**1. Introduction**

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In the fields of biochemistry and medicine there is growing interest to the crucial role of mitochondria in changing of physiological states and disease development. This interest is fueled by rapid progress in the modern branches of mitochondriology: Mitochondrial Physiology (MiP) and Mitochondrial Medicine [1-8]. Numerous investigations demonstrated mitochondrial dysfunctions in different experimental models of stress or disease. These results were obtained under clearly pronounced functional disorders induced by strong external stimuli or pathogenic mutations. These observations increased interest in the detection of mitochondrial dysfunctions in the organism as early biomarkers of pathogenic processes. The detection of mitochondrial dysfunction is crucial at subclinical stages of disease because it can lead to more effective prevention and earlier intervention. In spite of this benefit to date the available testing methods cannot properly measure the state of mitochondria in the organism in ex vivo observations. Past successes of respiration measurements in isolated mitochondria distracted attention from obvious, methodological shortcoming of the existing method of mitochondria isolation: intentional destroy of their native structure organization into entire network. It is known nowadays that the responses of mitochondria to various physiological challenges are based on the reversible dissociation

© 2012 Kondrashova et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of such subtle network structure. In the network state mitochondrial respiration proceeds at a low rate while excitation leads to the dissociation of the network and an increase in respiration. We demonstrated this shortcoming in simultaneous polarographic and microscopic studies of network fragments in KCl rat liver homogenate that contains a higher density of tissue than is usual (1:1 tissue:solution) [9-11].

Study of Succinate Dehydrogenase and α-Ketoglutarate Dehydrogenase in Mitochondria Inside Glass-Adhered Lymphocytes Under Physiological Conditions – The Two Dehydrogenases as Counterparts of … 237

An additional, clinical benefit of the CBCh method is the ability to measure mitochondrial function from a drop of blood, substantially reducing the invasiveness of muscle biopsy that is currently required for mitochondrial disease diagnosis. Besides, CBCh method abolishes problem of liquid blood sample changing during work with a group of patients. The state of glass-adhered cells is stable for several hours and after that all samples can be activated

In this review we briefly summarize our previous results on elaboration the method, focusing on recent data on its application in physiological and clinical studies. A special attention is paid to interpretation of the data because some unknown phenomena were

revealed. Perspectives of further development of our method will be also considered.

**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

simultaneously by insertion into media for measurement of enzyme activity.

**Succinate dehydrogenase and α-ketoglutarate dehydrogenase** 

**2. A brief description of the cytobiochemical method.** 

**Figure 1.** Images of human glass-adhered lymphocytes after SUC oxidation.

2000) equipped with a camera (Leica, DFC-425) connected to a computer.

Images were collected using an oil immersion lens (100x/1.25) under a light microscope (Leica, DM

SDH activity is registered by blue staining with formazan in mitochondria. Red staining is used for

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

smear.

nuclei visualization.

Since the first step of isolation of mitochondria is network destruction, this method artificially mimics the physiological signal of the activation of respiration. Thus, mitochondria isolated from non-active tissues using the standard methodology do not maintain low levels of respiration typical of the quiescent state and show rapid respiration.

A matter of great concern of biochemists is primarily not to inhibit the enzyme activity; therefore, rapid respiration of isolated mitochondria did not cause apprehension. However, the hyperactivation of functions as an initial step of their alteration preceding inhibition is well known in physiology. That is why artificially accelerated respiration of isolated mitochondria means in essence the LOSS of data for norm in biochemical investigations. The importance of preservation of the native mitochondrial network in ex vivo observations was recently highlighted in a special issue of IJBCB assembled under the editorial guidance of R. Rossignol [12].

The modern investigations clearly demonstrate strong correspondence in functional state of mitochondria and their native organization in the network. This is formulated by the impressive motto: "To be in good shape - to survive" [13]. At the beginning of mitochondria isolation coryphaei of mitochondriology considered possible loss of native properties caused by preparation. H. Krebs attracted the attention to the acceleration of respiration of mitochondria by dilution. He preferred to study mitochondria in homogenate as more native. A. Lehninger mentioned that effects observed in mitochondria in vitro can often reflect their "preparative story" but not native properties. N. Kaplan and collaborators carried out the study just of the SDH activity by the most sensitive function: the reversed electron transfer, which was at that time recently discovered by B. Chance [14]. They explained discrepancies between data of some leading laboratories by the dependence of the effects on the common experimental additions and conditions. Chance participated in discussion of results and all his life developed technics to measure mitochondria inside the organism.

