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.

[Influence of Microbial Metabolites of Phenolic Nature on the Activity of Mitochondrial Enzymes].

The aim of this work was to study the effect of microbial metabolites of phenolic nature on the activity of enzymes of the tricarboxylic acid cycle in isolated mitochondria, and determine metabolites of the tricarboxylic acid cycle as potential biomarkers of mitochondrial dysfunction in the blood of patients with sepsis. It is shown that microbial metabolites of phenolic nature have an inhibitory effect on the activity of dehydrogenases, determined by the reduction of dichlorophenolindophenol and nitroblue tetrazolium in liver mitochondria and liver homogenates. This effect is more pronounced in oxidation of the NAD-dependent substrates than succinate oxidation, and at lower concentrations of microbial metabolites than inhibition of respiration. By gas chromatography-mass spectrometry it was found that the content of the tricarboxylic acid cycle metabolites in the blood of patients with sepsis decreased compared to healthy donors. The data obtained show that the microbial phenolic acids can contribute significantly to the dysfunction of mitochondria and suppression of general metabolism, characteristic of these pathologies.

[Redox-dependent ferric oxide nanoparticles loaded with doxorubicin and their influence on the functions of mitochondria].

The spectral, fluorescent and functional properties of ferric oxide and ferric hydroxide nanoparticles loaded with doxorubicin and stabilized with citric acid or lysine were studied in comparison with free doxorubicin. Their effect on the opening of calcium-induced mitochondrial pore and the possibility of the controlled release of doxorubicin under the influence of redox stimuli were investigated. The data show that the effect of nanoparticles on mitochondria depends on the type of a stabilizer. The spectral and fluorescence methods used allow us to estimate the presence or absence of free doxorubicin in solution of nanoparticles and the:concentration of bound doxorubicin. It is shown that the dithiotreitol and glutathione increase the amplitude of absorption and fluorescence of doxorubicin during incubation with nanoparticles. It is assumed that this effect may be associated with the reduction of the oxidized iron by thiols with subsequent release of doxorubicin.

[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.

Mechanism of induction of oxidative stress in liver mitochondria by low concentrations of tert-butyl hydroperoxide.

The mechanism of the effect of tert-butyl hydroperoxide (tBHP) on the kinetics of decrease in liver mitochondrial ΔΨ (transmembrane electric potential) in response to successive additions of tBHP in low concentrations has been studied. FeSO(4) was found to increase significantly the damaging effect of tBHP; this effect was shown to increase in the presence of low concentrations of Ca2+ starting from 2 µM CaCl(2). Cyclosporin A prevents these effects. The data show that the damaging effect of low concentrations of tBHP in the course of pyruvate oxidation in isolated liver mitochondria is caused by the opening of the nonspecific Ca2+-dependent cyclosporin A-sensitive pore in the inner mitochondrial membrane. Application of a method of studying oxidative stress regulators, developed in this work, is illustrated by an example of the prooxidant action of ascorbate. This method is proposed for studying mitochondria in hemochromatosis, a pathology caused by excessive accumulation of iron.

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.

Mitochondrial models of pathologies with oxidative stress. Efficiency of alkalization to reduce mitochondrial damage.

Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentrations (5-20 µM) of tert-butyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies associated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mitochondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The experiments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mitochondria before the MPTP ("mitochondrial permeability transition pore") is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.2-7.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress.

[The role of thiol antioxidants in restoring mitochondrial functions, modified by microbial metabolites].

The effects of phenolic acids of microbial origin on mitochondrial functions and the possibility of removing their effects by thiol antioxidants dithiotreitol and N-acethylcysteine were studied. The action of some phenolic acids on the redox state of NADH, the membrane potential and calcium capacity of mitochondria is due to their interaction with thiol groups. The partial restoration of mitochondrial functions occurred in the presence of dithiotreitol and N-acethylcysteine, the full recovery (short-term duration) was promoted by the combined action of dithiotreitol and menadione (vitamin K3). It was found that the protective effect of thiol antioxidants became prooxidant one, if the medium contained free iron and compounds with a quinone structure, capable of entering into a redox cycle with thiols. It is shown that the interaction of thiols with iron and menadione is accompanied by absorption of oxygen to form superoxide anion. Prooxidant effect of thiol antioxidants may explain the absence of the protective effect at the later stages of sepsis and systemic inflammatory syndrome.

[Effect of steroid hormones on production of reactive oxygen species in mitochondria].

