Substrate-specific reduction of tetrazolium salts by isolated mitochondria, tissues, and leukocytes
- Авторлар: Fedotcheva N.1, Litvinova E.1, Zakharchenko M.1, Khunderyakova N.1, Fadeev R.1, Teplova V.1, Fedotcheva T.2, Beloborodova N.3, Kondrashova M.1
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Мекемелер:
- Institute of Theoretical and Experimental Biophysics
- Pirogov Russian National Research Medical University
- Negovskii Scientific Research Institute of General Reanimatology
- Шығарылым: Том 82, № 2 (2017)
- Беттер: 192-204
- Бөлім: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151259
- DOI: https://doi.org/10.1134/S0006297917020110
- ID: 151259
Дәйексөз келтіру
Аннотация
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.
Негізгі сөздер
Авторлар туралы
N. Fedotcheva
Institute of Theoretical and Experimental Biophysics
Хат алмасуға жауапты Автор.
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
E. Litvinova
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
M. Zakharchenko
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
N. Khunderyakova
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
R. Fadeev
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
V. Teplova
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290
T. Fedotcheva
Pirogov Russian National Research Medical University
Email: nfedotcheva@mail.ru
Ресей, Moscow, 117997
N. Beloborodova
Negovskii Scientific Research Institute of General Reanimatology
Email: nfedotcheva@mail.ru
Ресей, Moscow, 107031
M. Kondrashova
Institute of Theoretical and Experimental Biophysics
Email: nfedotcheva@mail.ru
Ресей, Pushchino, Moscow Region, 142290