Reference intervals for serum 24,25-dihydroxyvitamin D and the ratio with 25-hydroxyvitamin D established using a newly developed LC–MS/MS method

• Published in: The Journal of nutritional biochemistry Vol. 46; pp. 21 - 29
• Main Authors: Tang, Jonathan C.Y., Nicholls, Holly, Piec, Isabelle, Washbourne, Christopher J., Dutton, John J., Jackson, Sarah, Greeves, Julie, Fraser, William D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2017
• Subjects: Adolescent; Adult; Aged; Aged, 80 and over; blood serum; calcium; Calcium - blood; Child; Child, Preschool; Chromatography, Liquid - methods; Female; genes; Humans; Hypercalcemia - blood; Hypercalcemia - genetics; industry; Male; metabolism; Middle Aged; mutation; patients; phosphates; Reference Values; Reproducibility of Results; Tandem Mass Spectrometry - methods; vitamin D; Vitamin D - analogs & derivatives; Vitamin D - blood; Vitamin D Deficiency - blood; Vitamin D3 24-Hydroxylase - genetics; vitamin status
• ISSN: 0955-2863; 1873-4847; 1873-4847
• DOI: 10.1016/j.jnutbio.2017.04.005

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1. The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC–MS/MS method to determine: (1) the relationships between serum 24,25(OH)2D and 25(OH)D; (2) serum reference intervals in healthy individuals; (3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC–MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (P<.001) at 25(OH)D<50 nmol/L. The reference interval for 25(OH)D:24,25(OH)2D ratio in healthy subjects was 7–23. Measurement of serum 24,25(OH)2D can be used as predictor of vitamin D status, a concentration of>4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities.