**5.4. Addition of MAL: Endogenous SUC (ESUC = ES - MAL), reactive oxygen species (ROS) formation**

MAL is commonly used to exclude the contribution to oxidation of SUC, either added or endogenous (ESUC), formed in mitochondria from other substrates, particularly from KGL. Although the amount of ESUC is very low compared with that of added KGL, SUC dominates in competition for the respiratory chain due to the dominating reducing power of SDH. ESUC has additional advantage over added SUC, because it forms inside mitochondria and is better available to SDH [16]. In CBCh measurements, a decrease in formazan formation corresponds to the inhibition of respiration. The difference in reduction between sample ES and (ES+MAL) gives a measure of ESUC.

We observed an increase in ESUC content in animals excited by endogenous ADR (stress) or ADR administration. ESUC also increases in a spontaneously anxious individuals, animals or people. 12 examples of typical patterns of MAL effects on NBT reduction with ES are presented in Figure 6. They were selected from more than 20 volunteers examined. Among them we have found four types of repeating patterns and no more variations. Each type is presented by three examples. The form of pattern is well reproduced for each type. Therefore we believe the pattern to be a regular relationship but not random combination of characteristics. All volunteers were in the relatively calm state. Blood samples were taken in the laboratory between 9-10 a.m. after a very weak breakfast at about 8 at home. All of them were practically healthy, however, the level of health and the state on the day of examination varied. Probably, in accordance with these differences, we found four types of repeating patterns.

Ranging the patterns according to the increase in the SDH activity from low to high, we differentiated the following States: Deep Quiescence, Operative Quiescence, Activity, and Activation with Inhibition.

The decrease in NBT reduction, caused by MAL, is particularly pronounced in the subgroup Activation, in which the SDH activity is the highest. The members of this subgroup have personal reasons for anxiety and activity. The same type of pattern but with a lower amount of ESUC was found in the subgroup named Operative Quiescence, because the SDH activity was considerably lower than in the subgroup Activity. The decrease in NBT reduction caused by MAL evidences the contribution of ESUC to respiration when SDH is active.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 249

248 Dehydrogenases

influence of ADR.

repeating patterns.

Activation with Inhibition.

**species (ROS) formation** 

evidence for synergism between SUC and ADR.

between sample ES and (ES+MAL) gives a measure of ESUC.

SUC - 5 mM. The effects of signal concentrations were negligible or absent when measured with subtraction of sample with MAL in the intact animal. However, both effects rise similar to the SDH activity in ADR-activated animal. Under prolonged stress, they both decline also in accordance with SDH activity. These data are the first evidence of a great SDH activity in the presence of low, signal concentration of SUC, far below the substrate ones, under the

Signal concentrations of ADR act synergetically to SUC. These data serve as a further

MAL is commonly used to exclude the contribution to oxidation of SUC, either added or endogenous (ESUC), formed in mitochondria from other substrates, particularly from KGL. Although the amount of ESUC is very low compared with that of added KGL, SUC dominates in competition for the respiratory chain due to the dominating reducing power of SDH. ESUC has additional advantage over added SUC, because it forms inside mitochondria and is better available to SDH [16]. In CBCh measurements, a decrease in formazan formation corresponds to the inhibition of respiration. The difference in reduction

We observed an increase in ESUC content in animals excited by endogenous ADR (stress) or ADR administration. ESUC also increases in a spontaneously anxious individuals, animals or people. 12 examples of typical patterns of MAL effects on NBT reduction with ES are presented in Figure 6. They were selected from more than 20 volunteers examined. Among them we have found four types of repeating patterns and no more variations. Each type is presented by three examples. The form of pattern is well reproduced for each type. Therefore we believe the pattern to be a regular relationship but not random combination of characteristics. All volunteers were in the relatively calm state. Blood samples were taken in the laboratory between 9-10 a.m. after a very weak breakfast at about 8 at home. All of them were practically healthy, however, the level of health and the state on the day of examination varied. Probably, in accordance with these differences, we found four types of

