Extending the Scope of ¹H NMR Spectroscopy for the Analysis of Cellular Coenzyme A and Acetyl Coenzyme A

Abstract

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) are ubiquitous cellular molecules, which mediate hundreds of anabolic and catabolic reactions including energy metabolism. Highly sensitive methods including absorption spectroscopy and mass spectrometry enable their analysis, albeit with many limitations. To date, however, NMR spectroscopy has not been used to analyze these important molecules. Building on our recent efforts, which enabled simultaneous analysis of a large number of metabolites in tissue and blood including many coenzymes and antioxidants [Anal Chem. 2016, 88:4817-24; ibid 2017, 89:4620-4627], we describe here a new method for identification and quantitation of CoA and acetyl-CoA ex vivo in tissue. Using mouse heart, kidney, liver, brain and skeletal tissue, we show that a simple 1 H NMR experiment can simultaneously measure these molecules. Identification of the two species involved a comprehensive analysis of the different tissue types using 1D and 2D NMR, in combination with spectral databases for standards, as well as spiking with authentic compounds. Time dependent studies showed that while the acetyl-CoA levels remain unaltered, CoA levels diminish by more than 50% within 24 h, which indicates that CoA is labile in solution; however, degassing the sample with helium gas halted its oxidation. Further, interestingly, we also identified endogenous coenzyme A glutathione disulfide (CoA-S-S-G) in tissue for the first time by NMR and show that CoA, when oxidized in tissue extract also forms the same disulfide metabolite. The new ability to simultaneously visualize absolute concentrations of CoA, acetyl-CoA and endogenous CoA-S-S-G along with redox coenzymes (NAD + , NADH, NADP + , NADPH), energy coenzymes (ATP, ADP, AMP), antioxidants (GSH, GSSG) and a vast pool of other metabolites using a single 1D NMR spectrum offers a new avenue in the metabolomics field for investigation of cellular function in health and disease.

Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement

“…31 Phosphorous ( 31 P) is most frequently utilized to visualize concentrations of ATP, phosphocreatine, and inorganic phosphate, which can provide a dynamic measure of ATP production in the tissue. 13 Carbon ( 13 C) and proton NMR ( 1 H) can also be used to measure intermediates in the TCA cycle [ 33 , 34 ]. In contrast to NMR, PET imaging employs the detection of injected positron-emitting radionuclide tracers to measure the accumulation or consumption of metabolic intermediates [ 35 , 36 ].…”

Whole Body Distribution of Labile Coenzymes and Antioxidants in a Mouse Model as Visualized Using 1H NMR Spectroscopy

“…Even worse, the significantly varying peak intensities in the NMR spectra, from one sample preparation method to another (Figure ), has resulted in a gross misrepresentation of the levels and sometimes even the identities of labile metabolites in numerous published studies. Efforts during the last several years in our laboratory have established their identity in NMR spectra and has resulted in solutions to address the challenges associated with their quantitation. − Combining this knowledge, in this study, we have established the distribution of labile metabolites in different organs of one of the widely used strains of the mouse model (C57BL/6). This study is the first of its kind and provides useful baseline values of the whole body distribution of the labile metabolites.…”

“…In the last several years, we have focused our efforts on addressing factors that affect the stability of the compounds. Using 1 H NMR spectroscopy, specimen harvesting, extraction, and analysis conditions were optimized for mouse heart tissue for the analysis of ATP, ADP, AMP, NAD + , NADH, NADP + , NADPH, CoA, and acetyl CoA, all in one step. , Separately, a method was also developed for analysis of energy and redox coenzymes in human blood . More recently, this method was extended for analysis of the antioxidants GSH and GSSG using a chemical derivatization step. , These methods promise new avenues for analysis of the labile metabolites.…”

NMR Metabolomics Methods for Investigating Disease

“…Their levels are sensitive to sample harvesting, metabolite extraction, and analysis conditions. Recently, several factors that affect the stability were addressed, and some of these unstable metabolites were shown to be reliably quantifiable. − The notoriously unstable antioxidant, glutathione, however, was still outside the purview of such analysis. The active form (reduced glutathione, GSH) is notoriously unstable under ex vivo conditions, and it spontaneously gets converted to the oxidized form (oxidized glutathione, GSSG).…”