A.M. Gadzhiev, associate professor, Dr.Phys.Math.
S.A. Aliev, associate professor, Ph.D.
Institute of Physiology named after A.I. Karaev NAS of Azerbaijan, Baku, Azerbaijan
S.E. Agaeva, postgraduate
Azerbaijan State Academy of Physical Culture and Sport of Baku, Azerbaijan

Key words: physical loads, rate of oxygen consumption, reactive oxygen species, free radicals, lipid peroxidation, malondialdehyde, blood serum.

Introduction. In physiology and biochemistry of physical exercises and sport of today attention of researchers is focused on studying the role of free radicals in the processes of muscular activity. Free radicals are formed in skeletal muscles at rest, and their production increases during contractile activity. During intense physical loads such an increase in the formation of oxidants leads to a shift in the balance of pro- and antioxidant compounds in the working skeletal muscles, an oxidative stress develops - a phenomenon that presumably underlies muscle fatigue and various dysfunctions [6]. Animal studies indicate that muscle fibers adapt to an increased level of free radicals’ activity in order to counter the risk of oxidative damage to tissue, and that occurs due to adaptive changes of the endogenous antioxidant system of skeletal muscles under the influence of physical load [9, 12]. For a researcher of adaptive changes of the body to physical loads, particularly to the loads in elite sport, the issues of antioxidant adaptation of skeletal muscles should be important too. Speaking of this, we would like to draw attention to the aspects of oxidant-antioxidant relationship in the cells which could be useful for the study of antioxidant adaptation.

The characterization of sources of reactive oxygen species (ROS) and other free radicals in skeletal muscles during physical exercises and identification of features of their induction depending on the intensity and type of physical exercises are of great importance. The main source of ROS is the electron transport chain in the mitochondria, where part of utilized oxygen (from 1 to 4-5%) is released as superoxide anion [5, 16]. Naturally, an increase in oxygen consumption during physical exercise leads to an increase in ROS generation by mitochondria. The contribution of the mitochondrial source varies depending on the fiber composition (presence of fast and slow fibers) and the nature of physical loads (their duration and intensity).

Another source of ROS is xanthine oxidase reaction which is responsible for the degradation of purine nucleotides [8]. Under physiological conditions, the xanthine oxidase enzyme functions mainly as a dehydrogenase (using NAD⁺ for electron transfer), and in case of oxygen metabolism disturbances (e.g. during ischemia-reperfusion, intensive physical load) transfers electrons to О₂ forming superoxide anion radical. It is believed that skeletal muscles have low xanthine oxidase activity. Nevertheless, the xanthine oxidase pathway may become important if there is a significant deficit of adenine nucleotides in the skeletal muscle. This situation is quite possible in case of ischemic muscle contraction, isometric contractions, sprint exercises, exercises performed under hypoxic conditions as well as with the affect of blood supply due to vascular disease.

Neutrophils are also sources of free radicals for skeletal muscles [12]. Their activation usually begins with damage of muscles or other soft tissues caused either by oxidative damage involving ROS or by a simple mechanical action, straining. At present there is no evidence that neutrophils are involved in enhancing the production of ROS under normal dynamic exercising, so they can be considered secondary sources of ROS during the recovery period after strenuous physical load. 

It is necessary to classify endogenous antioxidants (enzymatic and non-enzymatic) in skeletal muscles, analyze their responses to acute and adaptation to chronic physical loads, to determine characteristics due to the nature of work and the type of working muscles. In order to fight the damaging effect of free radical attacks on the cell structures in skeletal muscles (as well as other organs) an antioxidant defense system was developed [11, 12]. This system consists of several enzymatic antioxidants and low molecular weight antioxidant substances. Glutathione peroxidase (GPO) neutralizes various peroxide compounds using reduced glutathione, hydrogen donor. Oxidized glutathione may be reduced by glutathione reductase in the presence of NADPH. Another important antioxidant enzyme, superoxide dismutase (SOD), converts superoxide radicals into hydrogen peroxide, which can then turn into water by means of GPO or catalase. Glutathione (a tripeptide - γ-glutathione) is the most common non-enzymatic antioxidant and is present in many tissues in significant amounts (up to millimolar concentrations). Glutathione can have reaction with oxidants directly or via glutathione enzyme system. Glutathione concentration in skeletal muscles correlates with oxidative capacity; its concentration is higher in muscles with predominantly oxidative type of fiber rather than muscles with glycolytic type.

Besides glutathione, non-enzymatic antioxidants include alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). Vitamin E is a fat-soluble compound that reacts with lipid peroxy radicals to form less reactive tocopheroxyl radicals. Vitamin C, as a water soluble antioxidant, is very important as a reductant of    tocopheroxyl radicals and can also react directly with superoxide and hydroxyl radicals in cytoplasm and extracellular fluid.

