Актуальная проблема современной передовой персонализированной медицины – определение состояния митохондрий (МХ) у человека. Принятые биохимические методы повреждают и весь организм (требуется биопсия), и, что не менее важно, сами выделенные МХ. Исследования в лаборатории биоэнергетики ИТЭБ РАН под руководством д.б.н., проф. М.Н. Кондрашовой позволили разработать метод оценки тяжести заболевания по измерению энергетических процессов в митохондриях лимфоцитов в капле крови. Оригинальный метод, названный цитобиохимическим (ЦБХ), позволяет оценить активность митохондрий и гликолиза в лимфоцитах крови пациента, создан специально, чтобы сохранить в выделенных препаратах нативную организацию митохондрий в сеть, что позволяет наблюдать в них регуляцию, имевшую место в организме. Работами М.Н. Кондрашовой было показано, что активность митохондрий связана с деятельностью общих физиологических систем регуляции, в частности симпатической и парасимпатической. Между симпатической и парасимпатической регуляцией в организме существуют взаимно противоположные, реципрокные отношения подавления активности противоположной системы при активации ее партнера. Подобная реципрокная регуляция существует и на митохондриальном уровне: в пределах физиологической нормы симпатическая активация усиливает энергетическое обеспечение физиологических функций за счет усиления распада жиров и углеводов. Однако симпатическая гиперактивация приводит к дисбалансу между процессами катаболизма и восстановительными биосинтетическими процессами, и это формирует дополнительный порочный метаболический круг развития патологии на уровне клеточного дыхания. Особенно выражены такие нарушения при хронических респираторных заболеваниях, что и послужило целью настоящего исследования: изучить показатели активности митохондриального фермента – сукцинатдегидрогеназы (СДГ) и фермента гликолиза – лактатдегидрогеназы (ЛДГ) у пациентов с атопической бронхиальной астмой (АБА). Это позволит получать новую информацию для исследователя и врача, на ранних стадиях развития патологического процесса распознать его и внести необходимые коррективы в тактику лечения и реабилитации пациентов с респираторной патологией. Рекомендации предназначены для специалистов клинико-биохимических лабораторий ЛПУ, терапевтов, пульмонологов, иммунологов-аллергологов.

Abstract—The effects of acetylating and deacetylating compounds on the activity of succinate dehydrogenase, as well as on the membrane potential and calcium retention capacity of the isolated liver mitochondria, supported by the oxidation of succinate, has been investigated. The chemical reagent N-acetylimidazole, the microbial metabolite phenylacetate, along with the drugs acetylsalicylic acid and N-acetylcysteine, were used as acetylating compounds. These compounds reduced succinate dehydrogenase activity to different extents depending on the concentration and incubation conditions. An inhibitory analysis using intermediate electron carriers has shown that the ubiquinone-binding site of the enzyme undergoes acetylation. The inhibition was partially eliminated or prevented by pre-incubation of the mitochondria with nicotinamide adenine dinucleotide, a co-factor for deacetylation, and with polyamine spermidine, an acceptor of acetyl groups.

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.

The cytobiochemical assay is described in the paper which is elaborated by three of its authors. This method possesses high sensitivity in detection of the state of mitochondria in the organism by measurement of enzyme activity inside blood lymphocytes in smears. Lactate dehydrogenase, succinate dehydrogenase and their ratio LDH/SDH were investigated. The authors proposed to use this ratio as a quantitative indicator of so called Warburg effect, typical of cancer cells. Whereas LDH/SDH is 5.5-8.8 in healthy children, aerobic glycolysis and the indicator rise up to 16.5-55.2 in young patients with leucosis. In children suffering myopathy aerobic glycolysis falls down and the ratio is lower than in norm: 4.5 and less than one. The authors suggested the decrease of aerobic glycolysis under myopathy to be a biomarker of restorative biosynthesis and cell growth weakening inherent in this disease.

The effect of a GSM 900/1800 mobile phone, which is a widespread source of electromagnetic radiation of the microwave frequency in the environment, on rabbits was studied at power densities of 5–7 μW/cm2. The biological effect was recorded by a sensitive method for the detection of the physiological regulation of enzyme activity inside lymphocytes in blood smears. Succinate dehydrogenase, which is the most powerful energy-supply enzyme in mitochondria, and lactate dehydrogenase, which is an enzyme of glycolysis, were measured. The lactate dehydrogenase to succinate dehydrogenase activity ratio was also calculated as an analog of the Warburg effect, which demonstrates the relationship between glycolysis and respiration. After 60 min of mobile-phone exposure each day for 11 days at a moderate dose, the emitted radiation induced a threefold increase in succinate dehydrogenase activity and a twofold decrease in lactate dehydrogenase activity. As a result, the lactate dehydrogenase/succinate dehydrogenase activity ratio falls from 15 to 5, thus indicating that respiration is predominant over glycolysis. The changes develop already after the first exposure and reach a maximum in 4 days. The predominance of respiration is usually considered as a beneficial state of an organism. However, continuous activation of respiration by mobile phone exposure may cause damage to the normal restorative processes that are supported by glycolysis during periods of rest.

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).

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).

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.

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.

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.

