**5.1. Endogenous substrates (ES), SDH true and SDH latent**

As mentioned in Methods, for the CBCh characteristic of SDH we used additional samples besides the basal with the addition of millimolar substrate concentrations of SUC. The main of them are shown in Figure 5 and 7, which present more completely the experiments demonstrated in Figure 4. Some additional samples are also given in Figure 6.

The first column from the left shows the reduction of NBT by only ES oxidation without addition of exogenous substrates. This indicator is ignored in most cytochemical investigations. Meanwhile its consideration provides additional valuable information.

The first important characteristic which is determined with the help of ES is a more precise measurement of SDH activity with subtraction of the contribution of substrates other than SUC.

As shown in Figure 5, in intact animal the value of reduction in sample SDH is close to that in the sample ES. When subtracting the data for ES from the data for SDH, as it is accustomed in biochemistry, it turned out that the addition of 5 mM SUC absolutely does not increase the reduction in the least. This means that the reduction in the sample with SUC is not caused by SDH activity but is due to other processes. These data show that SDH in physiological quiescence of the organism is not active, keeping a latent state. In contrast, after ADR administration reduction due to ES is lower, the SDH activity is considerably higher, and the difference SDH-ES is great. We call this calculated value true SDH activity (SDHtru) because the subtraction eliminates the contribution of other substrates. To distinguish this corrected value from the common SDH activity measured without subtraction we term the latter the total SDH (SDHtot). The loss of SDH activity in the quiescent state of the organism is well explained from the standpoint of physiology. In the quiescent state, the ADR regulation is switched off reciprocally to ACH regulation. Apparently, SDH as a target of ADR in mitochondria follows hormone signal, switching activity off. As shown in the last example under prolonged -24 h stress corresponding to the exhaustion stage, the activity of both DHs declined.

The state of non-active SDH can also be termed latent or "sleeping" because it does not function in spite of SUC addition. The finding of this state of SDH is one of the most important attainments of the CBCh method. Such a state is not known in biochemical investigations of mitochondria because they lose the quiescent state during preparation and measurements. Biochemists are familiar only with initially activated SDH. The wide range of states from quiescent state to activation is lost for current observation. The loss of the initial basal level of respiration**,** *i.e.* the quiescent state, is in essence the absence of data for norm in current biochemical investigations.

We have found that the crucial difference in SDH activity between healthy and sick people or between calm and anxious animals is just the loss of the quiescent state. This means that, in the state of relative physiological quiescence, sick people has no quiescent state in mitochondria. This will be demonstrated further.

**Figure 5.** CBCh patterns of mitochondria measured in glass - adhered lymphocytes from intact and stressed rats. Adrenaline administration, 30 min, and PES 24 h. NBT reduction is expressed as a mean area of formazan granules in 30 lymphocytes

Owing to a great number of objects calculated (200-800) the presented data are statistically significant at the level of 0.001 (99.99%)

## **5.2. KDH activity measurement. Addition of malonate (MAL)**

The pair of columns in Figure 5 next to SDH presents the KDH activity measurements without and with the addition of MAL. These samples contained also NAD+ as a soluble cofactor of KDH. It is worth reminding that MAL should be obligatory added for correct determination of KDH activity. It was well known but undeservedly forgotten in many polarographic measurements that SUC generated in mitochondria during KGL oxidation may amount to a considerable part of respiration in the presence of KGL added. The SUCdependent portion of respiration should be subtracted from total respiration with KGL. The data presented show that the SUC-dependent portion of KDH activity is absent in the intact state, when SDH is non- active. In contrast, this portion rises in excited animals. It is evident from this example that the own KGL oxidation is negligible under strong excitation. The elimination of the SUC-dependent portion of NBT reduction by MAL allows one to measure KDH activity more precisely. Reciprocal changes in KDH and SDH activities in the presence of MAL become also more evident.

### **5.3. Signal action of SUC and ADR**

246 Dehydrogenases

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

**5.1. Endogenous substrates (ES), SDH true and SDH latent** 

exhaustion stage, the activity of both DHs declined.

norm in current biochemical investigations.

demonstrated in Figure 4. Some additional samples are also given in Figure 6.

**5. Other CBCh characteristics of mitochondria besides SDH and KDH** 

As mentioned in Methods, for the CBCh characteristic of SDH we used additional samples besides the basal with the addition of millimolar substrate concentrations of SUC. The main of them are shown in Figure 5 and 7, which present more completely the experiments

The first column from the left shows the reduction of NBT by only ES oxidation without addition of exogenous substrates. This indicator is ignored in most cytochemical investigations. Meanwhile its consideration provides additional valuable information.

The first important characteristic which is determined with the help of ES is a more precise measurement of SDH activity with subtraction of the contribution of substrates other than SUC. As shown in Figure 5, in intact animal the value of reduction in sample SDH is close to that in the sample ES. When subtracting the data for ES from the data for SDH, as it is accustomed in biochemistry, it turned out that the addition of 5 mM SUC absolutely does not increase the reduction in the least. This means that the reduction in the sample with SUC is not caused by SDH activity but is due to other processes. These data show that SDH in physiological quiescence of the organism is not active, keeping a latent state. In contrast, after ADR administration reduction due to ES is lower, the SDH activity is considerably higher, and the difference SDH-ES is great. We call this calculated value true SDH activity (SDHtru) because the subtraction eliminates the contribution of other substrates. To distinguish this corrected value from the common SDH activity measured without subtraction we term the latter the total SDH (SDHtot). The loss of SDH activity in the quiescent state of the organism is well explained from the standpoint of physiology. In the quiescent state, the ADR regulation is switched off reciprocally to ACH regulation. Apparently, SDH as a target of ADR in mitochondria follows hormone signal, switching activity off. As shown in the last example under prolonged -24 h stress corresponding to the

The state of non-active SDH can also be termed latent or "sleeping" because it does not function in spite of SUC addition. The finding of this state of SDH is one of the most important attainments of the CBCh method. Such a state is not known in biochemical investigations of mitochondria because they lose the quiescent state during preparation and measurements. Biochemists are familiar only with initially activated SDH. The wide range of states from quiescent state to activation is lost for current observation. The loss of the initial basal level of respiration**,** *i.e.* the quiescent state, is in essence the absence of data for

We have found that the crucial difference in SDH activity between healthy and sick people or between calm and anxious animals is just the loss of the quiescent state. This means that, The last pairs of columns in Figure 5 presents the data on NBT reduction with the addition of 5 μM, "Signal" concentration of SUC or 5 μM of ADR with substrate concentration of

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 influence of ADR.

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

**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%)

Signal concentrations of ADR act synergetically to SUC. These data serve as a further evidence for synergism between SUC and ADR.
