Analysis of Coenzymes and Antioxidants in Tissue and Blood Using 1D 1H NMR Spectroscopy

Abstract

Cellular coenzymes including coenzyme A (CoA), acetyl coenzyme A (acetyl-CoA), coenzymes of redox reactions and of energy, and antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. The redox coenzymes include NAD+ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), NADP+ (oxidized nicotinamide adenine dinucleotide phosphate), and NADPH (reduced nicotinamide adenine dinucleotide phosphate); the energy coenzymes include ATP (adenosine triphosphate), ADP (adenosine diphosphate), and AMP (adenosine monophosphate); and the antioxidants include GSSG (oxidized glutathione) and GSH (reduced glutathione). Their measurement is important to better understand cellular metabolism. Recent advances have pushed the limit of metabolite quantitation using NMR methods to an unprecedented level, which offer a new avenue for analysis of the coenzymes and antioxidants. Unlike the conventional enzyme assays, which need separate protocols for analysis, a simple 1D 1H NMR experiment enables analysis of all these molecular species in one step. In this chapter, we describe protocols for their identification and quantitation in tissue and whole blood using NMR spectroscopy.