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

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.

LC-MS/MS application for urine free pyridinoline and free deoxypyridinoline: Urine markers of collagen and bone degradation.

Background: Pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of collagen degradation. Measurement of PYD and DPD can be used to evaluate changes in bone turnover in patients with metabolic bone disease and to monitor response to anti-resorptive treatment. Objective: To develop a method to extract and measure urine free PYD (fPYD) and free DPD (fDPD) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method was used to quantify urine samples from 172 healthy individuals and 63 patients diagnosed with metabolic bone disease. Method: Acidified urine samples were extracted using solid phase extraction with cellulose slurry. PYD and DPD were separated by reversed-phase, ion-paired chromatography prior to MS/MS detection. Results: The fully validated method showed good agreement with other laboratories in the UK National External Proficiency Scheme (UK NEQAS). The method was compared against two commercial immunoassays for fDPD and pyridinium cross-links, r 2 were 0.906 and 0.816 respectively. Urine concentrations of fDPD/Cr and fPYD/Cr were significantly higher in the patients than healthy individuals (p < 0.001). An average (±SD) fDPD:fPYD ratio of 0.29 (±0.08) was consistently observed across all subgroups. A markedly increased fDPD:fPYD ratio of 8.9 was observed in a patient with type VI Ehlers-Danlos Syndrome (EDS). Conclusion: Simultaneous measurement of two free pyridinium cross-links provides a valuable, cost effective assessment tool for use in the diagnostic work-up of patients with metabolic bone disease. Improvements in sample extraction efficiency have increased assay specificity and analysis throughput. The use of the fDPD:fPYD ratio can assist in the diagnosis of type VI EDS.