Associate professor, Dr.Biol. V.А. Kolupaev¹
PhD, Associate Professor I.Yu. Mel'nikov²
Dr.Med., Professor S.L. Sashenkov²
Honoured Scientist of the Russian Federation, Corresponding Member of the Russian Academy of Sciences, Dr.Med., Professor I.I. Dolgushin2
¹Ural State University of Physical Culture, Chelyabinsk
²South Ural State Medical University, Chelyabinsk

Key words: neutrophil phagocytic, lysosomal and NBT-activity, CD-lymphocytes, physical loads, entropy of leukocyte formula.

 Introduction. The role of leukocytes in protection against genetically unrelated aggression and realization of non-specific resistance mechanisms determines the relevance of studying the patterns of changes in the functional state of phagocytes and CD-lymphocyte count in athletes while adapting to environmental conditions and physical loads [1, 4, 7, et al.]. Therefore, the necessary task of the development of objective standards of adequacy of physical loads to the functional capacities of the body is to determine the standard values of the functional state of peripheral blood leukocytes (Leu) in athletes.

Objective of the research was to determine the range of natural variations in skiers’ indices of phagocytic, lysosomal and NBT activity and CD-lymphocyte count of the peripheral blood neutrophils in the off-season under the influence of low-intensity physical loads and favorable environmental conditions.

Research methods and structure. In order to determine the normal range of indices of neutrophil (Nph) phagocytic, lysosomal and NBT activity, as well as CD-lymphocyte count in athletes, out of the surveyed population with different levels of energy-supply of motor activity, we a posteriori selected the observations of apparently healthy racing skiers of 18-25 years of age with no complaints about overall health (n=50) made during the off-season of the annual macrocycle (the second half of April - June).

Leu content was detected by the conventional method using Goryaev chamber and air dried blood films flooded with Leishman's stain. The observation data were grouped depending on the presence of deviations of leukogram values from the norm ​​and its integral estimate - depending on the entropy of leukocyte formula (ELF) values in accordance with the Shannon entropy equation:

where a₁ - the percentage of basophils; a₂ - eosinophils; a₃ - banded Nph; a₄ - segmented Nph; a₅ - monocytes; a₆ - lymphocytes. Normal leukogram corresponds to relative entropy ranging between 56-67%, reversible adaptive response to environmental conditions or prenosological conditions corresponds to the range of 67-75%, and in case of relative entropy of over 75% a pathological state of the hematopoietic system can be diagnosed [5].

Phagocytic activity of Nph in athletes was studied using a traditional method based on the model of uptake of polystyrene latex with determination of the content and amount of phagocytizing cells, phagocytic index, phagocytic number and cumulative phagocytic activity of these cells. Neutrophil lysosomal activity was analyzed by means of luminescent microscopy by acridine orange vital staining of phagocyte granules, and oxygen-dependent metabolism was studied by means of spontaneous and latex-stimulated NBT-test [6]. The content of CD-subpopulations of Lym was determined by means of immunophenotyping with monoclonal antibodies anti-CD3, CD4, CD8, CD10, CD11b, CD16, CD20, CD25, CD34, CD56, CD95, CDHLA-DR of ICO series ("Medbiospektr", Moscow). The in vitro study was carried out at the premises of the Scientific Research Institute of Immunology of SBEI HVE "SUSMU" (head of the SRI of Immunology, rector of SBEI HVE "SUSMU", Honoured Scientist of the Russian Federation, Corresponding Member of the Russian Academy of Sciences, Dr.Med., Professor I.I. Dolgushin).

Results and discussion. At the initial phase of the study we assessed the functional state of leukogram indices in athletes during the off-season of the annual training macrocycle under the influence of favorable thermal environmental conditions and minimal physical loads.

Analysis of the results obtained revealed that, in an aggregate of these observations, Leu content in athletes spanned almost the entire range of normal values. At the same time, we observed moderate deviations of individual Leu populations from the norm in some of the surveyed. However, only 42% of skiers had normal ELF index, while in 6% of cases it was higher than normal, and in 52% of the surveyed it was characterized by low values.

While analyzing the observation results, out of the 729 possible combinations of the 6 studied populations of Leu in the leukogram state space, only 11 states were detected in the examined group. 14 observations fell under normal leukogram values, absolute lymphopenia was observed in 12 cases, absolute monocytosis - in 4 cases, eosinophilia - in 3 cases, as well as complex deviations of several leukocyte formula indices.

It is characteristic that some of the surveyed with no deviations of leukogram values from the norm have a low-level ELF. Thus, out of the 14 observations of athletes with normal leukogram normal ELF values are detected in 5 cases only, and in the rest 9 cases this index level is below normal. At the same time, out of the 21 observations with normal ELF level, in 16 cases we observed deviations in the content of some Leu populations: in 6 cases - absolute monocytosis, in 3 cases - moderate lymphopenia, in 1 case - eosinophilia, and in the rest of the cases – various complex deviations of leukogram values. At the same time, in 60% of athletes with normal leukogram values the ELF level was below normal, which is probably a reflection of the compensatory and adaptive changes in the peripheral blood system. So, hereafter we compared the studied indicators in these groups of skiers (see Fig.).