However, the convenient method to study mitochondria ex vivo was not found. Meanwhile the advantages of work with isolated mitochondria by rapid recording technics stimulated biochemists to wide studies and doubts on properties of preparations were neglected.

In order to overcome the crucial methodological shortcoming of traditional procedures, we have created a novel, CytoBioChemical (CBCh) method which preserves mitochondrial network *ex vivo* using glass-adhered blood lymphocytes. Studies of various physiological and pathological states using the novel CBCh method have shown greater responses of the mitochondria to functional changes in the organism compared with mitochondria isolated in the form of single granules.

Rossignol [12].

organism.

the form of single granules.

of such subtle network structure. In the network state mitochondrial respiration proceeds at a low rate while excitation leads to the dissociation of the network and an increase in respiration. We demonstrated this shortcoming in simultaneous polarographic and microscopic studies of network fragments in KCl rat liver homogenate that contains a higher

Since the first step of isolation of mitochondria is network destruction, this method artificially mimics the physiological signal of the activation of respiration. Thus, mitochondria isolated from non-active tissues using the standard methodology do not maintain low levels of respiration typical of the quiescent state and show rapid respiration. A matter of great concern of biochemists is primarily not to inhibit the enzyme activity; therefore, rapid respiration of isolated mitochondria did not cause apprehension. However, the hyperactivation of functions as an initial step of their alteration preceding inhibition is well known in physiology. That is why artificially accelerated respiration of isolated mitochondria means in essence the LOSS of data for norm in biochemical investigations. The importance of preservation of the native mitochondrial network in ex vivo observations was recently highlighted in a special issue of IJBCB assembled under the editorial guidance of R.

The modern investigations clearly demonstrate strong correspondence in functional state of mitochondria and their native organization in the network. This is formulated by the impressive motto: "To be in good shape - to survive" [13]. At the beginning of mitochondria isolation coryphaei of mitochondriology considered possible loss of native properties caused by preparation. H. Krebs attracted the attention to the acceleration of respiration of mitochondria by dilution. He preferred to study mitochondria in homogenate as more native. A. Lehninger mentioned that effects observed in mitochondria in vitro can often reflect their "preparative story" but not native properties. N. Kaplan and collaborators carried out the study just of the SDH activity by the most sensitive function: the reversed electron transfer, which was at that time recently discovered by B. Chance [14]. They explained discrepancies between data of some leading laboratories by the dependence of the effects on the common experimental additions and conditions. Chance participated in discussion of results and all his life developed technics to measure mitochondria inside the

However, the convenient method to study mitochondria ex vivo was not found. Meanwhile the advantages of work with isolated mitochondria by rapid recording technics stimulated

In order to overcome the crucial methodological shortcoming of traditional procedures, we have created a novel, CytoBioChemical (CBCh) method which preserves mitochondrial network *ex vivo* using glass-adhered blood lymphocytes. Studies of various physiological and pathological states using the novel CBCh method have shown greater responses of the mitochondria to functional changes in the organism compared with mitochondria isolated in

biochemists to wide studies and doubts on properties of preparations were neglected.

density of tissue than is usual (1:1 tissue:solution) [9-11].

An additional, clinical benefit of the CBCh method is the ability to measure mitochondrial function from a drop of blood, substantially reducing the invasiveness of muscle biopsy that is currently required for mitochondrial disease diagnosis. Besides, CBCh method abolishes problem of liquid blood sample changing during work with a group of patients. The state of glass-adhered cells is stable for several hours and after that all samples can be activated simultaneously by insertion into media for measurement of enzyme activity.

In this review we briefly summarize our previous results on elaboration the method, focusing on recent data on its application in physiological and clinical studies. A special attention is paid to interpretation of the data because some unknown phenomena were revealed. Perspectives of further development of our method will be also considered.
