Validation and pre-analytical considerations of a Liquid-Chromatography Tandem Mass Spectrometry 7a-hydroxy-4-cholesten-3-one assay, and investigation into its diagnostic utility for Bile Acid Diarrhoea

Abstract

Bile Acid Diarrhoea (BAD) is estimated to affect 5% of the population, however it remains significantly under-diagnosed. This is, in part, due to lack of awareness, and due to limited availability of diagnostic tests. 7a-hydroxy-4-cholesten-3-one (7aC4) is a precursor in the bile acid synthesis pathway and increases in BAD to replace bile acids lost in the faeces. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measurement of 7aC4 was optimised and validated. The impact of lipid-lowering therapy, diurnal variation and food intake on 7aC4 concentrations was assessed, and investigation into the clinical utility of 7aC4 measurement was performed comparing 7aC4 to the 23-seleno-25-homotaurocholic acid (SeHCAT) scan. The LC-MS/MS assay for 7aC4 was validated for routine use. 7aC4 demonstrated diurnal variation with increased concentrations at 12noon compared to 8am and 10am, indicating a requirement for blood collection between 8 and 10am. 7aC4 demonstrated no significant post-prandial response in the 3 and half hours following intake of a standardised meal, meaning fasting blood collection is not required for analysis. Concurrent lipid lowering therapy treatment decreased 7aC4 concentrations, and although further sample collection is required to fully elucidate the effects of lipid lowering therapy, caution should be exercised when interpreting 7aC4 concentrations when on these treatments. 7aC4 concentrations were significantly higher in patients with SeHCAT retention of 15%. Using a cut-off of 50nmol/L, 7aC4 demonstrated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 93%, 46%, 78% and 75%, respectively, whilst a cut-off of 131 nmol/L demonstrated sensitivity, specificity, PPV and NPV of 37%, 85%, 83% and 39%, respectively. The performance characteristics suggest 7aC4 may demonstrate utility in stratifying patients for SeHCAT referral.

Date of Award	1 Jan 1824
Original language	English
Awarding Institution	The University of Manchester
Supervisor	John Mclaughlin (Supervisor)