Morphological and Metabolic Parameters of Red Blood Cells after Their Treatment with Ozone

1 Национальный исследовательский Нижегородский государственный университет им. Н. И. Лобачевского, Россия, 603950, ГСП-20, г. Нижний Новгород, пр. Гагарина, д. 23 2 Нижегородская государственная медицинская академия Минздрава России, Россия, 603950, ГСП-470, г. Нижний Новгород, пл. Минина и Пожарского, д. 10/1 3 Нижегородская Городская клиническая больница No 40, Россия, 603083, г. Нижний Новгород, ул. Героя Юрия Смирнова, д. 71


Introduction
A loss of 30-50% of the circulating blood volume (CBV) at once is usually not an immediate threat to life, but signifies the onset of severe complications the body can't cope with.The body can compensate independently the loss of only not more than 25% of CBV due to protective and adaptive mechanisms.Transfusion of packed red cells and recovery of organ blood flow (perfusion) are the most effective measures in the case of a great blood loss due to achievement of a required CBV.The coefficient of the tissue oxygen utilization is also one of factors that compensate the consequences of anemia.
However, it is known that the storage of packed red blood cells causes a decrease in the concentration of ATP and 2,3 DPG in red blood cells and an increase in the content of potassium and lactate in them [1][2][3][4].The oxygen transport function of red blood cells deteriorates within 24 hours of storage [5].The deformability of red blood cells decreases by the second week of storage of the packed red cells [6].At this, the remaining red cells form microvesicles which contain phosphatidylserine thus facilitating the production of thrombin [7].In addition, the packed red cells lose osmotic resistance, thus leading to their destruction and the development of the «sludge syndrome» which, in turn, worsen tissue ischemia [8].Therefore, it is important to study methods improving the properties of the stored blood.
The purpose of the study was to assess the morphology of red blood cells and the association of morphological parameters with lipid peroxidation processes and the content of organic phosphates in red blood cells when treating packed red blood cells with the ozonized saline solution (with an ozone concentration of 2 mg/l) after different storage periods.

Materials and Methods
Packed human red blood cells stabilized with a blood preservative CPDA (sodium citrate + citric acid + sodium dihydrogen phosphate + dextrose + adenine) at a ratio of 1: 4 was the study object.The donor blood was taken at the blood transfusion station.The blood collection and production of its derivatives were carried out in accordance with «The Guidelines for the purveyance and preservation of blood» approved by the Ministry of Health of the Russian Federation on 29.05.99.The packed red blood cells were stored at t 4°С for 7, 14, 21 and 30 days.
During the study, 2 batches were formed: batch 1packed red blood cells (2 ml) was mixed with an equivalent volume of an ozonized 0.9% sodium chloride solution with an ozone concentration of 2 mg/l; batch 2 (reference)packed red blood cells (2 ml) mixed with an equivalent volume of an 0.9% sodium chloride solution.In each batch, 12 samples were taken.The saline solution was ozonized using and ozonizing therapeutic automatic device UOTA-60-01-Medozon (Russia).The device was manufactured in accordance with TU 9444-001-11441871-97 and could be used in healthcare institutions.Ozonization of the saline was performed immediately before mixing it with the packed red blood cells.The morphology of red blood cells, the malonic dialdehyde concentration (MDA), the catalase activity, and the concentration of ATP and 2,3 DPG were examined 60 minutes later.The physiological normal limits of the tested parameters were determined in the packed red blood cells on the day of the preparation.Measurements were performed in 12 samples.
Озонирование эритроцитной массы на этапе 7суточного ее хранения вызывало рост содержания ATP and 2,3 DPG were detected in the filtrate of hemolyzed RBC.While determining ATP, 1 mL of 2N HCl was added to 1 mL of TCA filtrate and hydrolysis was carried out in a boiling water bath for 7 min followed by cooling and neutralization with an equal volume of 2N NaOH.Inorganic phosphorus (IP) was determined before and after hydrolysis [16].To determine 2,3 DPG, nucleotides (ATP, ADP, AMP) were removed from the TCA filtrate of hemolyzed RBC by adsorption on the activated charcoal followed by centrifugation.In the supernatant (0.5 ml), IP (tube 1) was determined.A portion of the TCA filtrate (0.5 mL) was mixed with 0.5 ml of 5% magnesium nitrate solution, then it was boiled and cooled, then the contents of the tube were dissolved in 0.5 mL of 0.36N H 2 SO 4 .In 0.5 ml of the supernatant, IP concentration was measured (tube 2) in each tube by measuring the color density with the KFK-3 photometer (λ=660 nm).The concentration of PI was determined by means of a calibration curve using a standard KH 2 PO 4 solution.Calculation of the concentration of 2,3 DPG was carried out according to the formula (Р1 100 -Р2 10) / 2, where P1 is PI concentartion in tube 1 and P2 -concentration in tube 2 [17].
Mean arithmetic and standard errors of the mean were determined after proving that the experimental data belong to the normal distribution using the Shapiro-Wilk test.The t-test was used to compare the significance of difference between two subgroups.

