**4. Demonstration of high physiological sensitivity of the CBCh method in models of increased adrenergic regulation. SDH/KDH ratio as a distinct borderline between intensive functioning and early dysfunction**

Over a long initial period, the biochemical mechanisms of stress were studied under action of strong chemical or physical stressors, which are beyond the range of physiological adaptation. In spite of this, the responses of isolated mitochondria studied under pain immobilization stress for a long time, 24 h, were not pronounced, accounting for no more than 20% inhibition of succinate oxidation measured by the polarographic method [11, 50, 51]. The considerably higher sensitivity of the CBCh assay to changes in the physiological state is evident, as it reveals a great-range difference of SDH activity, 2-3 times and more, under the influence of much milder stressors compatible with life, such as the administration of ADR in submaximal dose or painless short-time restraint of rat in a narrow box for 30-120 min, psychoemotional stress (PES). A comparison of mitochondrial responses observed in isolated mitochondria and in mitochondria inside lymphocytes is convincing, considering that the state of animals after strong or physiological stress differed greatly. After strong stress, animals were exhausted and could not move, while animals after short-term stress looked like intact.

The changes in the SDH and KDH activities after the administration of ADR are presented in the upper panel of Figure 4. As shown, these activities are moderate and nearly equal in the initial quiescent state. Under the action of ADR, the initial acute phase of stress, an alarm reaction arises. It is clearly manifested by a pronounced (more than twofold!) increase in the SDH activity, and by a fall (about twentyfold!) in the KDH activity. Under 24-h restraint in a box, a stress phase of exhaustion develops. It is also clearly pronounced by a decrease in the SDH activity approximately to the initial level. It is very important that this decline does not mean a restoration of the initial state because the KDH activity nearly disappears. Therefore, the dynamics of SDH activity develops according to a bell-shaped curve with a maximum at early stages of stress. This corresponds to the classic stress dynamics followed by physiological data.

As mentioned, the SDH activity serves as a marker of ADR regulation, which supports the external work of tissues, while the KDH activity is a marker of ACH regulation which is responsible for restoration of expenditures for work and underlies the general immune resistance of the organism, including the participation of lymphocytes. Therefore, we consider the critical fall in the KDH activity as a manifestation of stress that exceeds the range of physiological adaptation. It is worth mentioning that the 24- h PES when so dramatic changes in the DH activity were observed by the CBCh method is not as severe as the widely investigated rough form of immobilization when only small changes in respiration were found in isolated mitochondria.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 245

**Figure 4.** Changes in SDH and KDH activity and their ratio in rats.

investigations and is Terra Incognita for mitochondriologists.

formazan granules in 30 cells.

formazan granules in 30 cells.

the level of 0.001 (99.99%)

Upper panel. Administration of moderately increased dose of adrenaline (50 μg/100 g) for 30 min and long-term (24-hour) psychoemotional stress in a box. SDH activity is expressed as a mean area (S) of

Low panel. Short-term psychoemotional stress in a box. SDH activity is expressed as a total area (S) of

Owing to a great number of objects calculated (200-800) the presented data are statistically significant at

As shown in the last example in the lower panel of Figure 4, the prolongation of PES to 180 min exceeds adaptive reserves as it leads to a fall in the activity of both DHs. This inhibition corresponds to the stage of exhaustion of stress. Thus the dynamics of SDH and KDH activity changes presented in both panels of Figure 4 for the first time clearly reveals

In contrast to the results obtained on isolated mitochondria, the CBCh method allows one to observe a broad range of mitochondrial states from deep quiescence up to strong excitation followed by pathological exhaustion in the model of wholly physiological stress. This area of states of mitochondria is not available under the conditions of standard biochemical

mitochondrial mechanisms that underlie the physiological development of stress.

An extremely interesting, quite novel area of real adaptive changes in the DH activity opens during even milder PES of shorter duration: 30, 60, 120 min. As seen in the lower panel of Figure 4, during 30 and 60 min a gradual hyperactivation of the both SDH and KDH activity are observed without the critical fall of the latter. Changes in the KDH activity are only of somewhat less extent than that of SDH. By 120 min hyperactivation of the both DHs is followed by the return to the quiescent state.

The status of DHs at 120-min PES is of crucial importance.

