Abstract
OBJECTIVE: LC-MS/MS-based metabolomics is an important tool for studying disease-related biomarkers. Conventionally, different strategies have been used to screen biomarkers. However, many studies for biomarker screening by different strategies have ignored the dose-response relationship between the biomarker level and exposure level, and no relevant studies have described and compared different strategies in detail. Phenobarbital (PHB) which belongs to the barbiturates, was selected as the typical representative of neurotoxins. Acylcarnitines have been promising candidates for diagnostic biomarkers for several neurological disorders and neurotoxicity. In this work, we aimed to use an acute PHB poisoning animal model to clarify PHB poisoning effects on plasma and brain acylcarnitine changes and how to rationally analyze data from LC-MS/MS. METHODS: The acylcarnitine profiles in plasma and brain regions in an actuate PHB poisoning animal model were utilized. The dose-response relationship between plasma PHB and carnitine and acylcarnitines (CARs) in plasma and brain were assessed by the variance analysis trend test and Spearman's rank correlation test. In different strategies, principal component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) screened the differential CARs, variable importance plots (VIPs) were utilized to select putative biomarkers for PHB-induced toxicity, and receiver operating characteristic (ROC) curve analysis then illustrated the reliability of biomarkers. RESULTS: Under the first strategy, 14 potential toxicity biomarkers were obtained including eight downregulated CARs with AUC >0.8. Under the second strategy, 11 potential toxicity biomarkers were obtained containing five downregulated CARs with AUC >0.8. Only when the dose-response relationship was fully considered, different strategies screen for the same biomarkers (plasma acetyl-carnitine (C2) and plasma decanoyl-carnitine (C10)), which indicated plasma acylcarnitines might serve as toxicity biomarkers. In addition, the plasma CAR level changes showed differences from brain CAR level changes, and correlations between plasma CARs and their brain counterparts were weak. CONCLUSION: We found that plasma C2 and C10 might serve as toxicity biomarkers for PHB poisoning disorders, and PHB poisoning effects on changes in plasma CARs may not be fully representative of changes in brain CARs.