Methods for the Measurement of Vitamin D Metabolites and Studies on Their Relationships in Health and Disease

Tang, Jonathan (2019) Methods for the Measurement of Vitamin D Metabolites and Studies on Their Relationships in Health and Disease. Doctoral thesis, University of East Anglia.

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The prevalence of vitamin D deficiency in the general population has become a major public health problem. Vitamin D deficiency might have significant consequences not only to bone health but possible to other autoimmune, infectious, cancer and cardiovascular diseases. The explosion of interests in vitamin D had sparked a massive increase in the number of laboratory requests for the measurement of serum 25 hydroxyvitamin D (25(OH)D). This in turn highlighted problems of the methodologies available for measuring vitamin D metabolites.

The aim of the study was to develop and fully validate quantitative assays for measuring serum vitamin D metabolites by Liquid Chromatography Tandem Mass Spectrometry (LC/MS-MS). The methods were used to perform analysis on samples collected for vitamin D research studies to establish relationships between the metabolites and determine reference intervals.

Using solid phase extraction to remove phospholipids in sample matrix and derivatisation to enhance sensitivity, assays were successfully developed for 25(OH)D3/D2, C3-epi-25(OH)D3/D2, and for the dihydroxyvitamin D 24,25(OH)2D3/D2 and 1,25(OH)2D3/D2. The performance characteristic of the assays satisfied industry standards for method validation. Results showed a high prevalence of C3-epi-25(OH)D3 (87.7%) in paediatric samples that resulted in misclassification of total 25(OH)D status in 10.4% of cases. Serum 25(OH)D showed a significant correlation with 24,25(OH)2D (r2=0.754, p<0.001), but not with 1,25(OH)2D (r2=0.1034). The reference intervals (2.5-97.5 percentile) for 25(OH)D:24,25(OH)2D ratio was established between 7-23. Loess fitting showed an increase in the 25(OH)D:24,25(OH)2D ratio at 25(OH)D <50 nmol/L; evidence of reduced catabolic activity during low vitamin D status. In contrast, when high dose vitamin D3 was supplemented, serum 24,25(OH)2D was found to be grossly elevated to counteract against excessive vitamin D and prevent toxicity. Using the 1,25(OH)2D:24,25(OH)2D/25(OH)D ratio model, this thesis was the first to demonstrate a relationship between the three metabolites, and the association with PTH concentration.

This thesis has provided new insights to the vitamin D metabolism that will further our understanding and appreciation of its role in health and pathophysiological conditions. The methods developed have provided an analytical platform for many large scale studies in musculoskeletal research and other areas of science; the publications and citations are a testament to the impact of this research.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 10 Jun 2020 11:17
Last Modified: 10 Jun 2020 11:17

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