**2. A brief description of the cytobiochemical method. Succinate dehydrogenase and α-ketoglutarate dehydrogenase as markers of adrenaline or acetylcholine regulation in the organism**

The CBCh method for the study of mitochondrial dehydrogenases was developed to avoid the loss of native mitochondrial network structure in *ex vivo* experiments. The CBCh method was based on the combination of cytochemical techniques of blood smear fixation on the glass with the use of modern biochemical media for incubation of mitochondria. The composition of the used medium was modified to resemble more closely the intracellular medium to better preserve ex vivo the quiescent state of mitochondria inside lymphocytes. We measure dehydrogenase activity by reduction of nitroblue tetrazolium (NBT) to blue formazan. The image of lymphocytes after succinate (SUC) oxidation is shown in Figure 1. Blue mitochondria are located by periphery. The bulk of the cell is occupied by large nuclei stained with neutral red after SDH measurement for identification of lymphocytes in the smear.

**Figure 1.** Images of human glass-adhered lymphocytes after SUC oxidation. Images were collected using an oil immersion lens (100x/1.25) under a light microscope (Leica, DM 2000) equipped with a camera (Leica, DFC-425) connected to a computer. SDH activity is registered by blue staining with formazan in mitochondria. Red staining is used for nuclei visualization.

In the search for the best conditions for network preservation, we have shown that common substances and conditions obligatory for the investigation of oxidative phosphorylation, such as inorganic phosphate, ADP, Ca2+, pH 7.4, evidently sucrose, as well as dilution of tissue are not only biochemical activators but they also dissipate assemblies of mitochondria [9-11]. Because the test of NBT reduction is more sensitive to electrons than oxygen consumption, the CBCh method first allowed us to measure low-rate respiration without artificial high concentrations of stimulators. This characteristic corresponds to a real Quiescent State of mitochondria in the cell. It is of great importance because additions distort the physiological regulation. For instance, ADP results in a shift of the ADP/GDP ratio in mitochondria and inhibits SDH. At the dawn of the polarography era, some leading mitochondriologists believed this extreme stimulation to be artificial, and the measurement of respiration without any external additions to be more physiological and desirable. However, the advantages of rapid-recording polarography were so attractive, compared with the previous monumental and slow manometric epoch that the polarographic method spread quickly over the world. Considering the aforesaid, the possibility to measure the real Quiescent State respiration is one more crucial advantage of the CBCh method. Therefore, besides the preservation of network by fixation, the quiescent state is protected by exclusion of the artificial addition of exogenous activators. Due to the combination of cytological and biochemical advantages, the novel method was called Cytobiochemical.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 239

**Figure 2.** Substrate - hormonal system. Participation of mitochondria in physiological regulation. Reciprocal adrenergic - cholinergic regulation as a whole with oxidation of succinate - α-ketoglutarate. Link between sympathetic and parasympathetic nervous system and mitochondria, or link between non

Therefore, the measurement of the activity of the chosen DHs, SDH and KDH, provides also information on the level of reciprocal ADR/ACH regulation in the organism. Substrates of the two DHs, SUC and KGL, were shown to influence the ADR and ACH level as well as support physiological functions under load and in recovery. It is of importance that the regulatory effects of SUC and KGL in the organism are well pronounced in concentrations far below than necessary for substrate supply. To differentiate these two activities of substrates, the low concentrations manifesting general regulatory action in the organism were called "signal" suggesting that it is realized via specific receptors [21-34]. It was an exciting event for our group when later on such receptors were found indeed, and also for only the two of many mitochondrial substrates. As the authors of the first paper stated, the cause of this selectivity was not clear [35-40]. However, it is well explained from the standpoint that just a pair of substrates is needed as a counterpart of also the paired ADR/ACH regulatory system. The novel mechanism of physiological regulation was named by our group Substrate-Hormonal System. It is presented in Figure 2. It serves as a link between the vegetative nervous system and mitochondria. This system may exist also inside cells with no innervation: in embryos, yeasts, plants, and as was shown recently, in

As was mentioned above, the SDH/KDH ratio reflects in mitochondria the balance of ADR

**3. Methodological meaning of statistical analysis in modern biomedical** 

Usual desire of physician to find unique for a certain patient individual diagnosis and treatment become to be an advanced direction of the modern probative medicine. Some statistical algorithms appeared which substantiate individual conclusions made earlier


lymphocytes [41-45]**.**

**investigations** 

and ACH regulation in the organism.

The measurements of the dehydrogenase (DH) activity are carried out using the cytochemical technique of glass-adhered cells by nitroblue tetrazolium reduction after 1-h incubation at 37 oC. The basal medium contains: 125 mM KCl, 10 mM НEPES, 1.22 mM NBT, рН 7.2 ± 0.05.

As distinct from the cytochemical method according to which only one sample with substrate is usually studied for DH activity measurement, the several selected samples with different additions are investigated according to CBCh procedure. The additions will be mentioned and explained further. These additions allow one to analyze the biochemical mechanisms of DH activity regulation. The regulation of the SDH activity by isocitric acid (ISC) and its combination with KGL activity were found to be most informative. A set of separate samples forms a CBCh pattern of DH activity and serves as a sensitive marker of the state of the DHs in the organism and, therefore, of the state of the organism itself. Examples of patterns and their interpretation will be described further.

