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Master Thesis

Mise au point et validation d’une méthode de dosage par HPLC-MS/MS de métabolites hydroxylés de la vitamine D

Abstract : Hypersensitivity to vitamin D is defined by major hypercalcemia with hypercalciuria, sharp decreased level of parathormon (PTH) and increase of circulating 1,25-dihydroxyvitamin D. Diagnosed in adults and children, up to 20% of cases are due to a decline in the 24-hydroxylase activity of the CYP24A1 enzyme, involved in vitamin D catabolism. The diagnostic of this deficiency is based on long and expensive molecular analysis, sometime difficult to interpret. The use of the ratio between the serum concentrations of the metabolites 25OH-vitamin D3 and 24,25(OH)2-vitamin D3 has recently been suggested to identify patients who might present 24-hydroxylase deficiency. This ratio, named Vitamin D Hydroxymetabolite Ratio (VHR), tends to increase in patients with CYP24A1 mutations. This work has the objective of developing a fast and simultaneous assay for 24,25(OH)2-D3 et 25(OH)-D3 using high performance liquid chromatography combined with tandem mass spectrometry. Method validation was conducted following the ISO 15189 guidelines. We set reference ranges for both compounds and VHR in a cohort of hypercalcaemic patients with decreased PTH. A threshold value of 31.1 for VHR allows to identify with maximal sensitivity and specificity patients with 24-hydroxylase deficiency from those without mutation on CYP24A1 gene. VHR could thus be a support for differential diagnosis of hypercalcemia when classical etiologies have been ruled out and allow a simple screening before molecular analysis of CYP24A1 gene
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Master Thesis
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https://dumas.ccsd.cnrs.fr/dumas-02451834
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Clémentine Gondolf. Mise au point et validation d’une méthode de dosage par HPLC-MS/MS de métabolites hydroxylés de la vitamine D. Sciences pharmaceutiques. 2019. ⟨dumas-02451834⟩

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