Lymph flow, ionic and biochemical indicators of lymph and blood during hypoxia

Fluxo linfático, indicadores iônicos e bioquímicos da linfa e do sangue durante a hipóxia

Abstract

In this study, the biochemical parameters and physico-chemical reactions of the body in experimental hypoxia, using a Sprague Dawley Rat Model. Hypoxia changed the dynamics and biochemical parameters of blood and lymph, as well as urine. During hypoxia, there was a change in the osmotic resistance of erythrocytes.

Hypoxic training was conducted in a hypoxic animal chamber for 15 days and 30 days for 40 minutes every day. Physical and chemical parameters of blood, lymph and its morphological composition were studied on a hematological analyser, oxygen tension and pH of blood and lymph on an OPTI CCA-TS2 Blood Gas and Electrolyte Analyser.

The value of osmotic pressure in the lymph changed slightly from 280.22 ± 2.07 to 293.3±3.1 and 285.6 ± 2.8 mOsm/l, respectively, 15 and 30 days of hypoxia. Urine osmotic pressure decreased by 15.1-10.4%, respectively, compared to the control group. After 15 and 30 days of hypoxia, ion exchange in the blood plasma showed a decrease in the concentration of K⁺, Cl^- ions and an increase in the concentration of Na⁺ ions in the blood plasma and lymph. Ca²⁺ concentrations decreased in blood plasma and increased in lymph and urine. The analysis of the osmotic resistance of erythrocytes showed its decrease. Lipid peroxidation of erythrocyte membranes showed a significant increase in the level of malondialdehyde and diene conjugates by 52.2% and 69.6%, as well as a decrease in the activity of superoxide dismutase and catalase by 32% and 29.7%.

Hypoxia leads to a decrease in erythrocyte resistance and lipid peroxidation in experimental animals. Shifts in pH on the side of acidosis and disturbances in physico-chemical properties in the blood and lymph were detected. As a result of developing hypoxia in the body, structural and functional rearrangements occur in the whole blood of experimental animals.

Keywords:
blood; hypoxia; ions; lymph; membranes

Resumo

Neste estudo, foram analisados os parâmetros bioquímicos e as reações físico-químicas do organismo em hipóxia experimental, utilizando um modelo de rato Sprague Dawley. A hipóxia alterou a dinâmica e os parâmetros bioquímicos do sangue e da linfa, bem como da urina. Durante a hipóxia, houve uma alteração na resistência osmótica dos eritrócitos.

O treinamento hipóxico foi realizado em uma câmara hipóxica para animais durante 15 e 30 dias, por 40 minutos diários. Os parâmetros físicos e químicos do sangue, da linfa e da sua composição morfológica foram estudados em um analisador hematológico. A tensão de oxigênio e o pH do sangue e da linfa foram medidos em um analisador de gasometria e eletrólitos OPTI CCA-TS2.

O valor da pressão osmótica na linfa variou ligeiramente de 280,22 ± 2,07 para 293,3 ± 3,1 e 285,6 ± 2,8 mOsm/l, respectivamente, após 15 e 30 dias de hipóxia. A pressão osmótica na urina diminuiu 15,1-10,4%, respectivamente, em comparação com o grupo controle. Após 15 e 30 dias de hipóxia, a troca iônica no plasma sanguíneo mostrou uma diminuição na concentração de íons K+, Cl- e um aumento na concentração de íons Na+ no plasma sanguíneo e na linfa. As concentrações de Ca2+ diminuíram no plasma sanguíneo e aumentaram na linfa e na urina. A análise da resistência osmótica dos eritrócitos mostrou sua diminuição. A peroxidação lipídica das membranas dos eritrócitos mostrou um aumento significativo no nível de conjugados de malondialdeído e dieno em 52,2% e 69,6%, bem como diminuição na atividade da superóxido dismutase e catalase em 32% e 29,7%.

A hipóxia leva a uma diminuição da resistência dos eritrócitos e da peroxidação lipídica em animais experimentais. Foram detectadas alterações no pH em direção à acidose e alterações nas propriedades físico-químicas no sangue e na linfa. Como resultado do desenvolvimento de hipóxia no corpo, ocorrem rearranjos estruturais e funcionais no sangue total de animais experimentais.

Palavras-chave:
sangue; hipóxia; íons; linfa; membranas

1. Introduction

One of the most life-threatening situations is a decrease in the supply of oxygen to vital organs. Interest in the effects of hypoxia is due, on the one hand, to the need to reveal the pathophysiological mechanisms of cardiorespiratory diseases, and on the other hand, to the importance of understanding adaptive changes in the body in response to a decrease in oxygen supply (Lucero García Rojas et al., 2021; Abe et al., 2017), but also to disruption of other functional systems and organs (McGarry, et al., 2018), that affecting the circulatory system (Semenza, 2014). In addition, the human need to explore water and outer space increases the incidence of hypoxia and cerebral ischemia in astronauts and aquanauts (Vernice et al., 2020; Baran et al., 2022). With ischemia, primary acidosis develops and participates in tissue damage (Levine, 1993; Tóth et al., 2020).

Hypoxia has a significant impact primarily on these systems and, accordingly, is a key pathogenetic link in most pulmonary, cardiovascular systems. In all these situations, it is the insufficient supply of oxygen to cells that can become the direct cause of death of the body (Fiskum et al., 2021). The process of adaptation of the body to hypoxia is realized at different levels of organization of the biological system (Rybnikova et al., 2020), and the authors found that systemic hypoxia can blunt oxidative DNA damage and induce cardiomyocyte proliferation (Nakada and Sadek, 2021). It is known from sources that the brain is most sensitive to hypoxia and disorders of its functions during oxygen starvation affect the activity of the most important organs and systems of the body (Peshkova et al., 2020; Hein et al., 2021; Li et al., 2022).

