CHANGES IN INDICES OF ENZYMATIC ACTIVITY OF PERIPHERAL BLOOD LYMPHOCYTES IN ELITE FEMALE ATHLETES
CHANGES IN INDICES OF ENZYMATIC ACTIVITY OF PERIPHERAL BLOOD LYMPHOCYTES IN ELITE FEMALE ATHLETES
M.F. Zakharova, postgraduate student. Ulyanovsk state university, Ulyanovsk.
S.P. Levushkin, professor, Dr.Biol.,director. Research institute of Russian state university of physical culture, sport, youth and tourism (SCOLIPC), Moscow
E.A. Lazareva, associate professor. Ulyanovsk state university, Ulyanovsk
Key words: enzymatic status, blood, lymphocytes, adaptation to physical loads.
Introduction. Human cell is an independent vital unit with all the properties of the whole, which is fully applicable to the peripheral blood cells - lymphocytes, neutrophils, platelets .
The biochemical properties of blood cells are interpreted by the activity of numerous enzymes characterizing different aspects of metabolism and energy exchange and breathing processes in leukocytes, particularly dehydrogenases. It was stipulated by the quantitative research method of enzyme activity of peripheral blood lymphocytes that promoted estimation of the average enzyme activity and considering of cell distribution parameters as characteristics in accordance with enzyme activity. The concept of the enzyme status of blood cells is the combination of these parameters. The lymphocyte enzymatic status as an indicator is selected, where it is proved that lymphocytes are the cells that perform the immunodefence function and also elements of a uniform information system, accurately representing the body condition and the process of its development . Nowadays it is established that mainly the structures and mechanisms of energy supply at the cellular level are involved in athletes’ body during urgent and long-term adaptation to physical exercises. The most common basic test to examine body state at various exposures is an indicator of the lymphocyte enzymatic status by the succinate dehydrogenase (SDH) and α-glycerophosphate dehydrogenase (α-GDH) activities. The preferable study of SDH activity is stipulated by the interest in movement - spatial body movement, of blood through circulatory system, of ions in cell membranes, etc., i.e. of the processes that need energy flow and most intensive under aerobic conditions. 
The purpose of the research was to study changes in the indices of enzymatic activity of peripheral blood lymphocytes in elite female athletes at muscle work of various intensity and duration.
Materials and methods. The lymphocyte enzymatic status was estimated using the quantitative research method of enzymes’ activity in peripheral lymphocytes, suggested by R.P. Nartsissov . The method is based on the ability of p-nitrotetrazolium purple to form the water-insoluble formazan round granules during an enzymatic reaction, which are calculated in 50 cells. The method contributes to not only determining the average enzyme activity, as it is common in biochemical studies, but also considering as characteristics the parameters of cell distribution according to enzyme activity, as well as the measure of cellular diversity. The concept of lymphocyte enzymatic status is defined by the combination of these parameters of distribution in accordance with enzyme activity. The enzyme activity (SDH, α-GDH) is estimated by the number of diformazan granules in the lymphocyte cytoplasm (50 cells). Microcopying of results of cytochemical reaction is followed by preprocessing with calculation of parameters (average activity, coefficients of variation, asymmetry, excess, measures of cellular diversity for succinate and glycerophosphate dehydrogenases), which is implemented by means of the software provided by professor R.P. Nartsissov (laboratory of cytochemistry, Institute of Pediatrics). 
The data were analyzed in Excel 6. The significance of differences was assessed using the Student t-test. the differences at 95% confidence were considered reliable (p <0.05).
The research results were processed using the common methods of statistical analysis .
The study involved 10 female athletes at the age of 17-20, candidates and masters of sport.
The subjects performed 3 types of physical loads:
1. The first type consisted of two one-week training microcycles. In this case, blood was tested before training sessions and at the end.
2. The second type included 40-minute cross. Blood was tested twice: before and after the workout.
3. The third type included the functional test with 20 sit-ups in 30 seconds and 15-second stationary run at maximum speed. Blood was tested three times: before the functional test, immediately after finishing it, and in 24 hours. The last blood test was made to compare with the initial indices as the complete leukogram recovery occurs within 24 hours.
Results and discussion. According to the pre-exercise cytochemical analysis of the enzyme activity of succinate dehydrogenase of lymphocyte mitochondrions in female athletes, the level of average SDH activity before the training session of the first type and after it was the same (13,54 ± 0,663 and 13,03 ± 0,350 respectively) and was within the normal range (12, 68-15, 93 granules per lymphocyte).
According to the study of the pre-exercise distribution of the members of lymphocyte population of the female athletes’ blood by the level of activity relative to the mean value, the skewness was 0,25 ± 0,008 (A <0), which indicates to the prevalence of low-active cells in the population along with single high-active lymphocytes. The post-exercise skewness of blood lymphocytes of athletes remained at the same level - 0,15 ± 0,007 (A <0). The analysis of the lymphocyte excess or deficiency along with the typical enzyme activity has not revealed any changes (E <0) in the kurtosis (E) before exercise (-1,04 ± 0,030) and after it (-1,02 ± 0,010), which indicates to the insufficient number of average-active cells. The study of the dispersion relative to the mean blood lymphocytes in athletes that shows the relative heterogeneity of cells revealed the difference in cell lymphocytes in respect to enzyme activity, as indicated by the coefficient of variation (V): before exercise - 47,03 ± 4,790 and after it - 49.14 ± 5,081 (V> 0).
The comparative analysis of the succinate dehydrogenase activity in lymphocyte mitochondrions of female athletes before and after the 40-minute cross has not revealed changes of the pre- and post-exercise index of the average SDH activity, conforming to the standard (12,64 ± 0,238 and 13,70 ± 0,392 respectively). The pre-exercise skewness amounted to 0,15 ± 0,022 (A <0), which indicates to the prevalence of low-active blood cell pool. The post-exercise skewness standardizes - 0,38 ± 0,007, meaning that cells with different enzyme activity have matched in lymphocytes. The pre-exercise lymphocyte kurtosis of female athletes was - 1,16 ± 0,013 (E <0), indicating to the deficiency of average-active cells in blood lymphocytes. The post-exercise kurtosis normalized to the index 0,81 ± 0,017, thus the number of average-active cells increased. The SDH activity coefficient of variation (V> 0) in the athletes’ lymphocytes remained unchanged after exercise.
The comparison of the succinate dehydrogenase activity in lymphocyte mitochondrions of female athletes after a short-term physical load (functional test) revealed the following changes: the average SDH activity before (14,24 ± 0,385) and after the test (13,70 ± 0,392) conforms to the standard. The pre-exercise skewness of lymphocytes was 0,16 ± 0,019 (A <0), indicating to the prevailing low-active cells along with single highly active lymphocytes. The athletes’ post-exercise A coefficient has changed to 0,30 ± 0,038, indicating to the balanced pool of cells with different enzyme activity. The kurtosis of lymphocytes before and after the functional test has not changed and amounted to -0,85 ± 0,099 and -0,66 ± 0,067 (E <0) correspondingly, indicating to the lack of average-active cells. The coefficient of variation of the SDH activity in athletes’ lymphocytes was 38,07 ± 4,605 and - 38,74 ± 5,011 before and after exercises respectively. This is above the standard (V> 0) and it means a significant difference between cells in enzyme activity.
Thus, the studies of the average enzyme activity of the SDH in lymphocyte mitochondrions in three types of exercises revealed the normal average SDH activity both before and after exercises in the comparative analysis of female athletes. The skewness and kurtosis after the first type of physical activity is deteriorated, which indicates to the predominance of low-active cells over highly active ones and the lack of average-active cells in blood lymphocytes. After the second and the third types of physical activity the skewness and kurtosis coefficients change and comply with standard indices, indicating to the matched cell pool relative to the SDH activity. The coefficient of variation, that characterizes the dispersion of options relative to the mean in all three types of exercise, exceeds the normal rate.
In this case the findings indicate to sufficiently high fitness level of female athletes, good adaptive response to physical load. However, one should pay attention to some deviation of the the skewness (A) and excess (E) coefficients from the norm. This decrease can be associated with the decrease of the number of mitochondrions, violation of the integrity of their membranes. Subsequently, this may lead to athlete’s fatigue.
The cytochemical analysis of the GDH activity in lymphocyte mitochondrions of athletes revealed the following changes: average GDH activity is 12,77 ± 0,223 and 13,78 ± 0,374 before and after the exercise of the first type respectively, which corresponds to the normal range. According to the analysis of the distribution of members of the lymphocyte population before exercise by the level of activity relative to the mean value, the skewness coefficient comprised 0,16 ± 0,022, its value is positive (A> 0), i.e. low-active cells are dominating, and after exercise - 0,38 ± 0,005, which corresponds to the normal range. The analysis of the excess or deficiency of lymphocyte cells with typical activity in athletes amounted to 1,13 ± 0,012 and 0,79 ± 0,027 before and after the exercise respectively, suggesting the lack of average-active cells. The analysis of dispersion relative to the mean value, which shows the heterogeneity of cells in blood lymphocytes of athletes showed that the pre- and post-exercise coefficients of variation do not change and correspond to the normal range (44,54 ± 6,161 and 42,02 ± 9,389 respectively).
According to the analysis of the athletes’ GDH activity in lymphocyte mitochondrions, the average GDH activity before the 40-minute cross, and after have not changed. The skewness was 0,24 ± 0,032 and 0,15 ± 0,039 (A> 0) before and after load. This indicates to the prevalence in the population of the low-active cell pool combined with single lymphocytes. The pre- and post-exercise lymphocyte excess coefficients of athletes are 0,77 ± 0,086 and 0,76 ± 0,045 correspondingly, where (E <0), indicating to the lack of average-active cells. The pre- and post-exercise coefficients of variation remain the same and correspond to the normal range.
The study of the athletes’ GDH activity in lymphocyte mitochondrions revealed no changes in the average GDH activity before and after the functional test (13,16 ± 0,927 and 13,28 ± 0,658 respectively), which correspond to the normal range (11-13 granules per lymphocyte). The pre-exercise skewness of lymphocytes in athletes is 0,01 ± 0,045 (A <0), the value is negative, indicating to the rise of the number of highly-active cells. The post-exercise skewness coefficient was 0,26 ± 0,016 (A> 0), i.e. the low-active cell pool is predominant along with single highly-active lymphocytes. The values of the coefficients of kurtosis and variation before and after the functional test matched the normal range.
As follows from the comparative analysis of the indices of distribution of the average activity of GDH in lymphocyte mitochondrions of female athletes, the average GDH activity is not correlated with the type of physical load. The skewness coefficient comes to normal after the first and third types of exercise. The skewness coefficient is below normal after the 40-minute cross, proving the prevalence of low-active lymphocyte cells over highly-active ones in the females’ blood. The role of the excess coefficient depends on the type of load, its indices are below normal. The number of average-active cells is insufficient in a lymphocyte population. The coefficients of excess and variation do not depend on the type of load and stay within the normal range.
Proceeding from the findings of the study, adaptive changes take place in athletes stipulated by physical loads. The findings indicating to the inclusion of compensatory mechanisms, are assumed to be prognostically unfavorable for the results in a competition . Thus, non-uniform activation of specific lymphocytes, accumulation and particularly lack of lymphocytes with typical activity of mitochondrial glycerophosphate dehydrogenase predetermine bad results in a competition. Thus, the discrepancy between frequency and intensity of physical loads can lead to tension of compensatory-adaptive responses in the body, and in extreme cases - to disruption of adaptation .
The researches of the cytochemical status of peripheral lymphocytes are extremely important for assessment of athletes’ adaptabilities and adequate correction of their chronic physical fatigue.
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