Among the targets of the steroid hormones are mitochondria, which as the main source of reactive oxygen species (ROS) in the cell play a central role in the development of various pathologies. We studied the effect of progesterone and its synthetic analogues on mitochondrial ROS production. It was found that progesterone activates the formation of superoxide anion and hydrogen peroxide in mitochondria during oxidation of complex I substrates of the respiratory chain and exerts no influence on production of ROS during oxidation of succinate, complex II substrate of the respiratory chain. Synthetic analogues of progesterone - medroxyprogesterone acetate, buterol, acetomepregenol, megestrol acetate, have different effects on ROS production, depending on their chemical structure. By the effectiveness of impact on ROS production in mitochondria all the steroids tested can be classified in the descending order as follows: progesterone > buterol > or = atsetomepregenol > medroxyprogesterone acetate > megestrol acetate. Activation of ROS production by progesterone and buterol has different mechanisms: progesterone acts as an inhibitor of NAD-dependent respiration, while buterol and acetomepregenol form noncovalently associated complexes by hydrogen bonds between the ester carbonyl at C3 and SH-groups of the respective targets.

[Exometabolites of some anaerobic microorganisms of human microflora].

Some exometabolites produced by basic representatives of human anaerobic microflora were investigated, detected by gas chromatography-mass spectrometry (GC-MS). In vitro besides lactic acid Bifidobacterium and Lactobacillus generate substantial amounts of phenyllactic and p-hydroxyphenyllactic acids. Clostridium produced 2-hydroxybutyric acid and to a lesser extent lactic and phenyllactic acids. In contrast to C. perfringens, C. sporogenes generates substantial amount of phenylpropionic and p-hydroxyphenylpropionic acids and less p-hydroxyphenyllactic acid. C. perfringens produced minor amounts of 2-hydroxyglutaric acid. Bacteroids are potent producers of succinic and fumaric acids; they also contribute to production of significant portion of lactic acid. E. lentum generate lactic, phenyllactic and succinic acids and form a characteristic only for ones (from studied microorganisms) 2-hydroxyhexanic and 2-hydroxy-3-methylbutyric acids.

Toxic effects of microbial phenolic acids on the functions of mitochondria.

Low-molecular-weight phenolic acids (PhAs) phenylacetate, phenyllactate, phenylpropionate, p-hydroxyphenyllactate, and p-hydroxyphenylacetate are essentially the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora. In sepsis, the concentrations of some of these acids in the blood increase tens of times. Assuming that these compounds can cause the mitochondrial dysfunction in sepsis, we examined their effects on respiration, the induction of pore opening, and the production of reactive oxygen species (ROS) in mitochondria. It was found that phenylpropionate and phenylacetate produce a more toxic effect on mitochondria than the other phenolic acids. At concentrations 0.01-0.1 mM they decreased the rate of oxidation of NAD-dependent substrates and activated the Ca2+- and menadione-induced opening of the cyclosporin A-sensitive pore and the production of ROS. The disturbances caused by these PhAs are similar to those observed in mitochondria in sepsis, and hence the rise in their level may be one of the causes of mitochondrial dysfunctions. Phenyllactate, p-hydroxyphenyllactate, and p-hydroxyphenylacetate inhibited the production of ROS and pore opening, acting as antioxidants. Thus, the ability of PhAs to affect the mitochondrial functions, as well as an increase in their concentrations in sepsis (the total concentration of these PhAs in the blood is close to 0.1 mM), suggests that PhAs can be directly involved in the development of mitochondrial failure.

Increase in the contribution of transamination to the respiration of mitochondria during arousal.

The involvement of transamination in the respiration of liver mitochondria in ground squirrels during hibernation and arousal has been studied. It was shown by HPLC that, in the presence of glutamate and malate, the formation of alpha-ketoglutarate (KGL), a transamination marker, and fumarate, a product of succinate oxidation, takes place. During arousal, the formation of KGL increased fourfold, and the respiration sensitive to the inhibitor of aspartate transaminase aminooxyacetate (AOA) increased threefold. The function of transamination upon arousal is related to the elimination of oxaloacetate, an endogenous inhibitor of succinate dehydrogenase. In addition, being more resistant to oxidative stress than oxidation, transamination is probably involved in the antioxidant defense required during the rapid rise of body temperature upon arousal. Our experiments showed an increase in the concentration of malonic dialdehyde (MDA), an end product of lipid peroxidation, in liver mitochondria in this state, which can reduce the activity of the enzymes of the tricarboxylic acids cycle. Under these conditions, the transamination contributes to the maintenance of a high respiration rate necessary for arousal.

Preservation of native properties of mitochondria in rat liver homogenate.

A protocol is developed for preparation of concentrated rat liver homogenate preserving assemblies of mitochondria in isotonic KCl under 0 and 15 degrees C. Assemblies preserve ability for self-organization during storage in homogenate. All key energy functions of mitochondria can be investigated in such a homogenate. Oxidative phosphorylation and membrane potential are stable for 5-7 h and can be still observed on the next day. Substrate-level phosphorylation is better pronounced for mitochondria in KCl than in sucrose medium while Ca2+ capacity is greater and lipid peroxidation is much lower. Sucrose addition impairs these functions. The rate of phosphorylating respiration is lower in large assemblies and higher in small. Transition from large to small assemblies corresponds to the transition from quiescent state of animal to adrenaline induced active state. The proposed method is particularly convenient for clinical investigations with small bioptates.

