M. V. Zakharchenko

Preservation of the in vivo state of mitochondrial network for ex vivo physiological study of mitochondria

Over the course of many years our laboratory has been engaged in the study of physiological functions of mitochondria ex vivo. We showed that the unavoidable destruction of mitochondrial-reticular network during traditional isolation of the mitochondria diminishes the observable ex vivo changes of mitochondrial processes in vivo. Comparing preparations obtained from quiescent and stressed rats, we found that the great difference in size of assemblies of mitochondria preserved in homogenate disappears when it is diluted for the measurement of respiration. This also leads to a decrease in the difference between respiration of mitochondria from quiescent and stressed animals. We developed a new method that provides ex vivo stable preservation of the in vivo network using a cytochemical procedure on glass-adhered lymphocytes in blood smear. We radically changed the incubation medium for the measurement of dehydrogenase activity that excludes an artefact of succinate dehydrogenase hyperactivation ex vivo by non-physiological components of the traditionally used solution. Our method made it possible to observe ex vivo two- to eightfold increase in succinate dehydrogenase activity by adrenaline in vivo, while the activity of alpha-ketoglutarate dehydrogenase changed reciprocally. The data obtained show that the structure changes of the network play an important role in physiological regulation of mitochondrial functions. Thus, it may be possible to correct mitochondrial dysfunctions in the organism by substances supporting the stability of mitochondrial network. The developed method is non-invasive, informative and, therefore, is convenient for clinical investigations, particularly of mitochondrial diseases.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

States of succinate dehydrogenase in the organism: Dormant vs. hyperactive (pushed out of equilibrium)

Using an original cytobiochemical method to study oxidation in mitochondria, preserving their native network organization within cells in a blood smear, we have revealed a hyperactive state of succinate dehydrogenase that arises in the organism under physiological stress. This is generally consistent with the notion of non-equilibrium state of enzymes during their activity. The mechanism moderating the succinate dehydrogenase hyperactivity is based on full-fledged functioning of α-ketoglutarate dehydrogenase, sup-ported by oxidation of isocitrate.

Importance of preserving the biophysical organization of isolated mitochondria for revealing their physiological regulation

A method has been elaborated that preserves the mitochondrial-reticular network in lymphocytes by immobilization of a blood smear on glass and its subsequent incubation in a medium close in composition to the physiological one. Physiological responses of mitochondrial respiration to excitation in the organism are well pronounced on these preparations. Detection of early responses of mitochondria to pathogenic agents in the organism is a topical problem of basic and medical research, since such alterations 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.

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].

The biological effects of near infrared radiation (850 nm) modulated by an acoustic frequency of 101 Hz were studied. The study was conducted on rats; the effect was registered by succinate dehydrogenase activity in lymphocytes in blood smears after the administration of an activating dose of adrenaline, which simulates the state of the organism at early stages of a pathogenic action (stress). A pronounced regulating effect of infrared radiation on the activity of succinate dehydrogenase in animals that were activated by adrenaline was shown. Infrared radiation has a normalizing effect via the reduction of the degree of inhibition or activation of the enzyme induced by adrenaline and has no effect on the control animals. Thus, by modulation of the activity of succinate dehydrogenase, infrared radiation regulates energy production in mitochondria that is provided by the most potent oxidation substrate, viz., succinic acid; the effect is especially pronounced under stress.

Changes in lymphocytes induced by oxidation substrates

Lymphocytes in blood smears of children diagnosed with cancer have been investigated and compared to those of healthy children (matching controls). The conventional hematological procedure for smear staining was used to assess the blood formula and lymphocyte indices, and a new staining method involving incubation of cells in a biochemical medium for 1 h was used to assess the activity of succinate dehydrogenase, lactate dehydrogenase, and the ratio of the activities of these enzymes characterizing the interaction of respiration and glycolysis. A dramatic increase of the size of lymphocytes obtained from cancer patients was detected when lactic acid was added to the incubation medium for the biochemical reaction. This effect was neither observed when succinic acid was added nor when blood smears from healthy children of the control group were tested. Structural changes in the patients’ cells were associated with changes in the activity of the enzymes under investigation. The cytobiochemical test that was developed allows increasing diagnostic sensitivity upon assessment of the patients’ state and can contribute to more efficient treatment of malignant tumors.

Cytobiochemical biomarkers of the state of mitochondria in humans. II. Small but clear differences between elderly people with bronchial asthma and these with no clinical symptoms or middle-aged examinees

Quantitative indicators of mitochondria functions and dysfunctions were estimated by the activity of dehydrogenases (DH): succinate DH (SDH), lactate DH (LDH), LDH/SDH as glycolysis-respiration ratio or Warburg effect (WEF) measure proposed by our group. The measurements were carried out by our advanced cyto-BIO-chemical method in mitochondria within lymphocytes in a smear of blood [1, 2]. In this study we have added α-ketoglutarate (KGL) DH (KDH) measurement by blue staining of nitroblue tetrazolium and estimation of KGL influence on red staining of nucleus (KNU) by neutral red (NR). Part I of the work showed that given the great differences between the states of examinees DH activity served as sensitive biomarker [3]. WEF reveals these changes with even greater amplitude. We have shown that DH do not distinctly reveal smaller differences between elderly people with chronic bronchial asthma and control group of the same age or with healthy middle-aged examinees. However, WEF detects the smaller distinctions clearly (Fig1). Strict distribution of examinees by this indicator completely corresponds to the physician’s opinion on the clinical data. But even WEF cannot reveal very mild differences between elderly and middle-aged people within the range of norm. However, KDH and KNU detect this differences (Fig2).

Cytobiochemical biomarkers of the state of mitochondria in humans. I. Great differences in children suffering from leukemia or myopathy as compared with healthy children of similar age.

Quantitative indicators of mitochondria functions and dysfunctions and physiological state of the organism were estimated by the activity of dehydrogenases (DH): succinate DH (SDH), SDH + isocitrate (ISC), lactate DH ( LDH), and LDH/SDH as glycolysis-respiration ratio or Warburg еffect measure proposed by our group. Our advanced cyto-BIO-chemical method was used to detect the state of mitochondria within lymphocytes in a smear of blood [1, 2]. The typical examples for the children examined are shown in the figure below. The pronounced rise of LDH activity and decrease in SDH are observed under leucosis. The dysfunctions are even more manifested in the LDH/SDH ratio. There is little difference between DH activity under myopathy and this in healthy children. However, the LDH/SDH ratio clearly reveals the decrease. The increase in Warburg effect is a beneficial property of cancer cells, which is essential for intensive biosynthesis and proliferation [3]. In contrast, the weakening of the restorative processes is typical for myopathy. Biomarkers of glycolysis ( LDH), respiration (SDH) and Warburg effect (LDH/SDH) in young patients suffering from leucosis (L) n=22 and myopathy (M) n=9, compared with healthy(H) n=25 children of similar age, measured by nitroblue reduction (NBR).