As seen from the data, the level of phagocytic activity and stimulated NBT-test of Nph in skiers was significantly higher in the athletes of Group 2 (with the signs of deviations of leukogram and ELF values from the norm) as compared to that in the skiers of Group 4, without any deviations of the studied indicators, and in the athletes with deviations of leukogram values, but with normal ELF values (Group 1). These differences were due to the different content of peripheral blood Nph. At the same time, phagocytic activity and phagocytic number of Nph, index of lysosomal activity and indicators of spontaneous and stimulated NBT-test in the athletes of the compared groups had no significant differences.

Fig. 1. Indices of phagocytic (a) and stimulated NST-activity (c) of Nph and CD₃₄ and CD₉₅ lymphocyte count (b, d) in skiers during off-season.

Legend: 1 - skiers with deviations of leukogram values from the norm; 2 - skiers with deviations of leukogram and ELF values from the norm; 3 - with deviations of ELF values from the norm; 4 - skiers with no deviations of leukogram and ELF values from the norm; P_1-2<0,0 ... - level of significance of differences between the indices of the examined groups estimated by the Mann-Whitney test.

Taking into account the relationship between the indices of phagocytic, lysosomal and NBT activity of Nph and monocytes and the content of CD₃₄ blood cells detected in the earlier studies [2], we compared the level of CD-lymphocytes in the skiers of the determined groups. When analyzing the pattern of Lym subpopulations, we observed a significantly lower level of the absolute content of CD₃₄ blood cells, as well as CD₃, CD₄, CD₈, CD₁₀, CD_11b, CD₁₆, CD₅₆ and CD₉₅ in athletes with deviations of leukogram values from the norm. At the same time, it is obvious that the low level of CD₉₅, containing Fas-antigens, is the cause of a smaller number of cells susceptible to apoptosis, which contributes to the maintenance of a sufficient number of fully functional cells at their low content in the blood.

As the indicators of neutrophil phagocytic, lysosomal and NBT activity, as well as CD-lymphocyte count in the skiers of the 3rd and 4th groups did not differ significantly, we combined them into one population to calculate standard values (Table 1).

Table 1. Standard values of neutrophil phagocytic, lysosomal and NBT activity in skiers in off-season of annual macrocycle

Legend. In the upper line - calculated values of 68.2% probability of normal distribution of indices within the interval , in the bottom line in brackets - median, minimum and maximum values of sampling population

As seen from the data submitted, the design norms for neutrophil phagocytic, lysosomal and NBT activity indices in skiers are characterized by a wide range of variations, as opposed to those not involved in sports. With that, the range of normal variations in the indices of phagocytosis of Nph are within the range of low values, and spontaneous NBT-test results - within the range of high values. In addition, the findings indicate that, in contrast to the regional standards for apparently healthy individuals not involved in sports [3], the spread of lognormal distribution of relative and absolute count of the studied CD-subpopulations of Lym in athletes is characterized by a more narrow range of variations.

Conclusion. The standard values of phagocytosis and NBT-test of Nph, determined based on the sampling of skiers with no deviations of leukogram values from the norm, were lower, and those of the absolute count of the studied CD-lymphocyte subpopulations - higher than those of the general population of the examined athletes. Herewith, in skiers with no deviations of leukogram indices from the norm the standard values of the content of CD₈, CD₁₀, CD_11b, CD₁₆, CD₂₀, CD₂₅, CD₃₄, CD₅₆, CD₉₅ and CD_HLA-DR were within the range of the lower limit of the regional standards, while the low values of 68.2 % probability interval of relative and absolute CD₃- and CD₄-lymphocyte count, as well as helper/suppressor ratio were below the lower limit of the regional standards for the population [3].

References

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Corresponding author: vitalico@mail.ru

Abstract
The standards of adequacy of physical loads to the functional capacities of the body were set by determining the normal range of variations in the indices of neutrophil phagocytic, lysosomal and NBT activity, as well as CD-lymphocyte count in cross country skiers in the off-season under the influence of low-intensity physical loads and favorable environmental conditions. We selected the observations of apparently healthy skiers of 18-25 years of age with no complaints about overall health (n=50) made during the off-season of the annual macrocycle (the second half of April - June).
It is shown that, as compared to non-athletes, the normal range of neutrophil phagocytic indices in cross country skiers is characterized by a wide range of natural variations within low values, and that of spontaneous NBT-test results - within high values. Skiers with no deviations of leukogram values from the norm have higher range of natural variations in the absolute count of the studied CD-lymphocyte subpopulations as opposed to the population of the surveyed athletes.