Endocrine Abstracts

Although a complex metabolic pathway for vitamin D exists, serum measurement of inactive 25-hydroxyvitamin D3 (25OHD3) continues to be the most common determinant of vitamin D ‘status’. However, several other metabolites contribute to the physiological role of vitamin D, notably the active form 1α,25-dihydroxyvitamin D (1α,25(OH)2D3), inactive 3-epi-25OHD3 and chiral 23R and 24R,25(OH)2D3 metabolites. Quantification of these additional metabolites could provide a more accurate assessment of vitamin D status, particularly when comparing active/inactive metabolite ratios. The aim of this study was to develop and apply a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to analyse multiple vitamin D metabolites in a cohort of human serum samples obtained as part of a study on hormones and ageing (n=116; age range=20–74). A Waters AQCUITY UPLC, using a Lux cellulose chiral column, coupled to a Xevo TQ-S mass spectrometer was used for separation and quantitation. Serum samples were prepared by supportive liquid–liquid extraction (SLE). Quantifiable measurement of the following metabolites where achieved using 0.2 ml serum: 25OHD3; 3-epi-25OHD3; 24R,25(OH)2D3; 1α,25(OH)2D3; 25OHD2. Concentrations of 25OHD3 ranged between 2.166 and 42.706 ng/ml. 3-Epi-25OHD3 and 24R,25(OH)2D3 concentrations ranged between 0.445–5.977 ng/ml and 0.084–9.514 respectively. Both 3-epi-25OHD3 and 24R,25(OH)2D3 correlated with increased 25OHD3 (r=0.714 and 0.883 respectively). It was possible to quantify 1α,25(OH)2D3 in 40% of samples measured. Close correlation was observed for 25OHD3 and 24R,25(OH)2D3 (r=0.714, P<0.001), and 25OHD3 and 3-epi-25OHD3 (r=0.883, P<0.001). However, correlation was not observed between 25OHD3 and 1α25(OH)2D3 (r=0.276, P=0.032), and 24R,25(OH)2D3 and 1α25(OH)2D3 (r=0.297, P=0.023). No differences in metabolite levels where observed between age groups, although some seasonal alterations in serum vitamin D was observed.

The high-throughput LC–MS/MS method developed provides a detailed profile of vitamin D metabolites, enabling a more comprehensive analysis of vitamin D function. This approach may be particularly informative for studies of vitamin D and human disease.