**6. Revealing fine physiological differences in the state of the organism in norm by the activity of dehydrogenases as measured with the CBCh method. Differences in the activity and responses of SDH in rabbits with opposite behaviour**

The CBCh method clearly detects some fine physiological differences in the state of the organism that were earlier not observed in mitochondria. Among them are some agerelated differences in rats at different stages of ontogenesis, namely, in newborn animals, depending on the time of feeding with mother milk, prior to or immediately after maturation, as well as individual differences of pattern types between healthy humans that are related to the current condition or temperament [48, 57]. Both age-dependent changes and differences of temperaments reflect different ratios of ADR/ACH regulation. In the earlier age, ADR regulation dominates; after maturation, it is more balanced by ACH.

measurements.

**opposite behaviour** 

ACH.

GTP-recoupling instead of usual OAA inhibition.

**measure of the rate of SUC oxidation in mitochondria** 

functions: stimulation, and restriction with restoration, respectively.

This is one more crucial advantage of CBCh measurements of DHs activity.

SDH regulation involving KDH-dependent GTP – recoupling - is not known in biochemical investigations because SDH is initially hyperactivated under the experimental conditions. The hyperactivation of SDH elevates the risk of inhibition by OAA due to the increase in its formation. Indeed, OAA inhibition is often observed, when SUC oxidation is accelerated, for example, by uncoupling. Therefore, OAA inhibition is considered to be the only mechanism of SDH control. However, this is true for in vitro conditions in biochemical experiments in isolated mitochondria. The preservation of the area more close to physiological conditions in the CBCh method opens Terra Incognita of unknown regulation of the SDH activity by

The last but not least, it is worth mentioning that the mutual reciprocal regulation of SDH and KDH activity is further evidence of ADR and ACH regulation in mitochondria. In essence, it corresponds to the reciprocal influence of ADR and ACH on physiological

Before continuation, let us put in a brief insertion to memorize the better approach of CBCh determination of SDH activity to mitochondrial processes compared to pure enzymatic

**Interlude: СBCh activity of SDH is not so much a characteristic of abstract DH, as a** 

What is the interest of physiologists and physicians in measurement of enzyme activities? Evidently they need to learn how the enzymes work in the organism in different states. Many purely enzymatic methods give exact information, although rather abstract in respect of the conditions in vivo. Preservation of the native state and native surrounding of SDH and KDH under CBCh conditions permit measurement of SUC oxidation, that approaches the real rate of respiration in mitochondria in vivo rather than an abstract activity of SDH.

**6. Revealing fine physiological differences in the state of the organism in** 

**method. Differences in the activity and responses of SDH in rabbits with** 

The CBCh method clearly detects some fine physiological differences in the state of the organism that were earlier not observed in mitochondria. Among them are some agerelated differences in rats at different stages of ontogenesis, namely, in newborn animals, depending on the time of feeding with mother milk, prior to or immediately after maturation, as well as individual differences of pattern types between healthy humans that are related to the current condition or temperament [48, 57]. Both age-dependent changes and differences of temperaments reflect different ratios of ADR/ACH regulation. In the earlier age, ADR regulation dominates; after maturation, it is more balanced by

**norm by the activity of dehydrogenases as measured with the CBCh** 

**Figure 8.** Responses of ES and SDH to activation by administration of adrenaline or succinate measured in lymphocytes of rabbits with different behavior.

A - calm rabbit, B - restless rabbit. SDH activity is expressed as a mean area of formazan granules in 30 cells. The mean of three measurements in different days for each rabbit are presented, M ± SEM. \* - р< 0.05, \*\* - p<0.01, comparing to the respective initial state, # - p<0.05, comparing to the calm rabbit.

As one of the models of domination of these regulation types we studied individual differences of CBCh patterns in rabbits with different behaviour.

One rabbit was calm during the experiment; another rabbit escaped and was anxious and restless even when it was not exposed to treatment. They represented characteristic types of a cholinergic and adrenergic, respectively. Figure 8 demonstrates significant differences between the calm and restless rabbits in the level of ES and in the SDH activity.

The reserve of ES in the calm rabbit is much greater than in the restless. After the injection of ADR, the level of ES in the calm rabbit decreases; however, this level is higher than in the restless animal under the influence of ADR. In the intact restless animal, the level of ES is 15 times lower than that of the intact calm rabbit and 5 times lower than in the calm animal after the injection of ADR. This indicates an enhanced expenditure of substrates in the restless animal.

