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 1H 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 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.

中文翻译：

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扩展1H NMR光谱学的范围，以分析细胞辅酶A和乙酰辅酶A.

辅酶A（CoA）和乙酰辅酶A（乙酰CoA）是普遍存在的细胞分子，介导数百种同化和分解代谢反应，包括能量代谢。尽管存在许多局限性，但包括吸收光谱和质谱在内的高灵敏度方法仍可对其进行分析。但是，迄今为止，尚未使用NMR光谱分析这些重要分子。在我们最近的努力的基础上，我们能够同时分析组织和血液中的大量代谢产物，包括许多辅酶和抗氧化剂（Anal。Chem。2016，88，4817-24;同上2017，89，4620-4627），这是一种在组织中离体鉴定和定量CoA和乙酰辅酶A的新方法。利用老鼠的心脏，肾脏，肝脏，大脑和骨骼组织，我们表明，简单的1H NMR实验可以同时测量这些分子。两种物种的鉴定涉及使用1D和2D NMR对各种组织类型进行全面分析，结合标准的光谱数据库，以及掺入可靠的化合物。时间依赖性研究表明，尽管乙酰辅酶A水平保持不变，但辅酶A水平在24小时内减少了50％以上，这表明辅酶A在溶液中不稳定。但是，用氦气对样品进行脱气可以阻止其氧化。此外，有趣的是，我们还通过NMR首次在组织中鉴定出了内源性辅酶A谷胱甘肽二硫化物（CoA-SSG），并表明当在组织提取物中被氧化时，CoA也形成相同的二硫化物代谢产物。同时可视化CoA绝对浓度的能力，