All energy transformations in the body are carried out with the participation of oxygen, therefore the hypoxic state, which changes the ratio between oxygen consumption and energy expenditure, plays an important role in the formation of adaptive mechanisms already at the earliest stages of the development of the animal world (Ramos-Campo et al., 2017; Millet et al., 2016).

Many publications are devoted to the influence of oxygen deficiency on the body of mammals and humans. Thus, during hypoxia, the tidal and respiratory minute volume increases (López-Barneo and Celeste Simon, 2020). An increase in respiratory volume during acute hypoxia is one of the adaptive reactions that promote increased oxygen transport to the tissues of the body (Pouyssegur and López-Barneo, 2016). During hypoxia, an increase in minute blood volume and heart rate, and a rise in blood pressure have been described (Johnson et al., 2007). It is believed that an increase in blood pressure during hypoxia is considered as a result of reflex vasoconstriction of blood vessels (Duennwald et al., 2013). Systemic hypoxia, lasting for 5 minutes and accompanied by a decrease in pO2 in arterial blood on average from 87 to 49 mm Hg (Lyamina et al., 2011), caused a decrease in blood pressure with a simultaneous increase in resistance in the caudal ventral artery (Naeije, 2010; Salameh et al., 2020).

Information about the effect of hypoxia on the lymphatic system in mammals is described. When the arteries in dogs (common carotid and vertebral arteries) are compressed, an increase in blood pressure, an increase in lymph flow, and a narrowing of the thoracic lymphatic duct are found (Abdreshov et al., 2011). According to the authors, pressure shifts in the area of the carotid artery reflexively changes the tone of the blood and lymphatic vessels (Ernens et al., 2017).

A relationship has been established between individual links of hemo- and lymphatic circulation, which is manifested in multidirectional reactions of resistive, capacitive and lymphatic vessels of different regions during the formation of a complex of adaptive reactions to hypoxia (Foskett et al., 2011). It has been established that the human internal carotid artery and rat portal vein cannot perform phasic contractions without extracellular calcium, although they are highly sensitive to hypoxia and hypercapnia. Lymphatic vessels are less sensitive to the content of extracellular calcium and have a higher resistance to the effects of hypoxia than venous and arterial vessels (Irigoyen et al., 2007). Under conditions of hypoxia, complete inhibition of the contractile activity of smooth muscle cells (SMCs) of the thoracic duct occurs after 1-1.5 hours, portal veins after 10-15 minutes, which indicates the great resistance of SMCs of the thoracic duct to a decrease in the partial pressure of oxygen (Wong et al., 2017). Thus, all basic, functional systems are involved in ensuring adaptive reactions to ischemia and hypoxia in mammals and humans.

It should be noted that despite the huge number of works devoted to the physiology and pathology of the cardiovascular and respiratory systems under conditions of acute and chronic forms of hypoxia in the body of mammals (Aalkjaer and Lombard, 1995), data on the state of the lymphatic system during the development of hypoxia are still insufficiently studied and very contradictory (Morfoisse et al., 2015; Becker et al., 2021). There is very little information in the literature about the state of the lymphatic system during oxygen starvation of organs and tissues. There is insufficient information in the literature about lymph flow with low oxygen levels in the blood. It is of interest to study the effect of oxygen deficiency in the inhaled air on the functions of the lymphatic system and water-salt homeostasis in an experiment. In this regard, the purpose of the research was to study changes in hemo-lympho dynamics, ionic and biochemical parameters in rats using experimental models of acute hypoxia.

2. Material and Research Methods

All experiments with animals were conducted in strict accordance with the rules developed and approved by the local Ethical Committee of the Institute of Genetics and Physiology, Protocol No. 12-314 of November 11, 2022, as well as in accordance with the rules of bioethics approved by the European Convention for the Protection of Vertebrates and the guidelines outlined in the European Union Directive, "On the protection of animals used for scientific purposes".

According to the purpose and objectives of the program, 75 adult male rats of the Spraque Dawley (SD) breed weighing 260-270 g were used in the experiments. The rats were fed according to the standard vivarium diet. Rats of group 1 (25 rats) were control. In the 2nd (25 rats) and 3rd group (25 rats) there were experimental (acute hypoxia). The experimental group was exposed to hypoxia for 15 days and 30 days 40 minutes every day. Hypoxia was achieved by supplying nitrogen gas into the box until the oxygen level reached 12-15%. The balloon was attached to the chamber to equalize the pressure of the compressed air, and the pressure inside was kept constant.

2.1. Study subject

An experimental acute model of hypoxia was obtained by once placing rats in a confined space for 1.5 hours/day, with a decrease in the O₂ content in the chamber to 8% by the end of exposure (Abdreshov et al., 2020). To implement the further experiment, a hypoxic cabin measuring 55x80x70 cm, Hypoxia isolation chamber Ox-100 series, manufactured (Shanghai TOW Intelligent Technology Co., Ltd., Shanghai, China, 2023) was purchased and used. The Ox-100 Animal Hypoxia Experimental System can control a continuous hypoxic environment in an animal experimental box to create appropriate hypoxia experimental models. The cabin is designed for experimenting with hypoxia at normal pressure, periodic hypoxia, etc. Sensors were installed in the cabin; it can monitor the concentration levels of oxygen and carbon dioxide, pressure, temperature and humidity and other parameters in real time. During continuous hypoxia experiments, the oxygen concentration uncertainty is 0.1%. The highly sensitive sensor operated in the oxygen concentration range from 0 to 25% (see Figure 1).

Figure 1
General view of the hypoxic chamber manufactured by Taiwan Technology, 2023.

