Substrate-Specific Reduction of Tetrazolium Salts by Isolated Mitochondria, Tissues, and Leukocytes.

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.

[Effects of Light Near-Infrared Radiation on Rats Assessed by Succinate Dehydrogenase Activity in Lymphocytes on Blood Smears].

Biological effects of light near infrared radiation (850 nm), with modulation acoustic frequency of 101 Hz, was studied. The study was conducted on rats, the effect was recorded by succinate dehydrogenase activity in lymphocytes on the blood smear after administration of the activating dose of adrenaline, which simulates the state of the organism in the early stages of the pathogenic effects (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals activated by adrenaline was shown. Infrared radiation has a normalizing effect reducing the degree of inhibition or activation of the enzyme induced by adrenaline and had no effect on the control animals. Thus, by modulating the activity of succinate dehydrogenase infrared radiation regulates energy production in the mitochondria supported by the most powerful oxidation substrate--succinic acid, which is especially pronounced under stress.

[Cytoplasmic superoxide dismutase activity is a sensitive indicator of the antioxidant status of the rat liver and brain].

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.

[Changes in lymphocytes under influence of oxidative substrates].

Blood lymphocytes of children with oncology diseases were studied in smears compared with healthy ones examined from the closely selected group. The following two methods were used: the traditional in hematology method of smear staining for detection of blood formula and lymphocyte indices and the staining with novel cytobiochemical method including 60-min incubation in biochemical medium for estimation of the activity of succinate dehydrogenase, lactate dehydrogenase and their ratio, that can serve as a measure of respiration and glycolysis--Warburg effect, typical of tumor. The great increase in lymphocyte size by addition of lactate was found in blood smears of patients with tumors. This effect was not pronounced by addition of succinate and in cells of healthy persons. Changes in the structure of the cells in patients were also related to the changes in the activity of enzymes studied. These changes can be considered as transition of metabolism and the structure of cells to the state in tumor. Cytobiochemical method allows for higher diagnostic sensitivity of patient examination and efficiency of medical care of patients with oncological diseases.

[State of succinate dehydrogenase in the organism--"unbalanced" or hyperactive].

Through the use of the original cytobiochemical method to study oxidation in mitochondria, preserving their native organization in network within cells in a blood smear, we revealed hyperactive state of succinate dehydrogenase, which is realized in the organism under physiological stress. It is consistent with the view of the non-equilibrium state of enzymes during activity. The mechanism of the succinate dehydrogenase hyperactivity moderation is based on the full functioning of alpha-ketoglutarate dehydrogenase, supported by oxidation of isocitric acid.

[Importance of preservation of biophysical organization of isolated mitochondria for revealing physiological regulation of their functions].

A method has been elaborated that preserves the mitochondrial-reticular network in lymphocytes in composition to the physiological one. Physiologicalby the immobilization of a blood smear on glass and its subsequent incubation in a medium closeresponses of respiration to excitation in the ition of early responses of ions. The recogn organism are well pronounced on these preparat mitochondria to pathogenic agents in the organism is a timely problem of basic and medicinal e- investigations since they play a leading role in the development of pathological states.

Hyperactivation of succinate dehydrogenase in lymphocytes of newborn rats.

We measured the activity of mitochondrial succinate dehydrogenase (SDH) within cells, in media with near-physiological composition, in lymphocytes immobilized in a blood smear on glass. SDH activity was studied in newborn rats characterized by natural hyperadrenergic status and also in adult animals injected with epinephrine. In most newborns very high activities were recorded, which exceeded the activities in adults at rest 7-8-fold or 3-fold according to the conventional calculation, or more than 30- and 6-fold according to our more precise calculation. The findings support our concept about a selective interaction between adrenergic stimulation and oxidation of succinic acid. According to this concept, epinephrine and norepinephrine specifically activate oxidation of succinic acid, whereas blood micromolar concentrations of the latter stimulate the release of catecholamines (the receptor-mediated signaling effect). This interaction is half of a substrate-hormonal regulatory system responsible for connection of vegetative nervous system with oxidation in mitochondria of the innervated organs. The increase in succinate oxidation by catecholamines includes activation of the faster pathways of succinate generation than the complete Krebs cycle, in particular, the glyoxylate cycle that is shown in the newborn rats in the present study.

Effect of carnosine on self-organization of mitochondrial assemblies in rat liver homogenate.

The effect of carnosine on self-organization of mitochondrial assemblies was studied in rat liver homogenate of quiescent and excited animals. It was shown in separate electron microscopy experiments with serial slices that under our conditions of preparation of homogenate, blocks of native mitochondrial-reticular network in the cell, assemblies of mitochondria, are kept. Carnosine was shown to prevent dissociation of assemblies during storage. Its effect is maximal for more dissociated assemblies from excited animals with decreased ability for self-organization. Prevention of disassembly of organelles by carnosine can serve as one of the mechanisms of carnosine-induced diminishing of muscle fatigue under prolonged work.

Role of the impairment of oxidative metabolism in pathogenesis of lower urinary tract symptoms with benign prostatic hyperplasia and their treatment by alpha1-adrenoblocker alfuzosin.

The role of impairment of general oxidative and energy metabolism in pathogenesis of lower urinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia (BPH) and their correction by (1-adrenoblocker alfuzosin was studied. One group of patients (N = 126) was examined by standard methods for determination of the severity of LUTS by IPSS and mean effective volume of urinary bladder (MEVUB). In the second group (N = 29) in addition to functional examinations, metabolic indicators in blood were measured: antioxidant activity (AOA) and succinate dehydrogenase activity (SDA). Severity of LUTS depends greatly on the MEVUB. It was the first to show a practically complete correlation between LUTS, AOA and SDA. Severity of LUTS exactly correlates with indicators of oxidative and energy metabolism. In patients with more heavy LUTS, lowest AOA and SDA values were found. In the course of effective treatment, both phenomena developed an improvement of clinical symptoms and a rise of biochemical parameters. Close correlation between functional and metabolic phenomena is evidence of an essential role of metabolic mechanisms in the pathogenesis of LUTS with BPH. This opens perspectives to use antioxidants and energy metabolism activators for correction of UB dysfunction in patients with BPH.