Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation.

The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D3 and 25-hydroxyvitamin D2) is currently used as an indicator of vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-(2)H6]hydroxyvitamin D3 as the internal standard.

Contemporary diagnosis and treatment of vitamin D-related disorders.

Plasma 25(OH)D has emerged as a valuable biomarker for the many varied health-related effects of vitamin D in the clinic mainly because of the recognition of the importance of the enzyme, CYP27B1, or the 25(OH)D-alpha-hydroxylase in the extrarenal, target cell production of calcitriol. This review briefly assesses current methodology for plasma 25(OH)D assay focusing mainly on currrent controversies surrounding the definition of the normal range and performance characteristics of the assay, separate measurement of both 25(OH)D(2) and 25(OH)D(3), and quality assurance tesing of laboratories offering the test. Clinicians have two main types of 25(OH)D assay based on either high-performance liquid chromatography with UV or mass detection or higher throughput kits based on protein (competitive protein binding assay or radioimmunoassay) binding.

Insights into Vitamin D metabolism using cyp24 over-expression and knockout systems in conjunction with liquid chromatography/mass spectrometry (LC/MS).

The development of novel gene expression systems for cytochrome P450s (CYPs) together with a revolution in analytical mass spectrometry with the emergence of liquid chromatography/mass spectrometry (LC/MS) has opened the door to answering some long-standing questions in Vitamin D metabolism. Our studies focused on: (1) elucidating the role of CYP24 in 25-OH-D3 and 1alpha,25-(OH)2D3 metabolism; (2) exploring how DBP influences this process; (3) measuring 25-OH-D3 metabolism in CYP24-knockout (CYP24-XO) cells and; (4) comparing 1alpha-OH-D2 metabolism in the CYP24-XO mouse in vivo and in vitro. Methodology employed CYP24 over-expression and knockout systems in conjunction with state-of-the-art analytical LC/MS, diode array, and radioisotopic detection methods.

Effects of 'non-calcaemic' vitamin D analogues on 24-hydroxylase expression in MG-63 osteoblast-like cells.

Background: New 'non-calcaemic' analogues of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are entering the clinical arena and some of them have been shown to have differential effects in bone. This may have a bearing on the evolution of bone lesions in uraemic patients receiving vitamin D therapies. A potential mechanism for differential effects of analogues lies in their target cell inactivation.

Use of vitamin D(4) analogs to investigate differences in hepatic and target cell metabolism of vitamins D(2) and D(3).

In this study, we used molecules with either of the structural differences in the side chains of vitamin D(2) and vitamin D(3) to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems-HepG2 and HPK1A-ras-to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) but not for 1alpha-OH-D(3) is also observed in both 1alpha-OH-D(4) and Delta(22)-1alpha-OH-D(3) metabolism.