For today it is known, that primary and secondary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of clinical manifestations and diseases. Therefore, the question about effective correction of various types of energy exchange disorders remains topical. Thus, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (EC 1.3.99.1) (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) (GPD2). The human lymphocytes assays were tested in vitro (22 donors). In negative control lymphocytes, cell culture normal saline solution was added. Normal saline solution with NaN3 was added in positive control lymphocytes cell culture. Experimental cell culture contained NaN3 and BAS. Our investigations had been revealed increase SQR activity in the experimental cell culture as compared with positive control culture throughout the time of experiment (measurements were carried out at 4, 24, 48, 72 h of incubation). The SQR activity of experimental cell culture and negative control lymphocytes cell culture was equal up to 24 h of experiment. It showed neutralization of NaN3 inhibitory effect (during 24 h) due to BAS influence. Activity of base glycerophosphate shunt ferment GPD2 of experimental lymphocyte cell culture was not different from GPD2 index in the negative control, but was lower than GPD2 activity in the positive control. It also indicated neutralization NaN3 inhibitory effect due to BAS influence. So we had found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro could effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link.
A key link of metabolism of any living system is an energy exchange. Both at the level of the whole organism, and at the level of an individual cell, the energy exchange is a grandiose complex process, which is regulated by multiplex and the subtlest manner and is organized in space and time.
Certainly the most important part of the energy metabolism is aerobic respiration. Due to this step energy exchange, organism receives most of the energy required for its life. Naturally, that practically any defects in the link of aerobic energy metabolism lead to significant structural and functional impairment, starting from the cellular level and ending with the whole organism. Currently, there are three type disorders of aerobic respiration: primary―a consequence of innate nDNA and mtDNA defects, disturbances caused by somatic mutations nDNA and mtDNA, and the so-called “energy-deficient diathesis” [
For today it is known, that primary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of nosological form. All of them first of all are dependent on violation aerobic respiration, as well as the degree of involvement in pathologic process of various tissues and organs. Mitochondrial medicine deals with issues of manifestation, prevention, epidemiology, diagnostic and therapy of mitochondrial diseases [
In the arsenal of doctors, there are several drugs able to activate specific inhibited reactions of energy exchange [
Therefore, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (GPD2).
RPMI-1640 medium, Dulbecco’s phosphate buffered saline (PBS), Histopaque-1077 Hybri-Max®, Fetal Bovine Serum (FBS), L-glutamine, penicillin-streptomycin, α-glycerol phosphate disodium salt hydrate, sodium succinate dibasic hexahydrate, potassium phosphate monobasic, sodium phosphate dibasic dihydrate and sodium hydroxide (Sigma-Aldrich Co., U.S.A.), ethylenediaminetetraacetic acid (EDTA) (Fluka Analytical, Switzer- land), Janus green B, sodium azide, acetone and hydrochloric asid (Merck KGaA, Germany), trypan blue stain (Gibco® by Life Technologis, U.S.A.), p-nitrotetrazolium violet (RPC “Sinbias”, Ukraine), consumables for cell culture, cytochemistry, etc. (Sarstedt AG&Co, Germany ), tubes for blood sampling Venosafe® (Terumo Medical Corporation, Japan ). Investigated complex of BASes (complex preparations Coenzyme Compositum®, Ubichinon Compositum®) in ultra low concentrations (“Heel”, Germany).
