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Abstract: Encephalic damage and disorders of the hepatobiliary system occur against the background of metabolic disorders. Metabolic processes largely depend on the activity, as well as the qualitative and quantitative characteristics of sirtuin-type proteins. Protein deficiency can be filled by artificially introducing them into the body through dietary supplements.
The aim of the study is to analyze the possibilities of using the dietary supplement OYOX for the treatment and prevention of encephalic damage and disorders of the hepatobiliary system.
Materials and methods. We used theoretical and practical research methods. In particular, the methods of analysis of literature sources, statistical data, results of clinical and preclinical studies were used. The method of generalization, analysis, systematization, classification of the obtained data was used.
Results and discussion. Deficiency of proteins of the sirtuin type negatively affects both the state of individual metabolic processes and the state of the body as a whole. Proteins SIRT1, SIRT3, SIRT6 are closely interconnected and their mechanism of action is largely intertwined. One of the methods for activating sirtuins and replenishing their deficiency in the body is the OYOX dietary supplement, which activates the production of sirtuins. A multicenter, open, randomized study of the efficacy and reliability of clinical effects, safety, immunogenicity of OYOX in humans was carried out according to a voluntary trial protocol. The results were evaluated 3 and 6 months after taking the dietary supplement. In the group of people taking OYOX, it was significantly noted: a statistically significant decrease in the percentage of short telomeres (p = 0.029); a decrease in the percentage of senescent cytotoxic (CD8 + / CD28-) T cells (by 1.5%; 4.4%; 8.6% and 7.5% after 3 and 6 months); improvement in lipid and carbohydrate metabolism glycosylated hemoglobin (HbA 1%) 4.9% (p = 0.01), total cholesterol 5.7 mmol / L (p = 0.003) low density lipoprotein (LCL-C) — 3.82 mmol / L (p = 0.0021) homocysteine-3.4 mmol / L (p = 0.001).
Conclusions. Dietary supplement OYOX is effectively used to eliminate the deficiency of sirtuin-type proteins SIR1, SIR3, SIR6.
Key words: orthomolecular composition, nutrigenomic preparation, OYOX, sirtuin-type proteins, SIRT1 replenishment; SIRT3; SIRT6, encephalic damage, diseases of the hepatobiliary system.
Relevance of the topic. Encephalic damage and disorders of the hepatobiliary system are very numerous and are characterized by a fairly high prevalence, especially in countries with a Western lifestyle (Europe, North America, Russia). These diseases are recorded with a frequency of 10-20%. Such a high frequency, in addition to the important contribution of genetic factors, is explained by dietary habits with the use of large amounts of simple carbohydrates. In Africa, Asia and Japan, the prevalence of these pathologies is lower — 3.5-5% .
The ratio of incidence among men and women is 1: 3. In recent decades, there has been an increase in the incidence of diseases of the hepatobiliary system in children and adolescents, an increase in diseases of the encephalic system is observed mainly in old and senile age. Among the various causal factors that contribute to the development of encephalic damage and violations of the hepatobiliary system in childhood, nutritional disorders are of great importance .
The risk of calculus formation in the biliary tract, even with relatively minor nutritional disorders, increases significantly already at an early age. It is no coincidence that balanced nutrition, including breastfeeding, is the cornerstone for preventing the development of disorders of the hepatobiliary system in childhood. Neurodegenerative disorders and various encephalic damage often occur against the background of metabolic disorders .
Research problem. Disorders of metabolic processes largely depend on the activity, as well as the qualitative and quantitative characteristics of sirtuin-type proteins . Deficiency of sirtuin-type proteins is a common cause of metabolic disorders, resulting in severe damage to the brain and hepatobiliary tract. Nevertheless, the deficiency of proteins can be compensated by artificially introducing them into the body through dietary supplements.
TARGETS AND GOALS
The aim of the study is to analyze the possibilities of using the dietary supplement OYOX for the treatment and prevention of encephalic damage and disorders of the hepatobiliary system.
METHODS AND MATERIALS
We used theoretical and practical research methods. In particular, the methods of analysis of literature sources, statistical data, results of clinical and preclinical studies were used. The method of generalization, analysis, systematization, classification of the obtained data was used.
RESULTS AND DISCUSSION
The role of sirtuins in maintaining metabolic processes. Sirtuins (silencer information regulator) are a family of proteins that have received a lot of attention in the last decade due to their central regulatory role in metabolic homeostasis in lower organisms and mammals. Proteins SIRT1-4 were first discovered in yeast as NAD + -dependent deacetylases, which, through suppression of gene expression, contributed to an increase in cell lifespan .
The subsequent discovery of homologous SIRTUIN (SIRT), a family of proteins in mammalian systems, soon led to the realization that these molecules have a regulatory effect on metabolism, aging, and the pathogenesis of diseases associated with aging . There are 7 representatives of the sirtuin classes in the human genome. SIRT-1, 6, 7 show their functions mainly by directly affecting the nuclear transcription of genes involved in metabolism, although a certain amount of SIRT1 is found in the cytosol. SIRT3–5 are found exclusively in mitochondria and regulate enzymes involved in the cycles of tricarboxylic acids, urea, oxidative phosphorylation, as well as in the production of reactive oxygen species [13, 14].
SIRT2 is cytoplasmic, although in certain situations it can also be found in the nucleus . The ability of sirtuins to influence metabolism and potentially life span is related to the ability of members of the SIRTUIN family to function as protein deacetylases. In addition to this function, SIRT4 can perform ADP-ribosylation of target proteins . Unlike other protein deacetylases, sirtuins require NAD + as a cofactor in the deacetylation reaction . The relationship between NAD +, NADH, and the biological effects of sirtuins has led to the opinion that this family of proteins acts as sensors of energy status .
In the deacetylation reaction carried out by sirtuins, 3 stages can be distinguished:
— hydrolysis of NAD + to ADP-ribose and nicotinamide;
— cleavage of the acetyl group from the lysine residue in the protein and the formation of deacetylated protein;
— transfer of the acetyl group to ADP-ribose with the formation of 2′-O-acetyl-ADP-ribose .
SIRTUIN1 (SIRT1) plays a central role in the regulation of metabolic processes in mammals among the sirtuin family, regulating such cellular processes as changes in chromatin structure and gene transcription, DNA repair and cell differentiation, metabolic homeostasis, inflammation, apoptosis, aging, and circadian rhythm [12, 20].
The variety of its physiological functions is associated with the variety of its substrates. SIRT1 targets are histones and non-histone proteins . The acetylation / deacetylation status of histone proteins determines whether chromatin is available for gene transcription. SIRT1 actively deacetylates a number of histones and facilitates chromatin condensation, regulates the transcription of silent genes .
SIRT1 non-histone substrates are molecules or enzymes that control signal transmission, metabolism, or gene transcription. Their diverse properties and cellular localization allow SIRT1 to play a dual regulatory role in different cellular processes or different phases of a particular process . For example, SIRT1 deactivates the expression of proinflammatory genes by deacetylation of NFκB / p65, but stimulates the anti-inflammatory transcription factors RelB and PGC-1α in acute inflammation .
SIRT1 inhibits apoptosis by deacetylation of p53, but stimulates the synthesis of fatty acids by deacetylation of acetyl-CoA synthase 1 . Specific binding sites allow SIRT1 inhibitors / activators to regulate the intensity of its deacetylase activity, but the availability of NAD + is primary in the activation of SIRT1. The NAD + / NADH ratio determines the movement of SIRT1 between cell compartments; an increase in the NAD + level leads to the activation of SIRT1. The product of NAD + hydrolysis, nicotinamide, inhibits the activity of SIRT1, competing with NAD + for binding sites in the active center .
SIRT1 activity can be controlled by various environmental signals that can alter the availability of NAD + to cells . For example, a state of low energy consumption during CR nutrition or physical exertion can increase the cellular level of NAD +, which stimulates SIRT1 activity . At the same time, the state of high energy consumption, caused, for example, by a diet high in fat or the development of acute inflammatory reactions, decreases the cellular level of NAD +, which, in turn, decreases the activity of SIRT1 .
In addition to the cellular NAD + level, the content and activity of SIRT1 is under the control of complex regulation, which turns on in response to hormonal and environmental signals . This regulation is carried out at different levels and is critical for maintaining an optimal SIRT1 level in response to various environmental stimuli .
In the liver and brain, SIRT1 plays an important role in the regulation of fatty acid metabolism. In particular, SIRT1 regulates lipid metabolism by activating the AMPK / LKB1 signaling pathway . AMPK (AMP-activated protein kinase) regulates lipid metabolism, glucose and cholesterol metabolism in the brain, neuroglia, liver, muscles, and adipose tissue . AMPK is the driver of the expression of nicotinamide phosphoribosyltransferase, which catalyzes the first step of NAD biosynthesis from nicotinamide. SIRT1, deacetylating, activates LKB1 kinase (Serine / threonine kinase 11 or liver kinase B1), which, in turn, activates AMPK . The influence of SIRT1 and AMPK can be mutual. SIRT1 activation stimulates fatty acid oxidation and indirectly activates AMPK . In hyperglycemia, SIRT1-mediated AMPK activation prevents lipid accumulation .
Liver SIRT plays an important role in the regulation of local and systemic metabolic homeostasis. SIRT is activated during negative energy balance, which occurs during fasting and restriction of the energy value of the diet .
In addition to the effect on gluconeogenesis in the liver, lipid metabolism, cholesterol synthesis SIRT promotes insulin production in pancreatic β-cells . In β-cells of the pancreas, SIRT regulates glucose-stimulated insulin secretion by affecting the synthesis of uncoupling protein 2 (UCP2).
UCP2 promotes longevity by enhancing the use of fatty acids as energy sources. SIRT1 inhibits UCP2 transcription by binding to its promoter . It was noted that transgenic mice with excess SIRT1 in pancreatic β-cells are characterized by lower levels of UCP2 and increased insulin secretion . SIRT1 promotes inhibition of adipogenesis and differentiation due to its combination with the transcriptional activator PPARγ, which regulates fatty acid metabolism, glucose metabolism and is considered one of the main regulators of adipocyte differentiation .
SIRT1 suppresses PPAR in white adipose tissue, thereby inhibiting the expression of adipose tissue markers such as adipocyte Protein 2, which promotes lipolysis and immobilization of fatty acids in response to caloric restriction. Another way of modulating lipolytic activity of SIRT1 in adipocytes involves deacetylation of FOXO1 and stimulation of transcription of adipocyte triglyceride lipase (ATGL) genes . Genetic elimination of SIRT1 from adipose tissue leads to increased obesity and insulin resistance .
Treatment of mice on a high-fat diet with resveratrol activates SIRT1 in cells directly or indirectly . These results show that SIRT1 acts in conjunction with transcription factors such as PPARγ, whose endogenous ligands are a number of lipid substances. This results in a change in gene transcription in white adipose tissue in response to changes in nutrient levels. It has been shown that impaired SIRT1 functioning in adipose tissue in ADIPO-H363Y mice leads to insulin resistance and hyperglycemia, dyslipidemia, but metabolic changes are restored with caloric restriction .
In addition to influencing glucose homeostasis and fat metabolism, SIRT1 interacts with and deacetylates key factors involved in stress responses, including the forkhead family of transcription factors (FOXO), by modulating their transcriptional activity. FOXO is a family of transcription factors that are important in the response to stress and cancer, since they are involved in cell cycle arrest, DNA repair, and apoptosis .
In response to oxidative or genotoxic stress, FOXO proteins are translocated from the cytoplasm to the nucleus, where they activate genes involved in cell cycle regulation, DNA repair, and apoptosis . Acetylation of FOXO decreases its binding to DNA and increases its phosphorylation and inactivation . SIRT1 deacetylates FOXO proteins, facilitating the transcription of FOXO targets involved in stress resistance and a decrease in the transcription of apoptosis genes .
For example, FOXO3, deacetylated by SIRT1, inhibits the induction of apoptosis after cellular stress and induces cell cycle arrest. Therefore, the combined action of SIRT1 and FOXO promotes survival after oxidative stress by inducing DNA repair .
SIRT1 has been identified as an important repressor of inflammation in many tissues / cells, including macrophages . For example, in mice with moderate overexpression of SIRT1, the inflammatory response is suppressed, while for the whole organism, SIRT1 deficiency induces systemic inflammation on a diet high in fat [10–12]. In addition, removal of SIRT1 in hepatocytes leads to an increase in local inflammation in the presence of a high dietary fat content .
