A Metabolomics-Based Study on the Discriminative Classification Models and Toxicological Mechanism of Estazolam Fatal...

Fatal intoxication with sedative-hypnotic drugs is increasing yearly. However, the plasma drug concentration data for fatal intoxication involving these substances are not systematic and even overlap with the intoxication group. Therefore, developing a more precise and trustworthy approach to determining the cause of death is necessary. This study analyzed mice plasma and brainstem samples using the liquid chromatography-high resolution tandem mass spectrometry (LC-HR MS/MS)-based metabolomics method to create discriminative classification models for estazolam fatal intoxication (EFI). The most perturbed metabolic pathway between the EFI and EIND (estazolam intoxication non-death) was examined, Both EIND and EFI groups were administered 500 mg of estazolam per 100 g of body weight. Mice that did not die beyond 8 hours were treated with cervical dislocation and were classified into the EIND groups; the lysine degradation pathway was verified by qPCR (Quantitative Polymerase Chain Reaction), metabolite quantitative and TEM (transmission electron microscopy) analysis. Non-targeted metabolomics analysis with EFI were the experimental group and four hypoxia-related non-drug-related deaths (NDRDs) were the control group. Mass spectrometry data were analyzed with Compound Discoverer (CD) 3.1 software and multivariate statistical analyses were performed using the online software MetaboAnalyst 5.0. After a series of analyses, the results showed the discriminative classification model in plasma was composed of three endogenous metabolites: phenylacetylglycine, creatine and indole-3-lactic acid, and in the brainstem was composed of palmitic acid, creatine, and indole-3-lactic acid. The specificity validation results showed that both classification models distinguished between the other four sedatives-hypnotics, with an area under ROC curve (AUC) of 0.991, and the classification models had an extremely high specificity. When comparing different doses of estazolam, the AUC value of each group was larger than 0.80, and the sensitivity was also high. Moreover, the stability results showed that the AUC value was equal to or very close to 1 in plasma samples stored at 4 °C for 0, 1, 5, 10 and 15 days; the predictive power of the classification model was stable within 15 days. The results of lysine degradation pathway validation revealed that the EFI group had the highest lysine and saccharopine concentrations (mean (ng/mg) = 1.089 and 1.2526, respectively) when compared to the EIND and control group, while the relative expression of SDH (saccharopine dehydrogenase) showed significantly lower in the EFI group (mean = 1.206). Both of these results were statistically significant. Furthermore, TEM analysis showed that the EFI group had the more severely damaged mitochondria. This work gives fresh insights into the toxicological processes of estazolam and a new method for identifying EFI-related causes of mortality.