The special interest to SDH is due to a great domination of SUC over other substrates in the rate of oxidation, known more than a century, and crown by the brilliant Chance's discovery of even greater domination in energy-dependent NAD+ reduction [15, 16].

As cited below, our group demonstrated numerous effects of stimulation of mitochondrial and physiological functions by SUC. The crucial role of SDH in tumor resistance and cardioprotection and disease development is recently reported [17-20]. The special properties of KDH are less known and will be considered further.

It was shown in previous studies by our group that the oxidation of SUC and selective activation of SDH are involved in adrenergic (ADR) regulation, while the oxidation of αketoglutarate (KGL) and selective activation of KDH is involved in cholinergic (ACH) regulation (see also for more details [10], section 3.2.).

рН 7.2 ± 0.05.

further.

[9-11]. Because the test of NBT reduction is more sensitive to electrons than oxygen consumption, the CBCh method first allowed us to measure low-rate respiration without artificial high concentrations of stimulators. This characteristic corresponds to a real Quiescent State of mitochondria in the cell. It is of great importance because additions distort the physiological regulation. For instance, ADP results in a shift of the ADP/GDP ratio in mitochondria and inhibits SDH. At the dawn of the polarography era, some leading mitochondriologists believed this extreme stimulation to be artificial, and the measurement of respiration without any external additions to be more physiological and desirable. However, the advantages of rapid-recording polarography were so attractive, compared with the previous monumental and slow manometric epoch that the polarographic method spread quickly over the world. Considering the aforesaid, the possibility to measure the real Quiescent State respiration is one more crucial advantage of the CBCh method. Therefore, besides the preservation of network by fixation, the quiescent state is protected by exclusion of the artificial addition of exogenous activators. Due to the combination of cytological and

The measurements of the dehydrogenase (DH) activity are carried out using the cytochemical technique of glass-adhered cells by nitroblue tetrazolium reduction after 1-h incubation at 37 oC. The basal medium contains: 125 mM KCl, 10 mM НEPES, 1.22 mM NBT,

As distinct from the cytochemical method according to which only one sample with substrate is usually studied for DH activity measurement, the several selected samples with different additions are investigated according to CBCh procedure. The additions will be mentioned and explained further. These additions allow one to analyze the biochemical mechanisms of DH activity regulation. The regulation of the SDH activity by isocitric acid (ISC) and its combination with KGL activity were found to be most informative. A set of separate samples forms a CBCh pattern of DH activity and serves as a sensitive marker of the state of the DHs in the organism and, therefore, of the state of the organism itself. Examples of patterns and their interpretation will be described

The special interest to SDH is due to a great domination of SUC over other substrates in the rate of oxidation, known more than a century, and crown by the brilliant Chance's discovery

As cited below, our group demonstrated numerous effects of stimulation of mitochondrial and physiological functions by SUC. The crucial role of SDH in tumor resistance and cardioprotection and disease development is recently reported [17-20]. The special

It was shown in previous studies by our group that the oxidation of SUC and selective activation of SDH are involved in adrenergic (ADR) regulation, while the oxidation of αketoglutarate (KGL) and selective activation of KDH is involved in cholinergic (ACH)

of even greater domination in energy-dependent NAD+ reduction [15, 16].

properties of KDH are less known and will be considered further.

regulation (see also for more details [10], section 3.2.).

biochemical advantages, the novel method was called Cytobiochemical.

**Figure 2.** Substrate - hormonal system. Participation of mitochondria in physiological regulation. Reciprocal adrenergic - cholinergic regulation as a whole with oxidation of succinate - α-ketoglutarate. Link between sympathetic and parasympathetic nervous system and mitochondria, or link between non - neuronal intracellular hormones and substrates.

Therefore, the measurement of the activity of the chosen DHs, SDH and KDH, provides also information on the level of reciprocal ADR/ACH regulation in the organism. Substrates of the two DHs, SUC and KGL, were shown to influence the ADR and ACH level as well as support physiological functions under load and in recovery. It is of importance that the regulatory effects of SUC and KGL in the organism are well pronounced in concentrations far below than necessary for substrate supply. To differentiate these two activities of substrates, the low concentrations manifesting general regulatory action in the organism were called "signal" suggesting that it is realized via specific receptors [21-34]. It was an exciting event for our group when later on such receptors were found indeed, and also for only the two of many mitochondrial substrates. As the authors of the first paper stated, the cause of this selectivity was not clear [35-40]. However, it is well explained from the standpoint that just a pair of substrates is needed as a counterpart of also the paired ADR/ACH regulatory system. The novel mechanism of physiological regulation was named by our group Substrate-Hormonal System. It is presented in Figure 2. It serves as a link between the vegetative nervous system and mitochondria. This system may exist also inside cells with no innervation: in embryos, yeasts, plants, and as was shown recently, in lymphocytes [41-45]**.**

As was mentioned above, the SDH/KDH ratio reflects in mitochondria the balance of ADR and ACH regulation in the organism.
