Simultaneous Analysis of Major Coenzymes of Cellular Redox Reactions and Energy Using ex Vivo 1H NMR Spectroscopy,Analytical Chemistry

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Simultaneous Analysis of Major Coenzymes of Cellular Redox Reactions and Energy Using ex Vivo 1H NMR Spectroscopy
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-04-14 00:00:00 , DOI: 10.1021/acs.analchem.6b00442
G A Nagana Gowda , Lauren Abell , Chi Fung Lee , Rong Tian , Daniel Raftery ₁

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Coenzymes of cellular redox reactions and cellular energy mediate biochemical reactions fundamental to the functioning of all living cells. Despite their immense interest, no simple method exists to gain insights into their cellular concentrations in a single step. We show that a simple ¹H NMR experiment can simultaneously measure oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD⁺ and NADH), oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate (NADP⁺ and NADPH), and adenosine triphosphate (ATP) and its precursors, adenosine diphosphate (ADP) and adenosine monophosphate (AMP), using mouse heart, kidney, brain, liver, and skeletal muscle tissue extracts as examples. Combining 1D/2D NMR experiments, chemical shift libraries, and authentic compound data, reliable peak identities for these coenzymes have been established. To assess this methodology, cardiac NADH and NAD⁺ ratios/pool sizes were measured using mouse models with a cardiac-specific knockout of the mitochondrial Complex I Ndufs4 gene (cKO) and cardiac-specific overexpression of nicotinamide phosphoribosyltransferase (cNAMPT) as examples. Sensitivity of NAD⁺ and NADH to cKO or cNAMPT was observed, as anticipated. Time-dependent investigations showed that the levels of NADH and NADPH diminish by up to ∼50% within 24 h; concomitantly, NAD⁺ and NADP⁺ increase proportionately; however, degassing the sample and flushing the sample tubes with helium gas halted such changes. The analysis protocol along with the annotated characteristic fingerprints for each coenzyme is provided for easy identification and absolute quantification using a single internal reference for routine use. The ability to visualize the ubiquitous coenzymes fundamental to cellular functions, simultaneously and reliably, offers a new avenue to interrogate the mechanistic details of cellular function in health and disease.

中文翻译：

使用离体 1H NMR 光谱同时分析细胞氧化还原反应和能量的主要辅酶

细胞氧化还原反应和细胞能量的辅酶介导对所有活细胞功能至关重要的生化反应。尽管他们非常感兴趣，但不存在简单的方法可以一步深入了解它们的细胞浓度。我们表明，简单的¹ H NMR 实验可以同时测量烟酰胺腺嘌呤二核苷酸的氧化和还原形式（NAD ⁺和 NADH）、烟酰胺腺嘌呤二核苷酸磷酸（NADP ⁺和 NADPH）的氧化和还原形式以及三磷酸腺苷（ATP）和其前体二磷酸腺苷（ADP）和一磷酸腺苷（AMP），以小鼠心脏、肾脏、大脑、肝脏和骨骼肌组织提取物为例。结合一维/二维核磁共振实验、化学位移库和真实的化合物数据，已经建立了这些辅酶的可靠峰身份。为了评估这种方法，使用小鼠模型测量了心脏 NADH 和 NAD ⁺比率/库大小，以心脏特异性敲除线粒体复合物 I Ndufs4基因 (cKO) 和心脏特异性烟酰胺磷酸核糖基转移酶 (cNAMPT) 过度表达为例。正如预期的那样，观察到NAD ⁺和 NADH 对 cKO 或 cNAMPT的敏感性。随时间变化的研究表明，NADH 和 NADPH 的水平在 24 小时内降低了约 50%；与此同时，NAD ⁺和NADP ⁺按比例增加；然而，对样品进行脱气并用氦气冲洗样品管可以阻止这种变化。提供分析方案以及每种辅酶带注释的特征指纹，以便使用常规使用的单个内参轻松识别和绝对定量。同时可靠地可视化对细胞功能至关重要的普遍存在的辅酶的能力，为探究健康和疾病中细胞功能的机制细节提供了新的途径。

更新日期：2016-04-14

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