Fedotcheva N I, Litvinova E G, Zakharchenko M V, Khunderyakova N V, Fadeev R S, Teplova V V, Fedotcheva T A, Beloborodova N V, Kondrashova M N
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochemistry (Mosc). 2017 Feb;82(2):192-204. doi: 10.1134/S0006297917020110.
Tetrazolium salts are commonly used in cytochemical and biochemical studies as indicators of metabolic activity of cells. Formazans, formed by reduction of tetrazolium salts, behave as pseudo-solutions during initial incubation, which allows monitoring their optical density throughout incubation. The criteria and conditions for measuring oxidative activity of mitochondria and dehydrogenase activity in reduction of nitroblue tetrazolium (NBT) and methyl thiazolyl tetrazolium (MTT) in suspensions of isolated mitochondria, tissue homogenates, and leukocytes were investigated in this work. We found that the reduction of these two acceptors depended on the oxidized substrate - NBT was reduced more readily during succinate oxidation, while MTT - during oxidation of NAD-dependent substrates. Reduction of both acceptors was more sensitive to dehydrogenase inhibitors that to respiratory chain inhibitors. The reduction of NBT in isolated mitochondria, in leukocytes in the presence of digitonin, and in liver and kidney homogenates was completely blocked by succinate dehydrogenase inhibitors - malonate and TTFA. Based on these criteria, activation of succinate oxidation was revealed from the increase in malonate-sensitive fraction of the reduced NBT under physiological stress. The effect of progesterone and its synthetic analogs on oxidation of NAD-dependent substrates by mitochondria was investigated using MTT. Both acceptors are also reduced by superoxide anion; the impact of this reaction is negligible or completely absent under physiological conditions, but can become detectable on generation of superoxide induced by inhibitors of individual enzyme complexes or in the case of mitochondrial dysfunction. The results indicate that the recording of optical density of reduced NBT and MTT is a highly sensitive method for evaluation of metabolic activity of mitochondria applicable for different incubation conditions, it offers certain advantages in comparison with other methods (simultaneous incubation of a large set of probes in spectral cuvettes or plates); moreover, it allows determination of activity of separate redox-dependent enzymes when selective inhibitors are available.
四氮唑盐在细胞化学和生物化学研究中常用作细胞代谢活性的指标。由四氮唑盐还原形成的甲臜在初始孵育期间表现为假溶液，这使得在整个孵育过程中能够监测其光密度。本研究探讨了在分离的线粒体、组织匀浆和白细胞悬浮液中，测量线粒体氧化活性以及硝基蓝四氮唑（NBT）和噻唑蓝（MTT）还原中脱氢酶活性的标准和条件。我们发现这两种受体的还原取决于氧化底物——在琥珀酸氧化过程中NBT更容易被还原，而MTT在NAD依赖底物氧化过程中更容易被还原。两种受体的还原对脱氢酶抑制剂比对呼吸链抑制剂更敏感。琥珀酸脱氢酶抑制剂——丙二酸和TTFA完全阻断了分离线粒体、洋地黄皂苷存在下白细胞以及肝脏和肾脏匀浆中NBT的还原。基于这些标准，在生理应激下，通过还原NBT的丙二酸敏感部分的增加揭示了琥珀酸氧化的激活。使用MTT研究了孕酮及其合成类似物对线粒体NAD依赖底物氧化的影响。两种受体也可被超氧阴离子还原；在生理条件下，该反应的影响可忽略不计或完全不存在，但在单个酶复合物抑制剂诱导超氧产生或线粒体功能障碍的情况下可被检测到。结果表明，记录还原NBT和MTT的光密度是评估线粒体代谢活性的一种高度灵敏的方法，适用于不同的孵育条件，与其他方法相比具有一定优势（在光谱比色皿或平板中同时孵育大量探针）；此外，当有选择性抑制剂时，它可以测定单独的氧化还原依赖性酶的活性。