Ranging the patterns according to the increase in the SDH activity from low to high, we differentiated the following States: Deep Quiescence, Operative Quiescence, Activity, and

The decrease in NBT reduction, caused by MAL, is particularly pronounced in the subgroup Activation, in which the SDH activity is the highest. The members of this subgroup have personal reasons for anxiety and activity. The same type of pattern but with a lower amount of ESUC was found in the subgroup named Operative Quiescence, because the SDH activity was considerably lower than in the subgroup Activity. The decrease in NBT reduction caused by MAL evidences the contribution of ESUC to respiration when SDH is active.

**5.4. Addition of MAL: Endogenous SUC (ESUC = ES - MAL), reactive oxygen** 

**Figure 6.** Four (and no more) types of specific CBCh patterns of SDH found in healthy volunteers and presented by three examples. Owing to a great number of objects calculated (200-800) the presented data are statistically significant at the level of 0.001 (99.99%)

Together with this traditional effect of MAL, we revealed the opposite action of MAL, namely, an increase in NBT reduction. It was most pronounced in the subgroup Activation with Inhibition, which was distinguished according to a decreased SDH activity relative to the Activation subgroup. One cause of the inhibition (example 20) could be an elder age and initial hypertension demanding the intake small doses of adrenoblockers, which also inhibits SDH. In this case, an increase in NBT reduction by MAL is the highest. This effect is also well pronounced relatively to ES in two other examples in this subgroup. This unknown effect of MAL on NBT reduction is also observed in Deep Quiescence, in which all activities are at the lowest level.

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 251

However, ISC preserves the ability to stimulate inhibited SDH under 180- min PES. Besides, we have found that ISC does not influence the SDH activity in the Quiescent State. The two opposite effects of ISL: an increase and a decrease in SDH activity are easily explained by the single antioxidant mechanism. The increase in NBT reduction by MAL at the beginning of PES shows the development of oxidative stress. This agrees with other observations. The level of oxidative stress is regulated by well-balanced interactions of several processes occurring in mitochondria. Among them are initial uncoupling and its secondary recoupling

Recoupling effect of GTP can explain our observation that preserving the KDH, which is a real source of GTP-formed in substrate-level phosphorylation, protects SDH from

**Figure 7.** CBCh patterns of mitochondria measured in glass -adhered lymphocytes from intact and

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

In this metabolic environment, protection of KDH from oxidation by ISC allows one to support GTP generation and to diminish the uncoupling that was a cause of initial SDH hyperactivation. This explanation agrees well with our repeated observations that the preservation of high KDH activity simultaneously with activation of SDH is typical of activity within physiological adaptation area. The "scissors" between SDH rise and KDH fall are observed in severe stress or pathology. Therefore, three different effects of ISL on SDH activity: suppression of hyperactivation, activation of inhibition, and absence of influence in norm, allow one to differentiate clearly functional states of SDH. The area of

NBT reduction is expressed as a total area of formazan granules in 30 lymphocytes.

by GTP [52].

hyperactivation followed by inhibition.

stressed rats under mild PES of different duration.

the level of 0.001 (99.99%)

Thus, MAL manifests the ability to increase NBT reduction in case of low SDH activity, in contrast to its traditional inhibition of respiration is great when SDH activity is high.

In our opinion, the increase in NBT reduction by MAL is a marker of ROS formation in mitochondria. As shown in Figure 7, ROS formation sharply rises at the beginning of stress. During stress, the rise of ROS formation declines as evidenced by our measurements with MAL. This agrees with other results [52]. As shown in the last example in Figure 7, ROS rise again by the exhausting stage. The biochemical mechanism of this effect of MAL is in deep inhibition of SDH, which leads to a fall of coenzyme Q reduction from full reduction to free radical form. Free radical form of Q produces superoxide in interaction with surrounding oxygen [53].
