Profiling Redox and Energy Coenzymes in Tissue and Blood Using NMR Spectroscopy
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Coenzymes of cellular redox reactions and cellular energy, as well as antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. Conventional analysis methods lack the opportunity to evaluate these important coenzymes including coenzymes of redox reactions, oxidized/reduced nicotinamide adenine dinucleotide (NAD+ and NADH) and nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH); coenzymes of energy, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP); and antioxidants, oxidized/reduced glutathione (GSSG and GSH). As an important alternative to tissue and serum/plasma metabolomics, we show here that a simple 1H NMR experiment can simultaneously measure the coenzymes and antioxidants in tissue and whole human blood. Owing to their unstable nature, or low concentrations, many coenzymes evade detection using established sample preparation protocols. Here we developed new methods to detect these interesting species without affecting other metabolites. Identities of the coenzymes and antioxidants in tissue and blood NMR spectra were established by combining 1D/2D NMR techniques, chemical shift databases, pH measurements and, finally, spiking with authentic compounds. Interestingly, while none of the coenzymes and antioxidants was detected in plasma, they were abundant in whole blood due to the high concentration of red blood cells. This is the first study to report identification of major coenzymes and antioxidants, and measure them, simultaneously and with high resolution, along with a vast pool of other metabolites in tissue and blood using NMR spectroscopy. Considering that the coenzymes and antioxidants represent a sensitive measure of human health and risk for numerous diseases, the presented NMR method potentially opens new opportunities in the metabolomics field.