The experiment ended after 15 and 30 days, and the rats were placed in the chamber at the same time every day and the experiments were carried out for 40 minutes, once a day (Kilic et al., 2022). The experimental procedure was continued by the same researcher. Reducing O₂ and maintaining it at the proper level was carried out using a 10L Nitrogen concentrator, and reducing CO₂ in the hypoxic chamber was carried out using a medical soda lime absorber.

At the end of the experiment, on the 15th and 30th days, samples of lymph, blood and urine were taken from rats under ether anaesthesia. Sedation was performed by ether inhalation through a mask. After anesthesia, an incision was made along the white line of the abdominal muscles, and then the thoracic lymphatic duct was dissected at the diaphragm, into which a graduated microcannula was inserted to determine the lymph flow, and lymph was collected for research, as well as arterial blood samples from the abdominal aorta were taken for blood testing. Urine was collected from the bladder of animals using microcannula. All groups of animals were in the same conditions of feeding and keeping in the vivarium.

2.2. Rheological property analysis and biochemical blood test

In all groups of animals, the clotting time of blood and lymph was determined using the generally accepted method according to Sukharev; the determination of the viscosity of whole blood, plasma, lymph and concentrated erythrocyte suspensions was studied using a capillary viscometer (Copley et al., 1959; Baskurt and Meiselman, 2003). Measurement of blood and lymph viscosity was determined with the capillary tube viscosimeter and compared to LS300 (KSP V-4, Rehomed GmbH, Aachen, Germany). Viscosity was determined by estimating the flow time of a known volume of liquid under the influence of applied pressure in a horizontal capillary with a working mouth radius of about 0.3 mm and thermostatically controlled at 37°C. Determination of hematocrit index (Hematocrit index) (Ht) was determined by the generally accepted method: hematocrit capillaries were filled with whole blood and centrifuged for 7 minutes at 12,000 g. Determination of osmotic pressure of lymph, blood plasma and urine was measured using an OMCK-01 osmometer (Russia). The operating principle of the device is based on changing the freezing temperature of a bio fluid depending on the concentration of soluble compounds contained in it. Physical and chemical parameters of blood, lymph and its morphological composition were studied on a hematological analyser SMEX KX-219 9 (Japan) and oxygen tension and pH of blood and lymph on an OPTI CCA-TS2 Blood Gas and Electrolyte Analyser (USA). In animals, the ion content was determined in the blood, lymph and urine electrolytes using an ABL 615/625 analyzer from Radiometer (Germany). Rats were placed in exchange cages with a wire floor to collect 24-hour urine. Determination of biochemical parameters and urine cells was carried out using a CL-50Urine Analyser High Technology (USA). In lymph and blood samples, the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the content of total protein, alkaline phosphatase, urea, creatinine were determined using a standard test system (Abdreshov et al., 2015), in accordance with the attached instructions with further processing of the results obtained on the analyser "COBOS INTEGRA 400" (USA).

2.3. Measurement of osmotic resistance of erythrocytes

The osmotic resistance of erythrocytes was studied according to generally accepted laboratory research methods (Waugh and Asherman, 1938; Penha-Silva et al., 2007), by determining the optical density of haemoglobin solutions resulting from the destruction of erythrocytes in a series of hypotonic sodium chloride solutions, which allows obtaining hemolysis dynamics curves. The osmotic resistance of erythrocytes was determined by the resistance of cells to hypotonic sodium chloride solutions. 25 μl of red blood cells were added to a series of tubes containing 2.5 ml of solutions with different concentrations of NaCl (0.85; 0.75; 0.65; 0.55; 0.50; 0.45; 0.35; 0.15; 0.1), mixed thoroughly and the samples were incubated at 37°C for 30 minutes. Hemolysis was stopped by adding an equal volume of solutions of the appropriate concentrations of sodium chloride required to restore isotonicity. After centrifugation for 5 min at 2000 rpm, measurements were taken in the samples using a spectrophotometer (Spectrophotometer Apel PD-303, APEL, Japan) at a wavelength of 540 nm, and the percentage of hemolysis was calculated using the formula (Walski et al., 2014; Salvagno et al., 2020), taking hemolysis in a sample containing 0.1% sodium chloride solution as 100%. To quantify the osmotic resistance of erythrocytes, we used the osmotic value corresponding to hemolysis of 50% of cells (C50), which is the centre of distribution of erythrocytes according to osmotic resistance.

2.4. Analysis determination of lipid peroxidation

The intensity of lipid peroxidation processes in erythrocyte membranes was assessed by the content of its products - diene conjugates (DC), malondialdehyde (MDA), and catalase and superoxide dismutase activity, in accordance with generally accepted laboratory research methods (Behn et al., 2007; Igbokwe and Igbokwe, 2016; Li et al., 2007). The optical density of the supernatant liquid was measured at a wavelength of 532 nm against distilled water. At high temperatures in an acidic environment, malondialdehyde reacts with 2-thiobarbituric acid, forming a colored trimethine complex with an absorption maximum at 52 nm. The content of thiobarbituric acid, the active products (TBA-AP) of lipid peroxidation, was determined by the fluorometric method (Wasowicz et al., 1993; Jo and Ahn, 1998).

2.5. Statistical analysis

The experimental results were processed by the method of variation statistics on a computer (Microsoft Corporation, Washington, DC, USA) using Student's t-test. The mean ± standard deviation (SD) was calculated and expressed as a percentage. Significance testing was performed using Fisher’s t-test and Student’s t-test to evaluate differences in mean values. The results were considered significant at p<0.01, p<0.05.

