Extending the Scope of 1H NMR-Based Blood Metabolomics for the Analysis of Labile Antioxidants: Reduced and Oxidized Glutathione

Glutathione is a ubiquitous cellular antioxidant, which is critically required to protect cells from oxidative damage and free radical injury. It is practically impossible to analyze glutathione in its native form after isolation from biological mixtures since the active form (reduced glutathione, GSH) spontaneously gets converted to the oxidized form (oxidized glutathione, GSSG). To address this challenge, numerous highly sensitive detection methods, including mass spectrometry, have been used in conjunction with derivatization to block the oxidation of GSH. Efforts so far to quantitate GSH and GSSG using the nuclear magnetic resonance (NMR) spectroscopy method have remained unsuccessful. With a focus on addressing this challenge, in this study, we describe an extension to our recent whole blood analysis method [Anal. Chem. 2017, 89, 4620-4627] that includes the important antioxidants GSH and GSSG. Fresh and frozen human whole blood specimens as well as standard GSH and GSSG were comprehensively investigated using NMR without and with derivatization using N-ethylmaleimide (NEM). NMR experiments detect two diastereomers, distinctly, for the derivatized GSH and enable the analysis of both GSH and GSSG in human whole blood with an accuracy of >99%. Interestingly, the excess (unreacted) NEM used for blocking the GSH can be removed from the samples during a drying step after extraction, with no need for additional processing. This is an important characteristic that offers an added advantage for simultaneous analysis of the antioxidants (GSH and GSSG), redox coenzymes (oxidized nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), reduced nicotinamide adenine dinucleotide phosphate (NADPH)), energy coenzymes (adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP)), and a large number of other blood metabolites using the same one-dimensional (1D) NMR spectrum. The presented method broadens the scope of global metabolite profiling and adds a new dimension to NMR-based blood metabolomics. Further, the method demonstrated here for human blood can be extended to virtually any biological specimen.