The object of the study were initial human lymphocytes, which are obtained from the blood of 20 adults, 10 men and 10 women, aged 30 to 64 years, without obvious clinical symptoms of disease, serious chronic pathologies and harmful habits. Blood sampling was carried out from the cubital vein into tubes with anticoagulant EDTA (K2). Preparation of primary blood lymphocytes were performed using a sedimentation method [
Obtained pool of primary lymphocytes from one donor (1.5 ml suspension) was divided into three equal portions of 0.5 ml which were added to 12 ml of the above medium and cultured as the negative control culture (K−), positive control culture (K+) and experimental culture. At the expiration of the six-hour cultivation in the culture of K+ and experimental culture of primary lymphocytes were added 0.5 ml of 448 mM solution of NaN3 in PBS without Ca2+ and Mg2+, and 0.5 ml of PBS without Ca2+ and Mg2+ into culture K. After 24 hours of culturing, from the moment of receiving primary lymphocytes, to the experimental culture of lymphocytes was added 1 ml investigated complex of BAS diluted in a 0.9% solution of NaCl. Simultaneously, to the cultures K+ and K− was added 0.9 ml 1% solution of NaCl. The final concentration of BAS in the respective cultures media of primary lymphocytes was 3 × 106/ml, NaN3 16 mM (
Name of the substance | The final concentration of substances in the culture medium (10−12 mol) | Name of the substance | The final concentration of substances in the culture medium (10−12 mol) |
---|---|---|---|
Coenzym A Ascorbic acid Thiamine hydrochloride Riboflavin sodium phosphate Pyridoxine hydrochloride Nicotinamide Cis-aconitic acid Сitriс acid Fumaric acid α-Ketoglutaric acid Malic acid Succinic acid Barium oxalsuccinate Sodium diethyl oxalacetate Sodium pyruvate Cysteine Sulfur copy | 0.47 223.11 113.20 106.89 191.18 321.75 2.05 1.86 3.08 2.45 2.66 3.03 0.01 170.06 3.25 294.68 11.25 | Adenosintriphosphat-dinatrium NAD Magnesium hydrogen phosphate Magnesium orotate dihydrate Cerous oxalate α-Lipoic acid Ubidecarenone Berberine Lactic acid Hydrochinon Hexaketocyclohexane octahydrate Antraqinone Naphthoqinone p-Benzoquinone Acetylsalicylic acid Histamine Magnesium D-gluconate-dihydrate and other | 0.01 0.55 236.88 96.39 0.55 173.28 0.01 40.43 396.43 0.32 0.01 0.01 0.02 0.03 0.01 0.01 0.01 |
aThis complex of biologically active substances in composition corresponds to the drug Ubiquinone compositum® and Coenzyme compositum® (Biologische Heilmittel Heel GmbH, Baden-Baden, Germany).
a result it was formed K−, K+ and experimental cultures next compositions (
Immediately before adding of the investigated complex BAS in experimental culture of primary cells and 0.9% NaCl solution to the control cultures and also after 4, 24, 48 and 72 hours after that, we obtained lymphocytes suspensions. Last were obtained by triple washing from the culture medium by PBS without Ca2+ and Mg2+ (centrifugation at 1200 g for 10 minutes.). In lymphocytes, using cytochemical methods of analysis, were determined the activity of key enzymes of energy metabolism-succinate dehydrogenase (SQR; EC 1.3.99.1) and mitochondrial glycerol-3-phosphate dehydrogenase (GPD2; EC 1.1.99.5) [
For carrying out cytochemical investigation we prepared smears of primary lymphocyte from suspensions obtained at different stages of the experiment. For this, in 3 µl of primary lymphocytes suspension were added 2 µl of authentic plasma. Received suspension mixture of primary lymphocytes and plasma authentic applied as small drops on a microscope slide. Touching upon drops, using cut glass and putting it at an angle of 45˚, we made smears.
Fixation of smears preceded air drying of them over 2 hours to reduce the solubility of cellular components. Dried smears were fixed for 30 seconds in 60% aqueous acetone solution saturated with EDTA. 60% aqueous solution of acetone saturated with EDTA were prepared by mixing 60 ml of acetone and 40 ml of water, then added EDTA to the termination its dissolvng. Fixed smears were washed with distilled water and dried in air at room temperature. Obtained smears were used to determine the activity of SQR and GPD2.