3. Results

Lymph flow from the thoracic duct in these animals was significantly lower, almost 1.32 times, compared to intact rats. The decrease in lymph flow from the thoracic duct to the diaphragm, which is 2/3 formed in the liver, is apparently associated with a decrease in blood supply to this organ, although it was below the initial level (as shown in Table 1). At the same time, a fairly high level of pCO₂ content in the blood of rats was noted (ranging from 64.20 ± 3.1^** and 53.61 ± 3.1^*) compared, respectively, to the intact groups. The data obtained indicate the formation of a state of hemic hypoxia in animals when exposed to hypoxia (as shown in Table 1). The results of the studies showed that after hypoxic exposure in experimental rats, there was a significant increase in hematocrit, a decrease in the osmotic stability of erythrocytes and an increase in blood viscosity compared to the control group of animals. The viscosity of whole blood reflects the nature of the intermolecular adhesion of the aqueous phase (plasma) with the components and structures dissolved in it that make up the blood cells, and is the most important characteristic of the state of the internal environment of the body. Thus, if in intact rats the hematocrit was 45.1 ± 1.02, then in experimental rats it was 52.3 ± 1.01 and 50.5 ± 1.04, the viscosity in the lymph increased from 2.6 ± 0.2 to 3 .3 ± 0.3 mPa·s (p<0.01) and, accordingly, blood viscosity increased by 30% (p<0.01).

Table 1
Physico-chemical parameters of blood and lymph in the control group and during hypoxia.

Indicators	Intact animals	Hypoxia 15 days	Hypoxia 30 days
Lymph flow, ml/min	0.31±0.02	0.19±0.01**	0.23±0.01**
рО2 mmHg	85.00±2.1	72.41±3.2*	80.41±3.2
рСО2 mmHg	49.30±1.1	64.20±3.1**	58.61±3.9*
Hematocrit, %	45.1±1.02	52.3±1.01	50.9±1.05
Hemoglobin, g/l	125.4±2.5	118.6±2.9**	122.1±2.3**
Blood, pH	7.40±0.05	7.29±0.03	7.33±0.03
Lymph, рН	7.56±0.08	7.42±0.02	7.49±0.02
Blood coagulation time, s	248±4.1	181±4.2**	201± 4.2*
Lymph coagulation time, s	332±5.7	268±5,7*	293±5,7*
Blood viscosity, mPa·s	4.3±0.1	5.7±0.04**	5.1±0.4*
Lymph viscosity, mPa·s	2.6±0.2	3.3±0.05**	3.0±0.06*

• Notes: Reliable in comparison with the control group.
• ^*
  p <0.05;
• ^**
  p <0.01.

In experiments, after hypoxic exposure in the blood and lymph, a decrease in pH towards acidosis was observed to 7.29 ± 0.03 and 7.42 ± 0.02, respectively. In our experiments, blood clotting in rats of the experimental group was observed within 181 ± 4.2 sec and 201 ± 4.2 sec. (p<0.01), in control group 248 ± 4.1 s., and lymph coagulability shortened by 19.3 and 11.7%, respectively (p <0.05) (as shown in Table 1, see Figure 2).

Figure 2
Shifts in pH, coagulability and viscosity of blood and lymph during acute hypoxia. Designations: along the ordinate axis - osmotic pressure, in mOsm/l, along the abscissa axis - stages of the experiment.

The value of osmotic pressure in the blood plasma changed slightly from 290.0 ± 5.1 to 299.8 ± 2.9 and 296.4 ± 2.1 mOsm/l, and in the lymph from 280.22±2.07 to 293.3 ± 3.1 and 285.6 ± 2.8 mOsm/l, respectively, 15 and 30 days of hypoxia. Urine osmotic pressure decreased by 15.1-10.4%, respectively, compared to the control group (see Figure 3, as shown in Table 2).

Figure 3
Changes in osmotic pressure in blood plasma, lymph and urine in the control group and in rats during acute hypoxia. Designations: along the ordinate axis - aminotransferases, µkat/L.

Table 2
Indicators of lymphodynamics, diuresis and biochemical composition in rats of the control group and after exposure to hypoxia.

Indicators	Intact animals	Hypoxia 15 days	Hypoxia 30 days
Blood plasma
Osmotic pressure, mOsm/l	290.0±5.1	305.8±2.9*	296.4±2.1
Total protein, g/l	65.4±2.4	53.1±1.5*	58.6±1.9
Glucose, g/l	5.4±0.6	7.8±0.8**	7.0±0.2*
ALT, µkat/L	0.14±0.02	0.32±0.03**	0.25±0.09**
AST, µkat/L	0.16±0.02	0.31±0.09**	0.24±0.09**
Alkaline phosphatase, U/L	238±14	327±16**	302±11*
Urea, mmol/L	65.7±2.8	54.6±2.2*	59.9±2.5
Creatinine, mmol/L	88.2±2.9	72.7±3.1*	79.8±3.9
Lymph
Osmotic pressure, mOsm/l	280.2±2.2	293.3±3.1*	285.6±2.8
Общий белок, г/л	41.5±1.5	33.4±1.7*	36.9±1.2*
Глюкоза, г/л	5.9±0.7	7.3±0.3**	6.7±0.3*
ALT, µkat/L	0.12±0.09	0.27±0.06**	0.23±0.06**
AST, µkat/L	0.17±0.02	0.33±0.09**	0.26±0.02**
Alkaline phosphatase, U/L	312 ±15	398 ±19**	369 ±16*
Urea, mmol/L	83.2±3.2	72.9±3.5*	76.4±3.7
Creatinine, mmol/L	69.5±2.9	57.2±3.1*	61.2±3.1
Urine
Diuresis µl/min	2.1±0.04	1.73±0.04*	1.93±0.08
Osmotic pressure, mOsm/l	566.7±15.9	481.6±12.2**	508.3±16.4
Urea, mmol/L	37.2±1.5	53.6±2.8**	49.7±2.4**
Creatinine, mmol/L	21.7±1.6	33.6±3.1**	28.9±2.9*

• Notes: Reliable in comparison with the control group.
• ^*
  p <0.05;
• ^**
  p <0.01.