[Monoamine oxidase activity in rat liver in vitamin B12 deficiency]. / Monoaminooksidaznaia aktivnost' v pecheni krys pri nedostatochnosti vitamina B12.

Changes in the activity of monoamine oxidase (MAO-B) in liver homogenates and mitochondria from normal and vitamin B12-deficient rats have been studied in various functional states of the mitochondria. Preincubation of liver preparation at 32 degrees C increased the MAO affinity for benzylamine in samples of normal (but not vitamin B12-deficient rats). Succinate added to the incubation medium decreased Km and increased Vmax in both normal and vitamin B12-deficient animals. A correlation was found between the decline of MAO-B activity under vitamin B12 deficiency, deceleration of succinate oxidation and reduction of the transmembrane potential.

[Effect of GDP and carboxyatractylate on potassium ion transport in brown fat mitochondria]. / Vliianie GDP i karboksiatraktilata na transport ionov kaliia v mitokhondriiakh burogo zhira.

It has been shown that the increase of the membrane potential in uncoupled brown fat mitochondria after addition of 1-2 mM GDP to a medium containing potassium acetate is accompanied by swelling of mitochondria which occurs in two phases. The first swelling phase is due to the electrogenic transport of K+ ions, while the second one is activated by carboxyatractylate and carnitine. The swelling is practically completely inhibited by a further rise of the GDP concentration irrespective of high values of the membrane potential. Unlike K+ transport, the potential-dependent transport of Na+ ions occurs more rapidly, is monophasic and insensitive to carboxyatractylate and carnitine. It is suggested that regulation of K+ ion transport in brown fat mitochondria is mediated by the action of nucleotides and fatty acid esters on the uncoupling protein and ADP/ATP antiporter.

[Metabolites of the propionate pathway as regulators of fatty and dicarboxylic acid oxidation in liver mitochondria]. / Metabolity propionatnogo puti kak reguliatory okisleniia zhirnykh i dikarbonovykh kislot v mitokhondriiakh pecheni.

The functioning of the propionate pathway of oxidation substrate metabolism in the norm and under vitamin B12 deficiency has been studied. This pathway has been shown to play an important role in oxidative processes occurring in normal organisms, for its inhibition in B12-deficient animals is associated with a reduction of respiration as well as with noticeable decreases in palmitoylcarnitine and succinate oxidation rates and oxidation phosphorylation coupling. Succinate, the end product of the propionate pathway, normalizes the respiration and restores the rate of palmitoylcarnitine oxidation in B12-deficient animals, which is suggestive of its crucial role in the propionate pathway. In vivo propionate inhibits, whereas methyl malonate stimulates palmitoylcarnitine respiration, however only in intact animals. In B12-deficient animals the sensitivity to these metabolites is decreased.

[Effect of propionate pathway metabolites on the oxidative activity of liver mitochondria under normal conditions and in vitamin B12 deficiency]. / Vliianie metabolitov propionatnogo puti na okislitel'nuiu aktivnost' mitokhondrii pecheni v norme i pri nedostatochnosti vitamina B12.

We performed a comparative investigation in vivo and in vitro of the propionate and methylmalonate effect on oxidative activity of liver mitochondria in control and vitamin B12-deficient rats and found that efficiency of the effects were less pronounced in vitamin B12-deficient rats. It is also shown that the rates of respiration and phosphorylation decreased in liver mitochondria of vitamin B12-deficient rats.

[The effect of metabolites of the propionate pathway on the oxidative activity of liver mitochondria]. / Vliianie metabolitov propionatnogo puti na okislitel'nuiu aktivnost' mitokhondrii pecheni.

Methylmalonate and propionate, the major metabolites of the propionate pathway of fatty and amino acid metabolism used at 1-4 mM cause selective inhibition of succinate and palmitoyl carnitine oxidation in liver mitochondria. Methylmalonate is more specific towards succinate, whereas propionate--towards palmitoyl carnitine oxidation. Methylmalonate is transported to mitochondria at a high rate with no effect on succinate transport. Being injected intramusculary methylmalonate has no inhibiting effect on the oxidative activity of mitochondria but is able to activate succinate and palmitoyl carnitine oxidation. The inhibiting effect of propionate on palmitoyl carnitine oxidation is a long-term one. Injections of these metabolites precursors, isoleucine, methionine and valine, produce an activating effect on succinate oxidation. Thus, propionate pathway metabolites may participate in the regulation of lipid-carbohydrate metabolism.

[Effect of antibodies against mitochondrial K+-transporting protein on K+ transport in rat liver mitochondria]. / Vliianie antitel k mitokhondrial'nomu K+-transportiruiushchemu belku na transport K+ v mitokhondriiakh pecheni krysy.

The K+ transport in rat liver mitochondria was studied by the immunochemical method. Antibodies to mitochondrial K+-transporting protein with molecular weight 60 kDa were obtained and used as possible inhibitor or K+ transport. Antibodies depressed the DNP-induced K+ efflux and energy-dependent swelling by 50% without causing changes in respiration and oxidative phosphorylation.