Results and Discussion
A significant ability of ozone to influence the ratio of abnormal forms of RBCs and discocytes of the packed red blood cells with the increase of the latter at all storage times was found during the study (table 1).Biconvex discocytes were prevalent in the RBC-containing medium on the 7 th day of the storage, whereas echinocytes and spherocytes count increased after 30 days of storage.Ozone restored pathologically altered forms of red blood cells.The restoration was most pronounced when packed red blood cells were treated with an ozonized saline solution from the 7 th to the 21 st day of storage.
Discocyte count increased from 60.5±1.97% to 97.4±2.97%, and the echinocytes and spherocytes count significantly decreased during ozonation of the packed red blood cells during the 7-day storage period (table 1) as compared to RBCs without treatment with ozone (fig.1).
By the 30 th day of storage of the packed RBC, along with a further growth of the echinocyte transformation, the spherocyte count also increased.In addition, erythrocyte aggregation was observed (fig. 1, d).
Studies of blood rheology emphasize that transfusion of packed RBC of late storage periods is associated with a great number of complications and worse clinical outcomes which may be explained by incapability of spherocytes to the transcapillary transport, as well as micro-clots and sludge obstruction of capillaries [19].Ozone was ineffective on day 30 as compared to previous study dates: there was an increase in morphologically transformed RBC to echinocytes and spherocytes in the packed RBC.
It is known that hydrogen peroxide is one of echinocyte agents [20].In addition, the formation of echinocytes and spheroeсhinocytes is associated with the emergence of hemin during the oxidative processes [19], leading to the dissociation of the spectrin-protein 4.1 -protein 3 [22].Transformation into the spherocyte which is considered a prehemolytic form of a RBC is possible with an increase in the lysophosphatidylcholine level [23].Therefore, the morphological changes in RBC during the storage and under the effect of ozone are most likely determined by a change in the ratio of pro-and antioxidant processes in red blood cells.This statement is confirmed by our study.A study Таблица 1. Морфологическая характеристика пойкилоцитоза консервированных эритроцитов после обработки озонированным и стандартным физиологическим раствором.Table 1.Morphological characteristics of poikilocytosis of packed RBC after their treatment with ozonized and standard saline.
The obtained results indicate that lipid peroxidation processes and the emergence of echinocytes and spherocytes increase with the increase of the storage time of the packed RBC.Morphological changes in RBC can be caused by modification of both lipid and protein components of erythrocyte membranes, for example, by destruction of bilayer sites enriched by double bonds upon activation of lipid peroxidation [24] or by cross-linking of proteins formed by MDA [25].The change in lipid-protein interactions in the erythrocyte membrane disrupts specific membrane-associated processes, including ion transport [26].Upon accumulation of Ca 2+ ions in the cells, membrane-associated phospholipases are activated, thus enhancing the phosphatidyl serine translocation to the outer surface of the membrane and formation of echinocytes [27].Changes in the shape of cells and oxidative damage to erythrocyte membranes adversely affect the viscoelastic properties of membranes and increases erythrocyte aggregation, which is a negative factor in transfusion of the erythrocytic mass [28].