In the experiments, a decrease in the concentration of total protein in the blood plasma by 18.3-11.2% (p<0.05) and in the lymph by 19.5-11.1% (p<0.05) was observed, respectively, compared to the control group. The observed decrease in the concentration of total protein in the blood and lymph is due to a decrease in the protein-synthetic function of the liver and this led to a decrease in the processes of filtration and resorption, and, consequently, to a decrease in the processes of lymph formation and a decrease in lymph flow from the intestinal lymphatic duct. Blood glucose level in rats under the influence of hypoxia, there was an increase in glucose levels both in the blood and in the lymph by 29.6–13.5%, respectively (p<0.05). Perhaps these dynamics in changes in glucose concentration is due to an additional reaction of the adrenal cortex and pituitary gland to hypoxia, the hormones of which stimulate the synthesis of glycogen in the liver, and carbohydrates, in addition to dietary intake, begin to be synthesized from protein. The content of urea and creatinine in the blood plasma decreased by 8.9-10.1% and in the lymph decreased by 8.2-12%, respectively, and in the urine increased by 33.6-33.1% (as shown in Table 2).

Diuresis in experimental animals under hypoxic conditions, there is a slight change in diuresis compared to intact animals. Diuresis in rats after the 30th day of hypoxia, compared with the control group, was decreased slightly by 8.1%. These indicators after hypoxia for 15 days were 1.73 ± 0.04^* ml/min, i.e. was reduced by 17.6% compared to the control group. Alkaline phosphatase is one of the most common and universal enzymes.

Alkaline phosphatase is an enzyme involved in the transport of phosphorus through the cell membrane and is an indicator of phosphorus-calcium metabolism. An increase in enzyme activity during liver damage occurs due to its release from hepatocytes.

The results show that the activity of alkaline phosphatase in blood plasma increases by 37.4-26.9% and in lymph by 27.6-18.3%, respectively, compared to the control group. It should be noted that changes in parameters (ALT and AST) in lymph were more pronounced than in blood plasma in all experiments. A more pronounced increase in ALT and AST in the lymph can be attributed to more severe damage to lymphatic microvessels compared to blood vessels. On the part of the enzyme system, during hypoxia in rats there was an increase in the levels of the enzymes ALT and AST both in the blood plasma and in the lymph - by 2.3 and 1.9 times, respectively (as shown in Table 2, see Figure 4). The predominance of an increase in the level of the ALT enzyme in the blood over AST indicates more pronounced damage to liver cells and the release of the desmoenzyme ALT into the blood and lymph.

Figure 4
ALT and AST indicators in rats’ blood plasma and lymph during acute hypoxia.

When exposed to hypoxia, a decrease in the osmotic stability of erythrocytes and a change in the activation of lipid peroxidation processes are observed. The erythrocyte membrane undergoes destructive changes under the influence of not only acid haemolytic, but also osmotic ones. The osmotic resistance of erythrocytes reflects the stability of cell membranes. Measuring the osmotic stability of erythrocytes is an important research method not only in in vitro experiments, but also as a diagnostic method in medicine and is used to study the mechanism of pathological processes and the action of certain drugs and biologically active compounds. Using data on the osmotic resistance of erythrocytes, it is possible to assess their physicochemical properties of blood to various influences. As a result of our research, specific features of the osmotic resistance of erythrocytes during experimental hypoxia on the body were discovered. As shown in Table 3 presents data on the state of osmotic resistance of erythrocytes in various concentrations of sodium chloride solution in animals of all observation groups. In the control group of animals, the onset of hemolysis corresponded to a 0.55% sodium chloride solution, erythrocyte lysis exceeded 10%. More than half (53%) of lysed erythrocytes were detected in a 0.45% sodium chloride solution. The onset of maximum hemolysis was observed in 0.40% sodium chloride solution (71%). Maximum (93.5%) hemolysis corresponded to a solution concentration of 0.2% (as shown in Table 3).

Table 3
Indicators of osmotic resistance of erythrocytes in various concentrations of sodium chloride solution in rats of intact and experimental groups are shown, %.

Concentration of NaCl solution	Relative content of lysed erythrocytes in the blood, %
	Intact animals	Hypoxia 15 days	Hypoxia 30 days
0.85	2.21±0.27	3.70±0.53	3.41±0.27
0.75	2.27±0.62	3.64±0.29	3.52±0.91
0.65	2.87±0.49	6.03±0.58**	4.24±0.65**
0.55	10.11±0.65	15.75±1.71*	5.98±0.64*
0.50	25.93±1.51	31.09±1.47*	19.49±1.09*
0.45	53.78±4.18	71.51±4.08**	28.64±2.81**
0.35	82.35±1.09	86.23±1.37	83.97±1.62
0.15	93.76±1.17	95.65±0.24	90.09±1.53

• Notes: Reliable in comparison with the control group.
• ^*
  p <0.05.
• ^**
  p <0.01.

After 15 days of hypoxia, the point of onset of hemolysis shifted towards a higher concentration of sodium chloride solution was 0.65% (percentage of hemolysis corresponded to 6.03, p<0.05). In a 0.55% sodium chloride solution, the percentage of hemolysed erythrocytes increased by 54.8% (p<0.05) compared to the intact group (as shown in Table 3). At the point of 50% hemolysis, the number of hemolysed red blood cells increased compared to the norm by 36.6% (p<0.05) and significantly approached the point of maximum hemolysis (70.05%). This indicates a decrease in the zone of maximum resistance. It can be noted that at points of sodium chloride solution concentration from 0.65% to 0.45%, the percentage of hemolysis exceeded that of the control group (p<0.05 at all points).