Several recent studies show that the beneficial effect of SIRT1 on metabolic disturbances is due in part to its ability to suppress the activity of NF-kB, a major regulator of the cellular inflammatory response in macrophages . It was shown that SIRT1 deacetylates the RelA / p65 subunit in NF-kB lysine, which leads to a decrease in the transcriptional activity of NF-kB, thereby reducing the production of proinflammatory cytokines and the expression of antiapoptotic genes .
Thus, moderate overexpression of SIRT1 in mice leads to a decrease in NF-kB activity , while SIRT1 knockout increases the secretion of tumor necrosis factor . Keeping mice on a high-fat diet induces degradation of the SIRT1 protein in adipose tissue, which occurs with the participation of inflammation-activated caspase-1 .
SIRT1 plays a role in the hypothalamo-pituitary axis as one of the regulators of energy metabolism. It has been shown that CR and a temporary decrease in energy consumption enhance the expression and activity of SIRT1 in the hypothalamus . In mice lacking SIRT1 in the brain, specific defects in the cells of the anterior pituitary gland, changes in pituitary signaling and motor activity in response to CR are revealed , while activation of SIRT1 in transgenic mice leads to an increase in the activity of hypothalamic neurons .
These results indicate that SIRT1 in the brain can function as a link between hypothalamic pituitary hormones and metabolic status. In the hypothalamus, neurons expressing proopiomelanocortin (POMC), which have anorexogenic activity, and neurons expressing agouti-linked protein (AgRP), are the main regulators of energy supply and expenditure . POMC neurons produce satiety peptides, thereby discouraging excess food intake, while AgRP neurons promote food intake in response to fasting and CR.
SIRT1 appears to exhibit different functions in these two populations of neurons. On the one hand, inhibition of hypothalamic SIRT1 activity increases FOXO1 acetylation, which increases POMC and decreases AgRP expression, thereby decreasing food intake and decreasing body weight gain. In accordance with these observations, AgRP neuron-specific destruction of SIRT1 decreases AgRP activity of neurons, which, by facilitating the effect of an inhibitory signal on POMC neurons, in turn leads to a decrease in food intake and a decrease in body weight .
On the other hand, the specific removal of SIRT1 in POMC neurons in mice causes a weakened response to leptin signaling and a decrease in energy expenditure, which leads to an increased risk of diet-induced obesity .
Although the physiological significance of the various functions of SIRT1 is still not clear, research evidence supports that SIRT1 is an important element in the peripheral and central feedback circuits that mediate the normal response to nutrient absorption. In general, it is believed that SIRT1 activity is important in the central regulation of nutrient sensitivity. Thus, it can be assumed that SIRT1 mediates the effects of CR on the body.
SIRT1 acts as a cellular energy sensor, and this determines its central role in the control and modulation of metabolic processes during changes in dietary habits. As one of the most important regulators of cellular and systemic energy homeostasis, SIRT1 may be of interest as a control link, the influence of which will allow changing metabolic processes, simulating the effects of calorie restriction, and influencing the key pathogenetic links of many diseases, including those associated with aging.
Possibilities of activating and replenishing the deficiency of sirtuin-type proteins by means of a dietary supplement OYOX. Deficiency of sirtuin-type proteins negatively affects both the state of individual metabolic processes and the state of the body as a whole. Proteins SIRT1, SIRT3, SIRT6 are closely interconnected and their mechanism of action is largely intertwined. However, a number of effects associated with the deficiency of each individual type of protein can be distinguished separately.
SIRT1 deficiency significantly reduces the rate of cell division, reduces the rate of epigenetic mechanisms, which is reflected in the slowing down of repair processes, a decrease in the rate of damage healing, and a decrease in tissue regeneration. The body’s ability to turn off inactive genes and repair DNA damage is significantly reduced. At the same time, the number of free radicals sharply increases, oxidative stress develops, which leads to premature aging and cell / tissue death. Also, a deficiency of this type of protein can lead to the suppression of the transcriptional activity of the tumor suppressor p53, as a result of which the processes of cell apoptosis are disrupted and tumor growth occurs. The protein’s ability to deacetylate HSF1 decreases, as a result of which the risk of cell death from heat shock increases, the amount of misfolded proteins accumulates.
The number of stress-resistant proteins decreases, as a result of which the cell cycle is suspended, and the amount of reactive oxygen species is significantly reduced. This increases the risk of developing oxidative stress, leading to premature cellular aging. Also, against the background of protein deficiency, the concentration of glucose in the blood can significantly increase, in connection with which the risk of developing diabetes mellitus and metabolic disorders increases sharply .
