Whole Blood Metabolomics by ¹H NMR Spectroscopy Provides a New Opportunity To Evaluate Coenzymes and Antioxidants

Abstract

Conventional human blood metabolomics employs serum or plasma and provides a wealth of metabolic information therein. However, this approach lacks the ability to measure and evaluate important metabolites such as coenzymes and antioxidants that are present at high concentrations in red blood cells. As an important alternative to serum/plasma metabolomics, we show here that a simple 1H NMR experiment can simultaneously measure coenzymes and antioxidants in extracts of whole human blood, in addition to the nearly 70 metabolites that were shown to be quantitated in serum/plasma recently [Anal. Chem. 2015, 87, 706−715]. Coenzymes of redox reactions: oxidized/reduced nicotinamide adenine dinucleotide (NAD+ and NADH) and nicotinamide adenine dinucleotide phosphate (NADP+ and NADPH); coenzymes of energy including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP); and antioxidants, the sum of oxidized and reduced glutathione (GSSG and GSH) can be measured with essentially no additional effort. A new method was developed for detecting many of these unstable species without affecting other blood/blood plasma metabolites. The identities of coenzymes and antioxidants in blood NMR spectra were established combining 1D/2D NMR techniques, chemical shift databases, pH measurements and, finally, spiking with authentic compounds. This is the first study to report identification of major coenzymes and antioxidants and quantify them, simultaneously, with the large pool of other metabolites in human blood using NMR spectroscopy. Considering that the levels of coenzymes and antioxidants represent a sensitive measure of cellular functions in health and numerous diseases, the NMR method presented here potentially opens a new chapter in the metabolomics of blood.

Recent Advances in Targeted and Untargeted Metabolomics by NMR and MS/NMR Methods

“…Metabolomics profiling by NMR has been often performed by one-dimensional (1D) 1 H NMR experiments [ 26 ]. The advantage of 1D 1 H NMR is its short acquisition time, which is typically less than an hour/sample and high sensitivity; it is able to detect metabolites down to single digit μM concentration [ 27 ]. When this is coupled to a manual peak fitting or a manual spike-in approach, 1D 1 H NMR is capable to provide absolute concentration of dozens to hundred of metabolites in metabolomics samples [ 28 ].…”

Profiling Redox and Energy Coenzymes in Whole Blood, Tissue and Cells Using NMR Spectroscopy

“…Among others, important metabolites identified in whole blood extracts were NAD + , NADH, NADP + , NADPH, ATP, ADP, AMP, GSH and GSSG. A comparison of whole blood and plasma NMR spectra revealed that the redox coenzymes, energy coenzymes and antioxidants were detected only in whole blood [ 13 ] ( Figure 8 ).…”

“…This new capability is significant in that there was no NMR or MS method that can enable analysis of major coenzymes and antioxidants in whole blood. The new NMR method is anticipated to pave new ways for blood metabolomics [ 13 ].…”

“…Another major challenge unconnected with measurement methods is the instability of several coenzymes including the NADH, NADPH and ATP due to their sensitivity to sample harvesting and metabolite extraction protocols. Nevertheless, in separate studies using different types of tissues, cells and whole blood, we have overcome these bottlenecks and provided simple NMR methods to quantify many coenzymes and antioxidants, simultaneously, in addition to a vast pool of other metabolites [ 12 , 13 ]. Studies were also focused on detecting coenzymes in subcellular levels.…”

Metabolic Changes in Synaptosomes in an Animal Model of Schizophrenia Revealed by 1H and 1H,13C NMR Spectroscopy

“…Protein precipitation by methanol, chloroform, or a mixture of organic solvents is an efficient method to remove the proteins from the system. The use of this method with biological samples as has been reported in the literature [33][34][35][36].…”