Let us remind that in the initial completely quiescent state, the activity of both DHs is low and nearly equal. This is time to note that the KDH activity is somewhat higher than SDH, which is well reproduced in the independent observations. This small excess of KDH activity over SDH is of great importance because it corresponds to the domination of ACH regulation over ADR regulation in the quiescent state. As shown in the lower panel of Figure 4, by 120 min both DHs return nearly to the initial level. Two wonderful details of this state should be noticed. The first is that a slight prevalence of KDH activity over SDH appears which is typical of the quiescent state. The second is that the level of both is somewhat higher than in the initial state before stress.

These specific features evidence a real return to the well balanced quiescent state in spite of continuation of stress. In essence this decrease in DH activity is a real adaptation to the load during training.

Such phenomenon is well known in physiological observations and only after repeated training.

But it has been never observed in a single short-term experiment, the more so for mitochondrial processes. A search for biochemical markers of such a desirable state was only a dream of physicians and trainers. The CBCh method revealed such changes in a short-term experiment and in a wide range.

A finding these two types of early changes in responses of mitochondria to the pathogenic influence detected for the first time a distinct borderline between stimulating, adaptive action of load and the transition to its adverse action, which increases the risk of pathology.

The reliable detection of this border is particularly important for the prevention of disease or a more effective intervention.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 245

244 Dehydrogenases

respiration were found in isolated mitochondria.

followed by the return to the quiescent state.

The status of DHs at 120-min PES is of crucial importance.

somewhat higher than in the initial state before stress.

short-term experiment and in a wide range.

a more effective intervention.

during training.

training.

As mentioned, the SDH activity serves as a marker of ADR regulation, which supports the external work of tissues, while the KDH activity is a marker of ACH regulation which is responsible for restoration of expenditures for work and underlies the general immune resistance of the organism, including the participation of lymphocytes. Therefore, we consider the critical fall in the KDH activity as a manifestation of stress that exceeds the range of physiological adaptation. It is worth mentioning that the 24- h PES when so dramatic changes in the DH activity were observed by the CBCh method is not as severe as the widely investigated rough form of immobilization when only small changes in

An extremely interesting, quite novel area of real adaptive changes in the DH activity opens during even milder PES of shorter duration: 30, 60, 120 min. As seen in the lower panel of Figure 4, during 30 and 60 min a gradual hyperactivation of the both SDH and KDH activity are observed without the critical fall of the latter. Changes in the KDH activity are only of somewhat less extent than that of SDH. By 120 min hyperactivation of the both DHs is

Let us remind that in the initial completely quiescent state, the activity of both DHs is low and nearly equal. This is time to note that the KDH activity is somewhat higher than SDH, which is well reproduced in the independent observations. This small excess of KDH activity over SDH is of great importance because it corresponds to the domination of ACH regulation over ADR regulation in the quiescent state. As shown in the lower panel of Figure 4, by 120 min both DHs return nearly to the initial level. Two wonderful details of this state should be noticed. The first is that a slight prevalence of KDH activity over SDH appears which is typical of the quiescent state. The second is that the level of both is

These specific features evidence a real return to the well balanced quiescent state in spite of continuation of stress. In essence this decrease in DH activity is a real adaptation to the load

Such phenomenon is well known in physiological observations and only after repeated

But it has been never observed in a single short-term experiment, the more so for mitochondrial processes. A search for biochemical markers of such a desirable state was only a dream of physicians and trainers. The CBCh method revealed such changes in a

A finding these two types of early changes in responses of mitochondria to the pathogenic influence detected for the first time a distinct borderline between stimulating, adaptive action of load and the transition to its adverse action, which increases the risk of pathology. The reliable detection of this border is particularly important for the prevention of disease or

Upper panel. Administration of moderately increased dose of adrenaline (50 μg/100 g) for 30 min and long-term (24-hour) psychoemotional stress in a box. SDH activity is expressed as a mean area (S) of formazan granules in 30 cells.

Low panel. Short-term psychoemotional stress in a box. SDH activity is expressed as a total 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%)

As shown in the last example in the lower panel of Figure 4, the prolongation of PES to 180 min exceeds adaptive reserves as it leads to a fall in the activity of both DHs. This inhibition corresponds to the stage of exhaustion of stress. Thus the dynamics of SDH and KDH activity changes presented in both panels of Figure 4 for the first time clearly reveals mitochondrial mechanisms that underlie the physiological development of stress.

In contrast to the results obtained on isolated mitochondria, the CBCh method allows one to observe a broad range of mitochondrial states from deep quiescence up to strong excitation followed by pathological exhaustion in the model of wholly physiological stress. This area of states of mitochondria is not available under the conditions of standard biochemical investigations and is Terra Incognita for mitochondriologists.

Further we shall describe some other unknown phenomena in this unexplored area.
