An Integrated LC-MS/MS Strategy for Quantifying the Oxidative-Redox Metabolome in Multiple Biological Samples

Abstract

The cellular redox balance plays a significant role in cell fate decisions and in the regulation of responses to various kinds of stress. In this study, we defined a novel concept of the oxidative-redox metabolome, and established a method for the simultaneous quantification of 23 metabolites involved in the oxidative-redox metabolome, covering NAD⁺ pathway, FAD pathway, GSSG pathway, and ATP pathway by using the AB SCIEX 5500 QTRAP LC/MS/MS system. Corresponding oxidative-redox metabolomics analysis was performed in plasma of humans, hamsters and mice, and hamsters were demonstrated to display a stronger resemblance than mice to humans. The known reductant dithiothreitol (DTT) and oxidant hydrogen peroxide (H₂O₂) were selected to treat A549 and HeLa cells to validate the current method, showing that DTT moderately increased while H₂O₂ greatly decreased most analytes. Antibiotic treatment may disturb the oxidative-redox balance in vivo. By comparing the oxidative-redox metabolome in antibiotic-fed hamsters with that of control hamsters, we demonstrated a substantial metabolic disparity between the two, further verifying the applicability and reliability of our method.