Incubation of lymphocytes to determine the succinate dehydrogenase. SQR activity was determined by cytochemical method. Smears of primary lymphocytes were incubated at 37˚C for 1 h in the medium of the composition described in
Incubation of lymphocytes for determining glycerol-3-phosphate dehydrogenase. As GPD2, SQR activity was determined by cytochemical method. Smears primary lymphocytes were incubated at 37˚C for 1 h in the medium of the composition described in
All smears that were incubated for determining the activity of SQR, and for determining the activity of GPD2,
Characteristics of the culture | Medium of cultivation (RPMI-1640, FBS, L-glutamine, penicillin and streptomycin), ml | Suspension of primary lymphocyte (3 × 106 cells/ml), ml | Solution of NaN3, (concentration 448 mM ), ml | PBS, ml | Complex of biologically active substances, ml | Solution of NaCl, (0.9%), ml |
---|---|---|---|---|---|---|
Culture of negative control (K−) Culture of positive control (K+) Experimental cultures | 12 12 12 | 0.5 0.5 0.5 | - 0.5 0.5 | 0.5 - - | - - 1.0 | 1.0 1.0 - |
Component | Quantity, ml | Quantity, mg |
---|---|---|
1/15 М solution of potassium dihydrogen phosphate 1/15 М solution of sodium dihydrogen phosphate EDTA p-nitrotetrazolium violet sodium succinate dibasic hexahydrate | 8 32 - - - | - - 11 11 340 |
Component | Quantity, ml | Quantity, mg |
---|---|---|
1/15 М solution of potassium dihydrogen phosphate 1/15 М solution of sodium dihydrogen phosphate EDTA p-nitrotetrazolium violet α-glycerol phosphate disodium salt hydrate | 8 32 - - - | - - 11 11 630 |
were washed with flowing water for 5 minutes and rinsed with distilled water. We carried out additional coloration of lymphocyte nuclei by 0.5% Janus green B solution for 5 seconds to relieve identification of the lymphocytes. Smears were washed with running water for 5 minutes and rinsed with distilled water. Smears were dried on the air. Cytochemical activity determination SQR and GPD2 based on the formation of formazan grains (purple granules) of p-nitrotetrazolium violet, which acts as a hydrogen acceptor from substrates, that fave been oxidated (sodium succinate dibasic hexahydrate for SQR and -glycerol phosphate disodium salt hydrate for GPD2). To determine the activity of SQR and GPD2 used cytochemical basic principle-the activity of these enzymes in the respective incubated cell is proportional to the square of formazan grains in it. Area of formazan grain was recorded using a light microscope AxioLab, A1 (Carl Zeiss, Germany ) and camera AxioCam ERc5s (Carl Zeiss, Germany ). Each smear was analyzed by 34 lymphocytes. These images are processed by a computer program UTHSCSA Image Tool, version 3.0 (The University of Texas Health Science Center, San Antonio , Texas , USA ). In each lymphocyte area of formazan grains was measured. As a result, we received the sum of 34 cells from a smear in mm2. After this, we calculated the enzyme activity (SQR or GPD2), which numerically differed by the average area of formazan grains in one cell. Activity was expressed in mm2/cell/hour.
To estimate statistical significance of changes in the experimental samples, in comparison to the control, as K+ and K− used nonparametric sign test G. The critical level of significance for statistical criteria taken, so, that, equals p < 0.05 [
One approach that provides the most objective picture of the state aerobic energy metabolism link are cytochemical methods of research activity of its enzymes in lymphocytes in vitro. They makes possible to evaluate the activity of enzymes in holistic cells, and mitochondrial enzymes, as in our case, in mitochondria, not destroying it. Because of this, cytochemical research techniques are highly sensitive, specific, informative, but most importantly the objectivity of the results [
In the experiment, for modeling of disorders in energy metabolism, six hours after obtaining of primary lymphocytes, in experimental culture and culture K+ NaN3 (final concentration of 16 mM ) was added. As is well known, this compound inhibits the complex IV work of the respiratory chain in mitochondria, which leads to dysfunction of the whole cell energy metabolism.
