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 redox and energy
coenzymes and
antioxidants in a single step. Major
coenzymes include redox
coenzymes: NAD⁺ (oxidized
nicotinamide adenine dinucleotide),
NADH (reduced
nicotinamide adenine dinucleotide), NADP⁺ (oxidized
nicotinamide adenine dinucleotide phosphate) and
NADPH (reduced
nicotinamide adenine dinucleotide phosphate); energy
coenzymes:
ATP (
adenosine triphosphate),
ADP (
adenosine diphosphate) and
AMP (
adenosine monophosphate); and
antioxidants:
GSSG (
oxidized glutathione) and GSH (
reduced glutathione). We show here that a simple ¹H NMR experiment can measure these
coenzymes and
antioxidants in tissue and whole blood apart from a vast pool of other metabolites. In addition, focused on the goal of identification of
coenzymes in subcellular fractions, we demonstrate analysis of
coenzymes in the cytoplasm using
breast cancer cells. Owing to their unstable nature, or low concentrations, most of the
coenzymes either evade detection or lose their integrity when established sample preparation and analysis methods are used. To overcome this challenge, here we describe the development of new methods to detect these molecules without affecting the integrity of other metabolites. We used an array of 1D and 2D NMR methods, chemical shift databases, pH measurements and spiking with authentic compounds to establish the identity of peaks for the
coenzymes and
antioxidants in NMR spectra. Interestingly, while none of the
coenzymes and
antioxidants were detected in plasma, they were abundant in whole blood. Considering that the
coenzymes and
antioxidants represent a sensitive measure of human health and risk for numerous diseases, the presented NMR methods to measure them in one step potentially open new opportunities in the metabolomics field.