Исследованы лимфоциты в мазках крови у детей с онкологическими заболеваниями в сравнении со здоровыми детьми подобранной близкой группы. Использованы принятый в гематологии способ окрашивания мазка для определения формулы крови и лимфоцитарных индексов и окрашивание по новый методике с часовой инкубацией клеток в биохимической среде для определении активности сукцинатдегидрогеназы, лактатдегидрогеназы и их отношения, характеризующего взаимодействие дыхания и гликолиза. При проведении инкубации в биохимических условиях обнаружено сильное увеличение размеров лимфоцитов при добавлении молочной кислоты в мазках крови больных с опухолями. Такой эффект не наблюдался при добавлении янтарной кислоты, а также в мазках крови здоровых детей из группы сравнения. Изменения структуры клеток у больных были связаны и с изменениями активности исследуемых ферментов. Предлагаемое цитобиохимическое исследование позволяет повысить диагностическую чувствительность при обследовании состояния больных и эффективность лечения при онкологических заболеваниях.

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.

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.

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.

The comparison of succinate dehydrogenase activity (SDH) was carried out in the presence of succinate (SUC) in commonly used millimolar ("substrate") concentrations that significantly exceeds the SUC content in tissues, and in micromolar concentrations really present in the organism, currently called "signaling" concentrations. This name reflects the function of SUC, a substrate of oxidation, as a signaling molecule regulating physiological processes. Regulatory signaling action of SUC occurs in significantly lower concentrations, at least 1000 times lower than "substrate" concentration. Signaling action is related to adrenergic regulation including SDH as its target. It is realized via specific SUC receptor, GPR91. Here the SDH activity was studied under different physiological conditions in rat and human using new cytobiochemical (CBCH) method, which sensitively reveals changes in the organism due to the preservation of the native state of mitochondrial network. SDH activity was measured in lymphocytes in blood smear by nitro blue tetrazolium (NBT) reduction due to oxidation of added and endogenous substrates. The high statistical validity is an additional advantage of the CBCH method, because every separate value is measured by videomicroscopic scanning of multitude of stained objects (mitochondria), 200-700 in one sample containing images of 30 lymphocytes of an individual. High sensitivity of CBCH method allowed detection of the SDH activity with SUC being in both millimolar and micromolar concentrations. Activation of SDH was revealed upon increase in adrenergic regulation in the norm (by adrenaline administration) and in acute phase of hypertension, followed by inhibition under arterial pressure stabilization by hypotensive preparations, which are adrenaline receptor blockers and internal regulators at chronic stages. The use of micromolar SUC concentrations reveals changes in SDH activity in the organism related to the differences in the physiological states more efficiently than the use of millimolar concentrations, which activate SDH and smooth over the development of inhibition under pathology progressing. While SDH activity measured at millimolar SUC concentrations was depressed, a significant rise in NBT reduction was observed in the presence of micromolar concentrations. This suggests that the effect of signal (but not substrate) concentrations can be realized through generation of superoxide and hydrogen peroxide. which possess vitally beneficial regulatory effects in micromolar concentrations in mitochondria.

Проведено сравнительное исследование активности сукцинатдегидрогеназы при добавлении янтарной кислоты в обычно используемых, сильно завышенных по сравнению с содержанием в тканях, миллимолярных, “субстратных” концентрациях (5 мМ), и реально существующих в организме микромолярных концентрациях (5–50 мкM), называемых в настоящее время “сигнальными”. Это название отражает вторую функцию субстрата окисления янтарной кислоты как сигнального вещества – регулятора физиологических процессов. Регуляторное, сигнальное действие янтарной кислоты проявляется в значительно более низких концентрациях, чем субстратное (не менее чем в 1000 раз). Сигнальное действие янтарной кислоты связано с адренергической регуляцией, включающей сукцинатдегидрогеназу как ее мишень. Оно реализуется через специфический рецептор янтарной кислоты GPR91. В данной работе определяли активность сукцинатдегидрогеназы крыс и человека новым цитобиохимическим методом, который выявляет изменения в организме благодаря сохранению нативного состояния митохондриальной сети. Активность сукцинатдегидрогеназы может быть измерена в лимфоцитах на мазке крови по восстановлению нитросинего тетразолия за счет окисления добавленных и эндогенных субстратов. Дополнительным достоинством метода является высокая статистическая значимость, так как каждая отдельная величина измеряется видеомикроскопическим сканированием множества (200–700) окрашенных объектов (митохондрий) в одном образце, содержащем изображения 30 лимфоцитов индивидуума. Высокая чувствительность цитобиохимического метода позволила измерить активность сукцинатдегидрогеназы в присутствии янтарной кислоты не только в миллимолярных, но и в микромолярных концентрациях. Выявлена активация сукцинатдегидрогеназы при усилении адренергической регуляции в норме (введением адреналина) и в острой фазе гипертонии с переходом к ингибированию при стабилизации артериального давления гипотензивными препаратами – блокаторами адренорецепторов и внутренними регуляторами при хроническом течении. Использование янтарной кислоты в микромолярных концентрациях позволяет с большей чувствительностью выявить изменения активности сукцинатдегидрогеназы, соответствующие различиям в физиологическом состоянии организма, чем при использовании субстратных концентраций, которые активируют фермент и маскируют развитие ингибирования при углублении патологии. При сниженной активности сукцинатдегидрогеназы, измеренной в присутствии янтарной кислоты в миллимолярной концентрации, наблюдается значительное повышение восстановления нитросинего тетразолия в присутствии микромолярных концентраций янтарной кислоты. Это явление указывает на то, что действие сигнальных концентраций, в отличие от субстратных, может реализоваться через образование в митохондриях супероксида и перекиси водорода, которые в микромолярных концентрациях обладают жизненно необходимыми благоприятными регуляторными эффектами.

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.