The SDH activity in the calm rabbit is low, and the injections of ADR or SUC strongly stimulates it. The stimulation of SUC is somewhat stronger, which increases the risk of the transition from hyperactivation to inhibition. By contrast to ADR, the injection of SUC also increases the level of ES.

SDH in the intact restless rabbit is hyperactivated nearly up to the level of calm under ADR influence. Thus, as judged by ES level and the SDH activity, the restless rabbit has no quiescence in the quiet state without external excitations. The same state of continuous internal metabolic excitation is inherent in all patients examined in our laboratory in contrast to healthy volunteers. The data will be considered in the next sections.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 255

**7. The changes in SDH and KDH activity in patients with metabolic** 

resistance, type 2 diabetes, risk of hypertension, heart diseases and others [1, 60-65].

**Figure 9.** True SDH without and with ISC in not severe patients suffering from food allergy

SDHtru is calculated as the difference between samples SUC 5 mM - MAL 5mM.

patient 1 (100%). The low row for SUC 5mM, the upper row for SUC 5 mM + ISC 5 mM.

Data are expressed in mean area (S) of formazan granules in 30 cells. Owing to a great number of objects calculated (200-800) the presented data are statistically significant at the level of 0.001 (99.99%)

The changes of SDH and SDHisc activity are presented in per cents respective to conditional norm of

accompanied by arterial hypertension.

Inscription above - types of patterns.

The stages of the SDH and KDH activity changes found under stress were observed in

Disturbance of normal metabolic flexibility between oxidation of glucose and fatty acids is the basis of many diseases. High dietary fat intake and low physical activity provoke this disorder. Complex of accompanied biochemical changes is called metabolic syndrome. Metabolic syndrome underlies various pathological symptoms, such as obesity, insulin

Metabolic syndrome is caused by increased intensity of life accompanied with persistent sympathetic hyperactivation and deficit of physical activity that leads to PES. That is why we have studied the participation of mitochondria in development of PES. The importance of mitochondria in development of metabolic syndrome is caused by their crucial role in intracellular oxidation. Aerobic fibers of skeletal muscle mitochondria make a major contribution to regulation of metabolic syndrome and support metabolic health of the whole

**syndrome and arterial hypertension** 

organism.

patients with metabolic syndrome and arterial hypertension.

At the background of the initially high SDH activity in restless rabbit ADR administration leads to only small stimulation, which only somewhat exceeds the level under the influence of ADR in calm rabbit. The influence of SUC, which was somewhat stronger activator when injected to the calm rabbit, leads to realization of risk of transition to inhibition in restless animal.

The difference in ADR and SUC effects on ES can be explained by different mechanisms of their influence on SDH activity. ADR only activates SDH through protein kinase. The influence of SUC is more complicated. SUC is an allosteric activator of SDH [58]. Simultaneously the rise of SUC level increases OAA formation, which inhibits SDH according to the mechanism of the negative feedback.

These effects are better pronounced in a sample without SUC addition, namely ES, because SUC excess partially abolish OAA inhibition. Therefore in sample ES SDH inhibition is better observed than in sample for SDH measurement, containing 5 mM SUC, because it is not masked by the addition of substrate. Probably, the main part of ES under SUC injection is ESUC, which is accumulated due to OAA inhibition. It should be considered that measurements are made for an hour. Insight into dynamics of the processes can be achieved by a comparison of data for calm rabbit, in which oxidation is slower with those for restless one, in which oxidation is accelerated. As shown for calm rabbit, at the beginning rapid oxidation of SUC occurred, resulted in an increase in OAA, which is evidenced by the initial accumulation of ESUC. The next stage is presented by data for restless rabbit in which stronger inhibition of SDH by OAA is already developed, which is not completely abolished by SUC addition and therefore ESUC accumulation is higher than in calm animal.

The data presented show great difference of the two CBCh properties of the state of mitochondria in animals with opposite behaviour, which corresponds in magnitude to the pronounced individual peculiarities in behaviour.

We have also found the pronounced difference in the SDH and KDH activity in rats before maturation, 6 week old with domination of ADR regulation, and just after maturation, 8 week old with increase in ACH regulation. These changes are close to differences between calm and restless rabbits.

It was shown also that complicated menopause in rats and women, which is manifested under the decrease in ADR regulation in the organism, is related to the increase in the SDH activity and can be corrected by metabolic regulators, containing SUC - "Amberen" [59].

The data considered support the view of existence of the link between activity of studied DHs and the level of ADR and ACH regulation.