Treatment of the packed red blood cells with ozone determines the optimization of pro-and antioxidant systems of red blood cells, which may be explained by the direct effect of ozone on the cells.In response to the introduction of ozone in red blood cells, ozonide cells are formed in the lipid bilayer membranes [29].Ozonolysis of the erythrocyte cell membrane leads to the formation of hydroxy-hydroperoxides through the cleavage of chains of unsaturated fatty acids.Hydroxy-hydroperoxides trigger different components of the antioxidant protection system [30].The activation of antioxidant systems restores the processes of peroxidation [31].
It should be noted that the shift of the oxidationreduction effect of ozone leads to the accumulation of oxidized glutathione [32], and, consequently, to the activation of the glucose-phosphate shunt and changes in the metabolic state of cells.In our experiments, it was found that the ozonization of the packed RBC caused an increase in the concentration of ATP and 2.3 DPG after 21 days of storage, while treatment of the packed red blood cells storred for 30 days with an ozonized saline was not accompanied by a statistically significant change in the concentration of ATP and 2,3 DPG in red blood cells (table 2).The observed effects of ozone on the content of the above phosphates in DOI:10.15360/1813DOI:10.15360/ -9779-2018-1-40-49 -1-40-49 Эспериментальные исследования образование эхиноцитов [27].Изменение формы клеток и оксидативное повреждение мембран эритроцитов негативно влияет на вязко-эластичные свойства мембран и увеличивает агрегируемость эритроцитов, что является отрицательным фактором при трансфузии эритроцитной массы [28].
Thus, the ozonization of the packed RBC promotes the recovery of the erythrocyte shape by optimizing the processes of lipid peroxidation of erythrocyte membranes and increasing the synthesis of ATP.Under the effect of ozonides, a mutagenic shunt of glycolysis is activated and 2,3 DPG is formed.2,3 DPG determines the strength of the hemoglobin binding to oxygen, facilitates oxygen release by oxyhemoglobin and, thus, improves the oxygen supply of tissues.The formed ATP is used in energy-dependent processes of RBC, including contractile proteins, thus improving the deformability of RBC.The restoration of these processes during ozonation of the packed RBC is observed when they are stored for 7-21 days.In this case, ozonization of the packed RBC stored for 30 days does not improve the morphometabolic characteristics of RBC studied.The obtained results indicate that the developed technology for treatment of the packed RBC stored for 7 to 21 days with an ozonized saline solution (with the ozone concentration of 2 mg/l) improves the morphometabolic status of RBC and can be used to correct the oxygen transport function of blood in patients in critical conditions.

Conclusion
An increase in the count of echinocytes, spherocytes, MDA concentration and a decrease in the catalase activity and the content of organic phosphates is observed with the increase of the shelf life of the packed RBC.
Treatment of the packed red blood cells stored for 7, 14, and 21 days with ozonized saline solution (with ozone concentration of 2 mg/l) optimizes the impaired state of pro-antioxidant systems in RBC, increases the concentration of 2,3 DPG and ATP and, as a result, promotes restoration of the shape of RBC (from spherocytes and echinocytes to discocytes) which increases their biological significance.
These processes under the effect of ozone do not appear on the 30 th day of storage of the packed RBC, which indicates the decompensation of intra-erythrocyte morphometabolic processes.

Indexes in series, % Values of indexes on the day of storage
44 w w w .r e a n i m a t o l o g y .c o m -эритромассы с физиологическим раствором на этапе исследования.