Plasma bile acid profile as a biomarker for drug-induced liver injury
Description
Description Bile Acid measurements (µM) in healthy volunteers, idiosyncratic drug-induced liver injury (DILI) and nonDILI cases for TransBioLine project. Background: The liver maintains bile acid (BA) homeostasis; circulating BA levels are used as a biomarker in certain cholestatic conditions. BAs can initiate processes in the pathogenesis of drug-induced liver injury (DILI), an unpredictable occurrence which can lead to liver failure. As such, this study aimed to explore whether changes in plasma BA profiles can serve as useful biomarkers for diagnostic and prognostic purposes in patients presenting with suspected DILI. Methods: In a prospective, nested case-control observational study, adult patients presenting with acute liver injury potentially due to DILI (at secondary care centres in 6 European countries) were sampled and followed through standard clinical care with severity and outcomes monitored. After review, cases were adjudicated as DILI (n=120) or nonDILI (alternate causes; n=49). Plasma BA levels and profile were quantified by University of Salamanca and compared to those in healthy volunteers (n=25). This is linked to publication 'Usefulness of plasma bile acid profile as a prognostic biomarker for drug-induced liver injury'. Values reported to 4 decimal places. Mean recovery (±SEM) for samples in the DILI group (n=120) was 89.0±0.8%, which was very close to that for the NonDILI group (n=49; 87.0±1.6%). The accuracy of calibration curves was calculated at an intermediate point of the concentration range, i.e., 5 µM. The results indicated that the average value (±SEM) was 91.6±0.8% for 230 calibration curves, with no significant differences among bile acid species. Reproducibility was calculated at two concentrations of the calibration curve for each of the 22 BAs analyzed. The coefficients of variation (CVs) were lower than 15% (range 2.8-14.5%) at 1 µM, and lower than 2% (range 0.8-1.8%) at 25 µM.
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EDTA-blood samples were collected at the study visit and separated by centrifugation and stored at -80ºC until analysis. A sub-set of DILI cases for analysis (n=120) were selected from those available at date of bile acid (BA) analysis in the prospectively-recruited TransBioLine study DILI cohort, (n=217) at random (randomisation.com), but with inclusion of all available cases with death/transplantation outcome or progression in severity. Plasma concentrations of 22 molecular BA species were determined by liquid chromatography tandem mass spectrometry (6420 Triple Quad LC/MS, Agilent Technologies, Santa Clara, CA) using an adaptation of a previously described method (Uriarte I, Santamaria E, López-Pascual A, et al. Biochim Biophys Acta Mol Basis Dis 2024; 1870(5): 167166; Ye L, Liu S, Wang M, Shao Y, Ding M. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences 2007; 860(1): 10-17). For patients, standard clinical biomarkers were determined at time of research visit as part of clinical care. For HV, these biomarkers were quantified in serum samples using standard clinical biochemistry assays (Cobas 6000 system, Roche) at MLM Medical Labs (Mönchengladbach, Germany). All analysis was done by researchers blinded to the study group. BAs were extracted from EDTA-plasma samples by using silica-based bonded phase cartridges (Sep-Pack Plus C18, Waters, Madrid)(Hernanz A, Codoceo R. Clinica Chimica Acta; International Journal of Clinical Chemistry 1985; 145(2): 197-203). Then, chromatographic separation was achieved with gradient elution using a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 µm) kept at 35ºC and a flow rate of 500 µl/min. Initial mobile phase was 73:27 methanol/water, both containing 5 mM ammonium acetate and 0.01% formic acid, pH 4.6, and it was changed to 97:3 methanol/water over 10 min and then returned to 73:27 for 1 min. Electrospray ionization (ESI) in negative mode was used, with the following conditions: gas temperature 350ºC, gas flow 11 l/min, nebulizer 45 psi, capillary voltage 2500 V. MS/MS acquisition was performed in multiple reaction monitoring (MRM) mode using the specific m/z transitions: [M-H]- ion to 80.2 for taurine-conjugated BAs and [M-H]- ion to 74 for glycine-conjugated BAs. As reported by others, free BAs do not generate characteristic ion fragments. Transition from unfragmented precursor molecular ions 407.1 to 407.1, 391.3 to 391.3 and 375.3 to 375.3 were selected for trihydroxylated, dihydroxylated and monohydroxylated unconjugated BAs, respectively. Transition 377.0 to 331.3 m/z was followed for the internal standard nor-DCA. The recovery of the whole analytical process, which included extraction from serum, followed by chromatographic separation using HPLC and quantification by MS/MS, was determined by the measurement of the added internal standard, i.e., nor-DCA.
Institutions
- Universidad de SalamancaCastilla y León, Salamanca
- Pfizer IncNY, New York
- University of NottinghamNottinghamshire, Nottingham
- Universidad de MalagaAndalucía, Malaga
- Universitat ZurichZH, Zurich