Hemamalini Ketha1, Rajiv Kumar2, Ravinder J Singh3. 1. Department of Pathology, University of Michigan Health System, Ann Arbor, MI; 2. Division of Nephrology and Hypertension, Department of Internal Medicine, and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN. 3. Department of Pathology, University of Michigan Health System, Ann Arbor, MI; singh.ravinder@mayo.edu.
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)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D 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)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D 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)2D 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)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. 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)2D. 25(OH)D/24,25(OH)2D of 7-35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99-467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS: Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.
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)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D 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)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D 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)2D 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)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. 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)2D. 25(OH)D/24,25(OH)2D of 7-35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99-467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS: Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.
Authors: Hemamalini Ketha; Tom D Thacher; Sara S Oberhelman; Philip R Fischer; Ravinder J Singh; Rajiv Kumar Journal: Bone Date: 2018-02-24 Impact factor: 4.398
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