After 30 days of hypoxia, completely different results were observed (as shown in Table 3). At the point of minimal resistance (0.55% solution), the percentage of hemolysed erythrocytes was lower compared to the intact group by 40.09% (p<0.05), and decreased even more significantly compared to experimental group 1 - by 65% (p<0.05). After 30 days of hypoxia, the point of minimal hemolysis shifted towards a lower concentration to 0.50% of sodium chloride solution. At the point of 65% hemolysis in the experimental group on the 30th day of hypoxia was 24.8%, which was significantly lower compared to the intact group (by 47.8%, p<0.05).

In sodium chloride solutions with concentrations of 0.50%, 0.45% and 0.40%, there was a significant decrease in the percentage of hemolysed erythrocytes compared to the experimental group after 15 days of hypoxia by 54.8%, 19.9% and 33% and the control by 10%, 25% and 53% according to the concentrations of the solution (p<0.05). In subsequent more hypotonic concentrations of sodium chloride solution, the indicators in all observation groups did not differ significantly. It has been shown that under the influence of 15 and 30 days of hypoxia, common mechanisms are revealed, expressed in a decrease in the parameters of osmotic resistance of erythrocytes.

In experimental hypoxia, POL was activated and antioxidant protection of erythrocytes was disturbed. Metabolic indices were changed in comparison with the norm. In erythrocytes of rats under hypoxia model MDA increased at 15 days by 52.2% and after 30 days’ hypoxia showed an increase by 26.7% compared to control group. The marker of free radical oxidation (diene conjugate) in the blood of animals after hypoxia, showed its increase during the whole time of the study compared to intact animals. Thus, on the 15th day the level of DC increased by 69.6%, on the 30th day on average by 42.8%. Erythrocyte catalase protects hemoglobin by removing more than half of the hydrogen peroxide formed in normal human erythrocytes that are exposed to significant concentrations of oxygen. The study evaluated the antioxidant properties of blood after hypoxia and showed a decrease in catalase, which was lower than the control values by 29.7% in group 2 and by 16.4% in group 3, reflecting the processes of antioxidant defense at the cellular level. The analysis of the study data indicates that the activity of catalase is one of the significant indicators of the activity of the antioxidant system in such a link as antiperoxidant defense (as shown in Table 4).

Table 4
Changes in indicators of lipid peroxidation and antioxidant activity in the membranes of erythrocytes in experimental animals under normal conditions and under hypoxic conditions.

Indicators	Intact animals	Hypoxia 15 days	Hypoxia 30 days
МДА, nmol/ml	0.85±0.009*	1.29±0.008**	1.08±0.001**
Diene conjugates (DC), nmol/ml	0.56±0.07	0.95±0.02**	0.80±0.03*
Superoxide dismutase (SOD), units/mL of red blood cells	27.62±1.25	18.78±1.11**	21.76±1.45*
Catalase, Mcat/L	16.29±0.46	11.45±0.19**	13.61±1.43*

• Notes: Reliable in comparison with the control group.
• ^*
  p <0.05;
• ^**
  p <0.01.

The activity of superoxide dismutase (SOD) was significantly higher in rats after hypoxia by 32 and 21.2%, respectively, compared to the control group. Thus, in comparison with the initial data, a significant increase in the level of malondialdehyde and diene conjugates in the membranes of erythrocytes in the control group of rats was noted by 52.2-26.7% and 69.6-42.8%, respectively, as well as a decrease in the activity of superoxide dismutase and catalase by 32-21.2% and 29.7-16.4%, respectively (as shown in Table 4).

An imbalance in the ionic composition is often provoked by diseases in the whole organism, therefore the content of these microelements in a liquid environment can reflect the functional state of the body under normal conditions and when exposed to extreme factors. In experimental animals in the blood, lymph and urine, the concentration of Na⁺ ions after hypoxia for 15 days increased by 5-7% (in the control, the concentration of ions Na⁺ was 138.7 ± 2.12 mmol/l in the blood, 135 ± 2.11 mmol/l in the lymph /l, in urine – 16.4 ± 1.02 mmol/l), respectively. After 30 days of hypoxia, the number of sodium ions was equal to 143.1 ± 3.24^* mmol/l in the blood, 139.7 ± 3.11^* mmol/l in the lymph, 16.9 ± 0.04 mmol/l in the urine and statistically differed by 3-3.2% from the values obtained in control groups (as shown in Table 5).

Table 5
Content of ions in the blood, lymph and urine during experimental acute hypoxia of sodium ions, mmol/L.

Indicators	Blood	Lympa	Urine
Sodium ions, mmol/L
Control group	138.7±2.12	135.4±2.11	16.4±00.2
Hypoxia 15 days	147.9±3.14*	143.3±3.17*	17.5±0.08
Hypoxia 30 days	143.1±3.24*	139.7±3.11*	16.9±0.04*
Potassium ions, mmol/L
Control group	3.89±0.20	3.63±0.10	3.14±0.10
Hypoxia 15 days	3.19±0.26*	2.90±0.12**	2.83±0.11*
Hypoxia 30 days	3.51±0.02*	3.28±0.12**	2.99±0.11*
Calcium ions, mmol/L
Control group	0.58±0.02	0.407 ±0.03	–
Hypoxia 15 days	0.43±0.08**	0.425±0.05	0.18±0.02**
Hypoxia 30 days	0.50±0.08**	0.423±0.01	0.13±0.02**
Chlorine ions, mmol/L
Control group	107.6±1.90	106.1±1.0	85.4±1.52
Hypoxia 15 days	104.9±1.23	102.6±1.38	81.8±1.48
Hypoxia 30 days	105.3±1.57	104.7±1.72	83.7±1.29

• Notes: Reliable in comparison with the control group.
• ^*
  p <0.05;
• ^**
  p <0.01.

