Lewis-Sigler Institute for Integrative Genomics and Department of Chemistry, Princeton University , Princeton, New Jersey.
Antioxid Redox Signal. 2018 Jan 20;28(3):167-179. doi: 10.1089/ars.2017.7014. Epub 2017 Jul 19.
Accurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.
We compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in C-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD, NADH, NADP, and NADPH concentrations in cells and mouse tissues were measured with this approach.
We found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP and NADH/NAD ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.
Extraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.
准确分析二核苷酸氧化还原辅因子烟酰胺腺嘌呤二核苷酸磷酸还原型（NADPH）、烟酰胺腺嘌呤二核苷酸（NADP）、烟酰胺腺嘌呤二核苷酸还原型（NADH）和烟酰胺腺嘌呤二核苷酸（NAD）对于理解细胞氧化还原稳态非常重要。在这项研究中，我们旨在开发一种简单的方法来淬灭代谢并提取 NADPH，避免还原形式和氧化形式之间的相互转化。
我们比较了七种不同的溶剂用于淬灭和提取培养的哺乳动物细胞和小鼠组织：一种常用于酶测定的冷水性缓冲液，有或没有去污剂，热水性缓冲液，以及冷有机混合物（80%甲醇、缓冲 75%乙腈和酸性 40:40:20 乙腈:甲醇:水，其中含有 0.02 M 或 0.1 M 甲酸）。提取物通过液相色谱-质谱法（LC-MS）进行分析。为了监测代谢物的相互转化，细胞在 C-葡萄糖培养基中生长，并将未标记的标准品掺入提取溶剂中。除了含有去污剂的酶测定缓冲液、80%甲醇和 40:40:20 乙腈:甲醇:水外，氧化形式和还原形式之间的相互转化非常显著，其中含有 0.1 M 甲酸的混合物相互转化最少，回收率最好。用这种方法测量了细胞和小鼠组织中绝对 NAD、NADH、NADP 和 NADPH 浓度。
我们发现，提取过程中还原形式和氧化形式之间的相互转化是准确测量 NADPH/NADP 和 NADH/NAD 比值的主要障碍。通过同位素标记细胞和掺入 NAD(P)(H)标准品可以监测这种相互转化。
用含有 0.1 M 甲酸的 40:40:20 乙腈:甲醇:水提取可以减少相互转化，因此适用于 LC-MS 测量氧化还原辅因子比值。这种溶剂也可用于一般代谢组学。样品提取后应立即中和，以避免酸催化降解。当没有 LC-MS 可用且相应地使用酶测定时，在水性提取缓冲液中加入去污剂可减少相互转化。抗氧化剂。氧化还原信号。28, 167-179。