Some studies show that acute physical load causes increased activity of SOD, GPO, glutathione reductase (GR) and catalase (Cat) in skeletal muscles. Long regular physical exercise leads to increased basic activity of some enzymes like SOD, GPO [12] and decreased activity of other enzymes like Cat, GR [3,12], i.e., adaptive changes of antioxidant enzymes in skeletal muscles are specific with regards to the enzyme itself, and possibly the type of muscle. Some enzymes respond to chronic loads by increasing their activity at rest (for example, SOD, GPO), others - by increasing their antioxidant capacity that can be induced by acute load (Cat, GR). These differences appear to be explained by post transcriptional and post translation features of expression of antioxidant enzymes in the process of adaptation to load; post transcriptional changes (translation stage) will occur in both groups of enzymes and post translation (protein folding stage) - for enzymes with low basic activity. In this aspect the study of induction of heat shock proteins (HSP) under the influence of physical loads is of special importance [14].

Is the antioxidant capacity of skeletal muscles enough to prevent the oxidative stress resulting from acute physical loads? The answer to this question also presumes studying the influence of exogenous antioxidants used as food additives on oxidation processes during physical exercise as well as physical working capacity of the body. It is known that today antioxidant supplements are widely used for general health promotion, however the influence of antioxidants intake on health effects of regular physical exercise is of interest both from a fundamental and practical points of view. In particular, the question of the influence of exogenous antioxidant supplements on the endogenous defense system against free-radical damage to muscles during strenuous physical exercise is important. In the literature there is evidence that antioxidant vitamin supplements lead to weakening of protective reactions of skeletal muscles to physical loads. According to the study of the effect of supplemental vitamin C, the adaptive antioxidant response of skeletal muscle (as well as lymphocytes) in humans weakens which is compensated by an increase in the basic activity of defense systems (SOD, catalase, heat shock proteins - HSP) [14]. According to our own studies [1] conducted on rats, a higher basic level of general antioxidant activity (AOA) in blood plasma and in skeletal muscles is observed in a vitamin C diet. At the same time adaptive reaction to physical load disappears in skeletal muscles and, on the contrary, appears in plasma. The effect of exogenous antioxidants with vitamin C taken as an example indicates that the adaptation of the endogenous system of antioxidant defense to physical loads may be modified (perhaps not always appropriately) with the help of exogenous antioxidants.

Oxidative stress caused by physical load in skeletal muscles may result in various changes in functional and structural properties of muscle fibers depending on the degree of oxidative shifts and, respectively, on the nature of the load. Relatively small shifts in the direction of increasing the formation of oxidants are normal physiologically significant phenomena that lead to adaptive changes in the antioxidant mechanism of muscles to resist future higher pressure of ROS. Significant oxidative shifts can cause more serious damage to cell structures; membrane structures of various cell organelles responsible for the implementation of the contractile function may be damaged, i.e. lipids and proteins involved in the processes of excitation - contraction - relaxation, their energy supply and regulation, may be oxidized. Such consequences of the oxidative stress may partly cause muscle fatigue, the process that is still not fully understood today. The joint effect of physical loads and antioxidant supplements is of interest.

The purpose of the study was to consider the effect of regular exercises on the oxidative and antioxidant indicators of skeletal muscle and blood plasma of rats on the background of antioxidant vitamins C and E. The level of lipid peroxidation (LPO) was analyzed as an oxidative indicator by assessing the intermediate product of lipid peroxidation of malondialdehyde (MDA), and general antioxidant activity (AOA) of tissues was selected as an antioxidant index.

Materials and methods. The experiments were conducted on 48 random bred white rats weighing 200-220 g that were kept under regular conditions of a vivarium. The animals were randomly divided into 2 equal groups: one group was subjected to regular training loads during 4 weeks, the other one was not. Both groups were in their turn divided into 2 subgroups that either received a vitamin supplement during 4 weeks of the experiment or didn’t receive it. The day after the experiment was over half of the animals from each subgroup was subjected to a single physical load, the other half was left in the state of rest. Immediately after the end of the physical exercise decapitation of animals was performed under light ether anesthesia. The training process was carried out by means of a running load in a roll with a diameter of 44 cm: 25 m/min, 20-30 min/day, 5 days/week. In groups with antioxidant supplements animals daily received 3 mg of vitamin C and 1.2 mg of vitamin E. Regular heparinized blood and calf muscle (m.gаstrocnemius) were used for the study. MDA content in the blood serum and muscle tissue homogenate was determined by reaction with thiobarbituric acid [2, 4]. АОА of samples was assessed by the degree of inhibition of peroxidation of Tween-80 in the ferrous ascorbate system to MDA. The statistical significance of comparisons between the indicators of different groups was assessed using the Student t-test.

Results and discussion. Table 1 presents data on changes in the concentration of lipid peroxidation product of malondialdehyde in blood plasma and skeletal muscle of rats exposed to regular physical loads against the background of receiving supplements of vitamins C and E as compared with the corresponding control animals. The values for the state of rest as well as after exercise are shown. In case of untrained animals that did not receive the vitamin supplements the concentration of MDA in blood plasma and muscle     undergoes significant growth (more than 2-fold) after a single exercise session as compared to the state of rest (p<0.01).

Table 1. Changes in the level of products of lipid peroxidation of MDA (nM/mg_protein) in skeletal muscle and blood plasma of rats under the influence of physical loads against the background of receiving the vitamin supplements (vitamin C + vitamin E), M±m, n=6

Here and below, * - indicates a significant difference between a state of rest and a post-exercise state; # - between groups of untrained and trained animals; ! - between groups that received and did not receive vitamins.

 Rats treated with vitamins for 4 weeks had a slight reduction in the MDA concentration while at rest: in plasma (18%) and muscle (15%), but these changes were not significant (p>0.05). However, the response of rats to physical load in this indicator of lipid peroxidation undergoes a significant change. After a single physical load, in contrast to the control rats, the increase of the MDA concentration in plasma is only 50% (p<0.05), and in muscle - 40% (low difference significance, p=0.06).

Regular physical loads lead to increased levels of lipid peroxidation in plasma and muscle of rats that did not get the vitamins, while they were at rest, by ~70% and ~30% (p<0.05) respectively. At the same time, these animals had no reaction of the lipid peroxidation level to loads that was observed in untrained rats, namely a sharp increase of in plasma and muscle. The differences in the MDA concentration both in plasma and in muscle before and after a single load proved to be insignificant. Against the background of receiving vitamins 4-week training showed no significant changes of the lipid peroxidation level related to the state of rest, although there appeared a reaction to the load again: after it the MDA concentration in plasma and muscle almost equally increased by 55% (p<0.05).

Table 2 presents data on the changes of OAO in blood plasma and skeletal muscle of rats exposed to regular physical loads against the background of the vitamin diet as compared with the corresponding control animals. In case of rats that did not take vitamin supplements regular training leads to an increase of OAO in plasma and muscle while at rest. The basic level of the OAO of plasma increases by ~40% (low significance, p=0.07), of muscle - by ~70% (p<0.05). The effect of training on the reaction of OAO to physical load proves to be different for plasma and skeletal muscle. Untrained rats demonstrate a decrease in both plasma and muscle under the influence of a single load (by 30% and 43%, respectively, p<0.05), although trained rats show a decrease only in plasma (40%, p<0.05), while in muscle OAO does not change.

Table 2. Changes in general antioxidant activity in skeletal muscle and blood plasma of rats under the influence of physical loads against the background of receiving the vitamin supplements (vitamin C + vitamin E), M±m, n=6

The4 weeks long vitamins intake results in an increase of over 80% in basic levels of OAO in plasma and muscle of both untrained and trained animals as compared to intact ones. However, while trainings without the vitamin supplements resulted in the leveling of the OAO reaction to the load in skeletal muscle (it remained in plasma), against the background of the vitamin supplements the effects of the AOA reduction in muscle resumed in the mode similar to intact animals. About 40% reduction of OAO in muscle after exercise is virtually the same as reduction in rats not subject to chronic loads and vitamin supplements. No significant reaction to the load was detected in the blood plasma of trained animals against the background of the vitamin supplements.

Why can exogenous antioxidants weaken the adaptive response of muscle to physical loads? Exercise-induced free radicals in skeletal muscles are considered not only as oxidation-damaging factors of cell structures and functions [12, 15]. Results of some recent studies indicate that free radicals that come to being as a result of physical loads, can act as signaling agents that stimulate specific adaptive responses of skeletal muscles that are very important for the functional integrity of the muscle [12]. It is known that the redox-sensitive transcription factor NF-κB is activated by exercise, leading to increased expression of SOD [12, 13]. Adaptive reactions caused by oxidants help to protect cells against the concentration of induced radicals that normally have a damaging impact. On the other hand, one can not ignore a very important and unexpected fact, that prior administration of antioxidants can weaken the muscle adaptive response to an oxidative stress caused by physical exercise. Prevention of cells’ adaptation to physical loads that is shown, in particular, in gene expression of antioxidant enzymes (SOD) was observed while suppressing oxidative stress induced with xanthine oxidase reaction in rats with allopurinol [7].

Thus, understanding the unique characteristics and regulatory mechanisms of various antioxidants can contribute to development of the right strategy of increasing antioxidant capacity of cells by means of physiological pathways and nutrition.

Conclusion. The findings in terms of lipid peroxidation and antioxidant activity suggest that regular exercises modify oxidant - antioxidant relationship in the blood plasma and skeletal muscles, ultimately causing positive changes in the body's response to strenuous physical load. The use of antioxidant supplements in the diet also enhances the antioxidant capacity of skeletal muscles and blood, thereby reducing the intensity of oxidative processes induced by physical loads. However, the positive effects of training loads may be somewhat reduced during treatment with antioxidant supplements, such as vitamins C and E, which is expressed in the increased "oxidative" and weaker "antioxidant" responses to strenuous exercises. Exogenous antioxidants, helping the body fight against harmful endogenous compounds (free radicals), at the same time attenuate the signal for adaptive changes in skeletal muscles to respond to future cases of more intense loads. We believe that regulation of doses of taken antioxidant supplements depending on the level of body fitness, the nature of the training loads, as well as the issues of recovery after intensive physical loads against the background of exogenous antioxidants are among the tasks to be studied first.

References

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Corresponding author: ahmed.hajiyev@yahoo.com