Deficiency of SIRT3 proteins is associated with disruption of the neural network and weakening of the signaling cascade, disruption of metabolic processes in the neuroglia, which increases the risk of neurodegenerative diseases. Deficiency of this protein is often associated with Alzheimer’s disease. Also, the ratio of AMP / ATP in cells is disrupted, as a result of which all processes of oxidative phosphorylation are disrupted, the rate of ATP synthesis decreases. Accordingly, the energy potential of the cell decreases, metabolic processes are disrupted. Against the background of protein deficiency, inhibition of the tricarboxylic acid cycle regulators occurs, oxygen metabolism is impaired .
Deficiency of SIRT6 proteins leads to impaired immune response, resulting in an increased risk of developing infectious diseases, an increased risk of malignant cell transformation, an increased likelihood of oxidative stress, which leads to premature aging. In addition, against the background of a deficiency of this type of proteins, lipid metabolism is disturbed, the risk of atherosclerosis and metabolic disorders increases .
One of the methods for activating sirtuins and replenishing their deficiency in the body is a dietary supplement OYOX. By their function, sirtuins are control proteins that regulate the sequence of synthesis and the activity of other proteins, which provide the cell with the ability to adapt to various kinds of tasks, factors and stresses. Sirtuins correctly reject molecules, even with insignificant errors, thereby achieving the correct and, more importantly, sequential synthesis of protein molecules. The flawless work of cellular processes leads to a balanced work of the systems of the whole human body at all levels.
OYOX activates the production of sirtuins, replenishing their deficiency, and prevents the development of various pathologies associated with age-related intracellular disruptions and disorders. Having achieved balance, the human body is capable of producing optimal levels of neurotransmitters, enzymes, hormones. Ready for physiologically normal regeneration of tissues, collagen, elastin and flawless functioning of the body as a whole, characteristic of a young age.
OYOX has undergone repeated testing on laboratory models and clinical studies — in contrast to many dietary supplements. OYOX has been convincingly proven to be highly effective and safe. By creating OYOX, DANDA Pharma united the world’s leading R&D centers, modern pharmaceutical and biotechnological production. As a result, the work of scientists was crowned with the invention of a unique method of producing substances, due to which their activity increases. This is how the key element of OYOX was born, the unique molecule «CYC-8», which supports protein synthesis and metabolic rate. In addition, DANDA Pharma has implemented artificial intelligence technologies to further process data from clinical trials to determine the optimal course of OYOX and analyze long-term use.
The effectiveness of the use of a dietary supplement OYOX for the treatment and prevention of encephalic damage and disorders of the hepatobiliary system. A multicenter, open, randomized study of the efficacy and reliability of clinical effects, safety, immunogenicity of OYOX in humans was carried out according to a voluntary trial protocol. Natural activator SIRT1; SIRT3; SIRT6 as part of a health maintenance program .
The aim is to evaluate the clinical effects of OYOX. Within the framework of this study, the telomere length was determined. Lymphocytes and granulocytes were used as research material. The FlowFISH method was used. The main attention was paid to the study of biological indicators of aging processes. The subjects underwent a blood test with a detailed study of the leukocyte formula, immunogram. Additionally, the functional state of the kidneys, liver, endocrine and cardiovascular systems, bones and skin was assessed. Among the biochemical indicators, special attention was paid to determining the level of cholesterol and glucose. The study involved 980 people aged 30-87 years, of whom 58% were men, 42% were women.
During the study, the participants were divided into 2 groups: experimental and control. The experimental group took OYOX, the control group took a placebo. Comparative results of the study carried out before the start of taking the drug and after 3 and 6 months are presented in tables 1 and 2.