Chlorine ions, along with sodium ions, play an important role in the ionic balance of the body, since they predominate in the extracellular fluid. The ratio of sodium and chlorine in the extracellular fluid is 1.4:1. Chlorine and sodium account for the vast majority of blood plasma ions. The concentrations of sodium and chlorine in the blood, expressed in mmol/l, are strictly related to each other and have practically the same diagnostic value. The concentration of chloride ions in blood, lymph and urine compared to the control group decreased slightly both after 15 days of hypoxia and after 30 days of hypoxia, respectively 2.1%, 1.3% and 2.0% (as shown in Table 5). According to the literature, the concentration of chlorine depends on the bicarbonate content, i.e., on the acid-base balance and about 70% of chlorine is found in the extracellular fluid. A certain amount of it is found in the cells of various organs and tissues, and chlorine is released almost entirely in the form of sodium chloride (Leviel et al., 2010). 46 During the process of ultrafiltration of blood plasma, a liquid similar in composition to the extracellular fluid enters the lumen of the nephrons, in which chlorine and sodium ions predominate, and their reabsorption significantly exceeds the reabsorption of all other filtered substances combined (Velazquez and Wright, 1986; Greger, 2000).

A study of the content of potassium ions showed that in a series of experiments after 15 days of hypoxia, more pronounced changes in the content in the above-mentioned biological fluids were noted; this indicator decreased by 18%, 20.2%, 10%, respectively, both in blood plasma and in lymph and urine (as shown in Table 5). After 30 days of hypoxia, the amount of potassium ions was equal to 3.51 ± 0.02^* mmol/l in the blood, 3.28 ± 0.12^* mmol/l in the lymph and 2.99 ± 0.11^* mmol/l in the urine (p>0.01).

Ca⁺² ions in the blood decreased slightly compared to the initial data, and Ca⁺² ions in the lymph increased slightly. There are no Ca⁺² ions in the urine or only trace amounts. The relative concentration of electrolytes in the urine of rats after 15 and 30 days of hypoxia, the Ca⁺² concentration in the urine increased from 0.18 ± 0.02^** mmol/l and 0.15 ± 0.02^** mmol/l, respectively. After 15 days of acute hypoxia, the content of Ca⁺² ions in the blood decreased, showing 0.43 ± 0.08^** mmol/L, and in the lymph the content of Calcium ions increased by 4.4% (as shown in Table 5).

After hypoxia for 15 days, the concentration of Na⁺, K⁺ and Cl^- ions, respectively, was: 186.7 ± 10.6 mmol/l, 7.9 ± 0.8 mmol/l and 86.3 ± 5.7 mmol/l, with 30 days of hypoxia – 162.2 ± 12.7 mmol/l, 6.71 ± 0.4 mmol/l and 96.8 ± 6.4 mmol/l. As can be seen from the presented data, with 15 days of hypoxia, there was a decrease in the concentrations of K⁺, Ca²⁺, Cl^- and an increase in the concentration of Na⁺ ions, in contrast to the indicators we obtained with 30 days of hypoxia, where there was a tendency for the indicators to return to their original values. The data obtained show significant changes in the ion balance of the blood when exposed to hypoxia on the body in rats. In the plasma, an increase in osmotic pressure was noted with an increase in the first 15 days of hypoxia; its value, in the control, was 287 ± 2.62 mOsm, reaches 299.8 ± 2.9^* mOsm (p<0.05). At the same time, the content of cations in the blood plasma of animals in the control groups, the concentrations of Na⁺, K⁺ and Ca²⁺ were, respectively, 138.7 ± 2.12, 3.89 ± 0.20, 0.58 ± 0.02 mmol/l and the concentrations of Cl^- anions were 107.6 ± 1.90, then in experimental animals observed only an increase in Na⁺ content from 6.6% and 3.2%, respectively, days of hypoxia. At the same time, the ion composition of the plasma changed little: only an increase in the Na⁺ concentration was observed by 4.4, 13.3 and 22.2%. In turn, hypoxia caused a significant decrease in the concentration of K⁺ ions by 18-10%, Ca²⁺ by 26-15% and Cl^- concentration by 2.5-2.1%. In the lymph and urine, all showed similar patterns as in the blood, only the calcium content increased in the lymph by 4.4-3.9% and in the urine by 0.18 ± 0.02^** and 0.13 ± 0.02^** mmol/l, respectively. Due to the fact that 15 and 30 days of hypoxia, the detected change in blood ion balance, apparently, cannot be a consequence of O₂ deficiency in the body.

4. Discussion

In rats, oxygen tension in the blood and organ lymph flow decrease, viscosity increases and the clotting time of blood and lymph accelerates, the pH of the blood and lymph shifts towards acidosis. Leads to a change in microcirculation (Jung et al., 2016), lower hemoglobin levels and increased hematocrit to blood cell ratio result in lower blood viscosity and coagulation (Gregersen et al., 1965), it is logical to a lack of oxygen transport by the organism and a change in the osmotic pressure of organism fluids. The authors showed that oxygen concentration affects the rheological properties and blood flow in two healthy donors, a man and a woman, which indicates compensatory reactions under hypoxia conditions, leading to a decrease in blood viscosity (Valant et al., 2016). The proportion of formed elements in the blood increases as a compensatory reaction of the blood system in response to hypoxia, and this process requires an influx of additional fluid into the blood. Under these conditions, the release of lymphocytes from the lymph nodes into the lymph is associated with a compensatory reaction of the lymph nodes, which supply the bulk of lymphocytes to the blood. According to researchers, lymphocytes enter the bloodstream mainly from lymphoid organs. Yelmen and his colleagues conducted experiments on the effect of (CLTIH) on rheological blood parameters, which was studied in the control group and in CLTIH. The authors found that the effect of (CLTIHH) on blood rheological parameters was higher than in the control group and there was no significant difference in the oxygen delivery index between the groups (Yelmen et al., 2011), it has an erythropoietic effect (Núñez-Espinosa et al., 2014) and chronic intermittent hypoxia had a greater effect on blood rheological properties than chronic continuous hypoxia (Kang et al., 2016). After exposure to intermittent hypobaric hypoxia, hypoxia alone or in combination with cold, significant changes in blood were observed in animals compared to the control group (Santocildes et al., 2021).

