiv; Singh, Ravinder J
• Is Part Of: Clinical chemistry. Volume 62:Number 1(2016)
• Page Start: 236
• Page End: 242
• Publication Date: 2020-01-06
• Subjects: Clinical chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Biochimie -- Périodiques; Diagnostics biologiques -- Périodiques; Biochemistry; Clinical chemistry; Pharmaceutical chemistry; Biochemistry; Laboratory Techniques and Procedures; Klinische chemie; Periodicals; 616.075605
• DOI: 10.1373/clinchem.2015.244459
• Languages: English
• ISSNs: 0009-9147

Abstract

Abstract: 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], increased 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 (D3 and D2 ) 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 D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24, 25(OH)2 D2 . Intra- and interassay imprecision was 4%–15% across the analytical measurement 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 patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS: Increased 25(OH)D/24, 25(OH)2 D supports 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 unknown etiology.