Table 1. — Results of the OYOX study (before priming and after 3 months)
|Before taking||After 3 months||Before taking||After 3 months|
|measurement of telomere length. kb||4,4||5,4||10,1||9,4||4,8||4,7||4,8||4,5|
|CRB mg / l||4,3||4,8||3||2,9||4,4||4,9||4,5||4,8|
|total cholesterol. mmol / l||5,2||5||4,8||4,6||5||4,6||5,3||4,9|
|LPNV. mmol / l||2,8||3,1||2||2,2||2||2,4||2||2,3|
|HDL. mmol / l||0,7||1||1,5||1,7||0,9||1,9||1||1,9|
|glycosylated hemoglobin (HbAlc.%)||5,8||5,7||4,6||5,1||5,9||5,6||5,8||5,8|
|creatine μmol / l||98||96||80||77||98||105||100||101|
|total bilirubin. μmol / l||10,8||12,2||9,6||9,8||11,3||11,9||11,2||11,5|
|ALT U / L||23||26||20||19,2||28||31||28||31|
|AST U / L||29||30||28||20||23||28||24||27|
Table 2. — Results of the OYOX study (before priming and after 6 months)
|Before taking||After 3 months||Before taking||Before taking|
|measurement of telomere length. kb||7,9||8,1||12,3||10,4||7,3||7,9||7,5||7,2|
|CRB mg / l||4,2||4,1||2,5||2,9||4,3||3,8||5,4||2,6|
|total cholesterol. mmol / l||5,4||5,8||4,8||5||5,9||4,9||6||5,4|
|LPNV. mmol / l||2,8||2,6||2||1,8||2,8||1,9||3||1,8|
|HDL. mmol / l||1,3||1,4||2||1,8||1,5||1,6||1,4||1,3|
|glycosylated hemoglobin (HbAlc.%)||5,8||5,6||4,9||5||5,3||5,9||5,5||5,4|
|creatine μmol / l||100||98||90||85||112||99||111||102|
|total bilirubin. μmol / l||12,8||10,7||10,2||9,4||11,6||10,9||11,8||10,2|
|ALT U / L||25||19||22||16||22||28||23||27|
|AST U / L||22||21||19,5||20||25||26,7||25,2||27,1|
Thus, in the group of people taking OYOX, it was reliably noted:
— Statistically significant decrease in the percentage of short telomeres (p = 0.029);
— Reduction in the percentage of senescent cytotoxic (CD8 + / CD28-) T cells (by 1.5%; 4.4%; 8.6% and 7.5% after 3 and 6 months);
— Improvement of lipid and carbohydrate metabolism indicators glycosylated hemoglobin (HbA 1%) 4.9% (p = 0.01), total cholesterol 5.7 mmol / L (p = 0.003) low density lipoprotein (LCL-C) — 3, 82 mmol / L (p = 0.0021) homocysteine-3.4 mmol / L (p = 0.001).
Among the various causal factors that contribute to the development of encephalic damage and disorders of the hepatobiliary system, nutritional disorders are of no small importance. Neurodegenerative disorders and various encephalic damage often occur against the background of metabolic disorders. Disorders of metabolic processes largely depend on the activity, as well as the qualitative and quantitative characteristics of sirtuin-type proteins. Protein deficiency can be filled by artificially introducing them into the body through dietary supplements.
We have analyzed the possibilities of using the dietary supplement OYOX for the treatment and prevention of encephalic damage and disorders of the hepatobiliary system. In the liver and brain, sirtuins play an important role in the regulation of fatty acid metabolism. In particular, they regulate lipid metabolism by activating the AMPK / LKB1 signaling pathway. Deficiency of proteins of the sirtuin type negatively affects both the state of individual metabolic processes and the state of the body as a whole. Proteins SIRT1, SIRT3, SIRT6 are basic, they are closely interrelated and their mechanism of action is largely intertwined.
One of the methods for activating sirtuins and replenishing their deficiency in the body is a dietary supplement OYOX. The drug activates the production of sirtuins, replenishing their deficiency, prevents the development of various pathologies associated with age-related intracellular disruptions and disorders. A multicenter, open, randomized study of the efficacy and reliability of clinical effects, safety, immunogenicity of OYOX in humans was carried out according to a voluntary trial protocol.
The results were evaluated 3 and 6 months after taking the drug. There is an improvement in lipid and carbohydrate metabolism indicators glycosylated hemoglobin (HbA 1%) 4.9% (p = 0.01), total cholesterol 5.7 mmol / L (p = 0.003) low density lipoprotein (LCL-C) — 3, 82 mmol / L (p = 0.0021) homocysteine-3.4 mmol / L (p = 0.001). The decrease in systolic and diastolic blood pressure of the dynamics was 17.1-4.2 mm Hg. (p = 0.006 and 0.002, respectively).