In our research, at the initial stage of the experiment, after 18 hours of cultivation in the presence of NaN3, lymphocytes of the cultures K+ (
To identify the effect of the investigated complex of BAS on the energy exchange, this complex was added to the experimental culture of lymphocytes that have been exposed NaN3. As a result, after only 4 hours of cultivation
activity of SQR in experimental lymphocyte culture increased lymphocyte activity to a level K−, thus significantly exceeded the SQR activity of culture K+ by 2.6-times (
A similar picture was also observed after 24-hour exposure to of the investigated complex of BAS. SQR activity of lymphocytes in the experimental samples was at the level of lymphocytes K− SQR activity, thus exceeding this index of cultures K+ by 2.5 times. However, as seen in
Changes in SQR activity were followed by no less significant dynamic activity of GPD2 Thus it is necessary to note that NaN3 can substantially activate mitochondrial GPD2. At the initial stage of the experiment, after 18 hours of cultivation in the presence of NaN3, lymphocytes cultures K+ (
After adding into the experimental lymphocytes culture of the investigated BAS in ultra low concentrations, there was a significant inhibition of GPD2. As a result, after only 4 hours of cultivation, the activity of GPD2 lymphocytes in experimental culture decreased to the level of activity in lymphocytes K− and became lower by 1.6 times than activity of GPD2 culture K+ (
Even more significant changes in the activity of GPD2 lymphocytes were identified at the end of 24 hour cultivation in the presence of ultra low concentrations of the experimental BAS. At this stage of the experiment, the activity of GPD2 lymphocytes experimental culture was 2.2 times lower than in cultures K+ (
As it is known, SQR and GPD2 catalyze some of the most important reactions of aerobic energy metabolism link. Thus SQR in the citric acid cycle catalyzes the reversible oxidation of succinic acid to fumaric acid. The electrons are transferred from the SQR directly to the respiratory chain complex II, and then on coenzyme Q, which makes SQR a major enzymes of energy exchange. Mitochondrial GPD2 is the main component of the glycerophosphate shuttle transfer mechanism of recovered equivalents from cytosol NADH to the mitochondrial FAD [
Conducted researches have allowed to reveal significant change in the activity of SQR and GPD2. At the initial stage of the experiment, the inhibitory effect of NaN3 on aerobic respiration was realized through substantial reduction of SQR activity with parallel activation of GPD2. These changes were observed in the K+ culture and experimental culture after 18-hour cultivation with NaN3, before entering to the last of the experimental BAS. Of course, such changes in the activity of enzymes in aerobic respiration were adaptive-compensatory response. Reduced SQR activity in response to the impaired function of cytochrome C oxidase by azide undoubtedly wore a compensatory character. It is known that the normal functioning of all the components of the respiratory chain cannot be performed unless it is accompanied by phosphorylation of ADP. That is why, it is logical to inhibit SQR in response to an impaired function of cytochrome C oxidase in the initial stage of the experiment (
Adding to the experimental lymphocyte culture of the investigated complex BAS and their subsequent cultivation for 4 hours led to significant changes in the activity of SQR and GPD2. Thus in the experimental lymphocyte culture has been rapid increase of SQR activity on the background of simultaneous inhibition of GPD2 (
This pattern was preserved in the next phase of the experiment. 24-hour of experimental lymphocyte culture cultivation with the investigated complex of BAS has led to stabilize SQR and GPD2 activity to the level K−. At the same time, in spite of significant excess activity of SQR in experimental culture, SQR activity in the culture K+ had tendency for the relative decline in the first (
Several other changes SQR activity was identified by us in the 48-hour action on experimental lymphocytes culture of the investigated complex BAS used in ultra low concentrations. Although, as in the previous stage of the experiment, the activity of SQR experimental culture exceeded that index in culture K+, but it was significantly lower than SQR activity of culture K− (
At the final stage of the experiment (72-hour incubation of experimental culture with a complex of BAS) observed similar changes in the activity of the investigated enzymes, as in the previous stage. SQR activity in experimental cultures exceeded this index in culture K+, but at the same time the SQR activity was lower than in culture K− (
So carried out researches have allowed to reveal the growth of SQR activity in the experimental lymphocyte culture relatively culture K+ during the entire experiment. At the same time, activity of the main of enzyme glycerophosphate shuttle mechanism-mitochondrial GPD2 in the experimental lymphocytes culture is significantly lower relative to the corresponding index in K+ culture. All of this suggests powerful energotropic effect of ultra low concentrations of experimental BAS in the lymphocyte aerobic element of energy exchange. It is also important to note relative decreasing of this effect starting from 48-hour incubation of experimental culture with the complex of BAS. This is evidenced by decreased activity of SQR in the experimental lymphocyte culture, relative to the corresponding index culture K−.
So we have found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro, can effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link. Certainly, a hard research work should be done for understanding the mechanism of ultra low doses action of different substances and the chosen BAS complex in particular. We hope that our research will shed light on solving the fundamental question, namely how such ultra low doses substances can lead to significant biochemical, physiological, immunologic and other effects.
Sergii V. Girin,Iryna V. Savinova,Iryna V. Antonenko,Natalia V. Naumenko, (2016) The Effect of Ultra Low Concentrations of Some Biologically Active Substances on the Aerobic Respiration. CellBio,05,1-13. doi: 10.4236/cellbio.2016.51001