The metabolomics of acetaminophen toxicity observed in human biofluids and cultured primary human hepatocytes

The mechanisms of acetaminophen toxicity are well-established. However, the use of metabolomics to identify small molecule (< 1 kD) biomarkers of acetaminophen toxicity in human biofluids is novel. This research establishes the first pharmaco-metabolomic study of acetaminophen in a population of humans. This method makes use of multivariate statistical techniques to elucidate changes in the metabolome before clinical manifestation of acetaminophen toxicity. Furthermore, prior to this experimental analysis, another study was performed which demonstrated that the human metabolome normalized within 2 days of a standardized diet in an inpatient hospital setting.The use of 13C-labeled nutrient tracers to identify off-target enzyme (> 10 kD) inactivation in primary human hepatocyte cultures is original. By tracking the metabolism of 13C tracers, a metabolomic surrogate of enzyme inactivation due to acetaminophen toxicity was discovered. The enzyme inactivation is likely via arylation by the cytochrome P450 bioactivated acetaminophen metabolic product N-acetyl-para-quinonimine. Furthermore, it was observed that the human hepatocytes appeared to be in a stressed metabolic state, due to the lack of glycolysis or glutaminolysis, even in the presence of high insulin and glucose concentrations. This metabolism was compared to that of primary rat hepatocyte cultures, which did not exhibit these features, likely due to absence of stress inducing hormones prior to hepatocyte isolation. This has yet to be described in the literature, likely because this is the first report of the use of 13C-labeled nutrients in primary human hepatocyte cultures.