One of the signs of cell damage can be an increase in the content of aminotransferase enzymes in the blood and, which is especially interesting, in the lymph flowing from organs (Abdreshow and Demshenko, 2009), that have undergone spasm. The peak increase in transaminases also occurred in the second week after hypoxic exposure. Increase in ALT and AST this is due to the peculiarities of the blood supply to organs, since lymphatic vessels receive nutrition and oxygen exclusively through the vasa vasorum, while blood vessels of the same diameter can receive everything they need directly from the blood that flows through them (Babashev et al., 2023; Abdreshov et al., 2021). For this reason, in the event of a decrease in blood supply, the lymphatic vessels suffer accordingly to a greater extent.

From the data obtained it is clear that during acute hypoxia in rats, caused by a decrease in the oxygen content in the chamber where the rats were kept, at 15 and 30 days of hypoxia there was a decrease in lymph flow, the volume of circulating plasma, a decrease in urination and blood viscosity, the ionic composition of urine and its osmotic pressure changed. An increase in the content of potassium ions in the blood plasma and especially significant in the lymph was noted. The content of sodium ions in the blood and lymph increased unreliably.

Thus, we can conclude that when exposed to hypoxia, when the oxygen concentration in the chamber decreases to 8%, it led to a state of hypoxic stress, which resulted in a redistribution of blood flow and the formation of tissue hypoxia. Hypoxia is a condition that results from insufficient oxygen in body tissues. However, the effect of hypoxia on diuresis and its underlying mechanism remain unclear. Diuretic effect may alter the cellular composition of blood, which may be the underlying initial increase in hematocrit and decrease in rheological properties of blood and lymph, moreover, pH is altered by acidosis. The result of this could be damage to the lymphatic microcirculation and liver.

There is one of the most important mechanisms for ensuring homeostasis while the conditions of the body’s existence change are maintaining the optimal ionic composition of the blood. Based on our data, we can assume that when exposed to hypoxia, the electrolyte balance in the body changes, this leads to a change in the ratio of cations and anions on both sides of the membrane and, as a consequence, a change in the charge of the membrane. This causes the development of a chain of pathological reactions leading to metabolic disorders in the body. Moreover, the effect of hypoxia after 15 days on the body of laboratory animals turned out to be more pronounced and lasting than the effect of hypoxia after 30 days. A decrease in the concentration of K⁺ and an increase in the content of Na⁺ in the blood plasma during hypoxia, which is usually accompanied by the release of water from the cells and a decrease in the forces of intermolecular adhesion between water molecules and lipophilic molecular structures. Obviously, there is an increase in the amount of free water in the extracellular fluid and plasma leads to a decrease in blood micro viscosity, which was recorded by us. In this regard, the micro viscosity of whole blood can be used to assess the state of the internal environment of the body under extreme environmental factors. However, the connection of this indicator with biochemical and physiological changes in the body when exposed to extreme factors requires further study.

Thus, the analysis of the mechanisms of ionic homeostasis in the blood of rats under hypoxic conditions shows that they are extremely complex. Hypoxic effects lead to significant disturbances of homeostasis in animals, characterized by significant activation of lipid peroxidation processes, as well as a decrease in the activity of antioxidant defence, especially in rats 15 days of hypoxia. In our experiments it is shown that after 15 days of hypoxia the activity of catalase in blood increases by 29.7%. SOD activity is usually sufficient to inactivate reactive oxygen species at the site of their formation, preventing diffusion in the environment of tissue macromolecules (Debevec et al., 2015; Ga Won Jeon et al., 2019). The identified changes in the antioxidant defence system indicate that the processes are aimed at maintaining the necessary balance characteristic of the physiological metabolic reactions of the body. The increase of the general level of antioxidant balance on the background of hypoxia was also confirmed by the data of MDA and DC determination. Thus, changes in antioxidant status under hypoxia indicated a decrease in oxidative potential at the systemic level (blood) and could be one of the causes of weakening of the organism's resistance and reduction of its immunoreactivity. A change in the permeability of membranes to electrolytes during hypoxia leads to their redistribution between extra- and intracellular spaces, which causes disturbances in the acid-base balance of the blood and water-electrolyte metabolism in the body.

5. Conclusions

Hypoxia leads to a decrease in erythrocyte resistance and lipid peroxidation in experimental animals. Shifts in pH on the side of acidosis and disturbances in physico-chemical properties in the blood and lymph were detected. Characteristic metabolic changes in the blood system during hypoxia are excessive accumulation of superoxide dismutase and catalase with a simultaneous decrease in the activity of malondialdehyde and diene conjugates. The obtained data testify to the objectivity of the revealed regularities and allow to emphasize the important role of free radical oxidation and antiradical protection processes after hypoxia. As a result of developing hypoxia in the body, structural and functional rearrangements occur in the whole blood of experimental animals, which apparently affect both its hydrophilic and lipophilic components and are expressed in an increase in antagonism between them, a decrease in the degree of molecular ordering of the internal environment of the body over time by reducing metabolic “bound” and increasing “free” water. This imbalance, obviously, is the basis for functional disorders associated with oxygen deficiency in the body.

Acknowledgements

We are particularly grateful to all the people who have given us help on our article.This work was supported by the Program-targeted funding for scientific, scientific-technical programs BR18574139 Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan.

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Publication Dates

• Publication in this collection
  07 Feb 2025
• Date of issue
  2024

History

• Received
  07 Mar 2024
• Accepted
  01 Oct 2024