Matrix and Sampling Effects on Quantification of Protein Biomarkers of Drug-Induced Liver Injury

Abstract

Macrophage colony stimulating factor 1 receptor (MCSF1R), osteopontin (OPN), high-mobility group protein B1 (HMGB1), glutamate dehydrogenase (GLDH), keratin 18 (K18), and caspase-cleaved keratin 18 (ccK18) are considered promising mechanistic biomarkers for the diagnosis of drug-induced liver injury. Here, we aim to elucidate the impact of the sample matrix and handling on the quantification of these emerging protein biomarkers. We investigated effects such as time from collection to centrifugation during serum (± gel) or EDTA plasma preparation on two assay platforms: immunoaffinity liquid chromatography mass spectrometric assays and sandwich immunoassays. Furthermore, we measured GLDH activity with an enzymatic activity assay. Matrix effects were observed particularly for HMGB1 and MCSF1R. HMGB1 levels were higher in serum than in plasma, whereas higher concentrations of MCSF1R were observed in plasma than in serum. A comparison of sample collection to centrifugation time ranging from 15 to 60 min demonstrated increasing levels of HMGB1 in serum, while MCSF1R, OPN, GLDH, and ccK18 concentrations remained stable. Additionally, there was a poor correlation in HMGB1 and ccK18 levels between serum and plasma. Considering the observed matrix effects, we recommend plasma as a matrix of choice and cross-study comparison studies to be limited to those using the same matrix.

Study design for development of novel safety biomarkers of drug-induced liver injury by the translational safety biomarker pipeline (TransBioLine) consortium: a study protocol for a nested case–control study

“…3 ; Table 4 ). High mobility group box 1 (HMGB1), osteopontin (OPN), macrophage-colony-stimulating factor 1 receptor (MCSF1R) and GLDH will be analysed by multiplex immunoprecipitation coupled to nano-liquid chromatography and tandem mass spectrometry read-out (IP-LC–MS/MS) (Ultimate 300 coupled to Q-Exactive Plus, ThermoFisher) and quantified by adding 13 C/ 15 N -labelled peptide standards to the enzymatically fragmented sample as described by Anselm et al [ 28 ]. The candidate biomarkers cytokeratin-18 (K18) and caspase-cleaved K18 (ccK18) will be determined by sandwich immunoassays as described by the manufacturer (Peviva, Sweden).…”

In vivo antiviral efficacy and immune dampening effect of zapnometinib emphasize a dual therapeutic potential against COVID-19

“…Serum samples from hamsters representing pre-(0 dpi) and post-infection (4 dpi) and treatment timepoints were analyzed to assess the levels of different candidate safety biomarkers. Serum proteins were enzymatically fragmented by proteolysis, and protein-specific peptides were targeted for quantification of the corresponding protein biomarker levels by immunoaffinity liquid chromatography tandem mass spectrometry (IA-LC-MS/MS) performed as described previously 27, 28 . For details see supplementary.…”