Leukocyte activation, erythrocyte damage, lipid profile and oxidative stress imposed by high competition physical exercise in adolescents

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Abstract

Background: The aim of this study was to evaluate and to compare the lipid profile and the levels of leukocyte activation, red blood cell (RBC) damage and of oxidative stress in two groups of adolescents, with similar body mass index: high competition swimmers and adolescents practising moderate regular physical exercise. Methods: As markers of leukocyte activation, we measured plasma lactoferrin, elastase and granulocyte–monocyte colony stimulating factor. We studied RBC membrane band 3 profile and membrane-bound hemoglobin, as markers of RBC damage and aging; total and differential leukocyte count and RBC count, hematocrit, hemoglobin concentration and hematimetric indexes were also measured. Lipid profile included the evaluation of triglycerides (TG), total cholesterol (Chol), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), apolipoproteins AI and B (Apo AI and B), and lipoprotein (a) (Lp(a)). To evaluate oxidative stress, lipoperoxidation products and total antioxidant capacity were measured. Results: We found that high competition adolescents presented increased plasma levels of leukocyte activation products, increased RBC damage suggesting aging and premature removal, and higher oxidative stress. Lipid profile showed some risk and some protective changes. Conclusions: Our data suggest that high competition exercise, by imposing a higher and sustained oxidative and proteolytic stress, may contribute in the future to a higher risk of cardiovascular disease. We believe these findings warrant a reevaluation of current views in the intensity, duration and regularity of physical exercise, and that the evaluation of leukocyte activation products, RBC damage, oxidative stress and lipid profile may represent good markers to establish putative protective thresholds.

Introduction

Moderate physical exercise has been accepted as a cardiovascular protector [1], [2], though the underlying mechanism is not well established. For intense exercise some controversy about the protective effect still exists [3].

Atherosclerosis is very gradual in its development. It begins in the first or second decade of life and the clinical manifestations appear decades later. Atherosclerotic vascular lesions are commonly reported in coronary arteries and aorta before age 20 [4]. Besides the well known cardiovascular risk factors (lipids and lipoproteins), epidemiologic studies suggest that increased blood viscosity, which is determined by physical conditions (temperature, blood flow and shear forces) and by the composition of blood (hematocrit, leukocyte count), is related to cardiovascular risk and to atherogenesis [5], [6], [7]. Physical exercise triggers both an increase in blood flow and in blood viscosity. Actually, during physical exercise, red blood cells (RBCs) must deliver oxygen to tissues at a higher flow rate in a more viscous fluid, due to a reduced plasma volume and to a leukocytosis [8]. Other changes imposed by physical exercise, such as the rise in cellular metabolism and in hemoglobin (Hb) turnover, may favour reactive oxygen species (ROS) production within the RBC, activation of leukocytes and the development of oxidative stress [9], [10]. Whenever ROS are produced outside the RBCs, resulting from leukocyte activation or from cellular metabolism, they are also able to diffuse across the RBC membrane enhancing the oxidative stress within the cell.

Activated white blood cells (WBCs) are important sources of ROS and proteases, both of which may impose oxidative and proteolytic changes to plasma constituents and to neighbouring cells, such as circulating RBCs [11], [12], [13], [14], [15], [16], [17], [18]. Leukocyte activation products are known to modify RBC membrane proteins altering their antigenicity, and to induce Hb damage, membrane lipid peroxidation or even hemolysis. The RBC, with a very limited biosynthesis capacity and poor repair mechanisms, whenever it is exposed to unusual physical or chemical stresses, may suffer and accumulate physical and/or molecular modifications, which may underlie an accelerated senescence or even premature removal [19], [20]. Band 3, a RBC transmembrane protein, known as the anion channel, seems to mark the RBC for death by triggering the binding of a specific auto-anti-band 3 antibody, and complement activation [18], [21], [22], [23], [24], [25], [26], [27]. The antigenicity of band 3 may result from its cleavage, clustering or even from exposure of unusual epitopes.

Considering the modifications associated with physical exercise, it seems reasonable to wonder whether intense and sustained physical exercise, such as that performed in high competition sports, imposes enhanced levels of continuous oxidative and proteolytic stress.

The aim of this study was to evaluate and to compare the lipid profile and the levels of WBC activation, of RBC damage and of oxidative stress, imposed by a high competition sport and by a moderate and regular physical exercise, such as that performed by adolescents in their school physical education classes. The latter group was used as a control, since in Portugal it is obligatory at this age to attend physical education classes. The lipid profile included triglycerides (TG), total cholesterol (Chol), high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc), apolipoproteins AI (Apo AI) and B (Apo B) and lipoprotein (a) (Lp(a)); as markers of WBC activation, we evaluated plasma lactoferrin, elastase and granulocyte–monocyte colony stimulating factor (GM-CSF) concentration; as markers of RBC aging or damage, we evaluated the band 3 profile (% of band 3 monomer, aggregates and fragments) and the membrane-bound hemoglobin (%MBH); the hematologic study included the total and differential WBC count; RBC count; hematocrit (Ht); Hb concentration; hematimetric indexes—Mean Cell Volume (MCV), Mean Cell Hemoglobin (MCH), and Mean Cell Hemoglobin Concentration (MCHC). To evaluate the oxidative stress, we measured the total plasma lipoperoxidation products and the total plasma antioxidant capacity.

Section snippets

Subjects

This study was performed in two groups of adolescents aged 12–16 years, including a similar number of males and females with similar body mass index (BMI). One group included 40 high competition swimmers who trained more than 20 h/week; the other group included 42 adolescents, exercising for 2–4 h/week during physical education classes.

Collection and preparation of blood samples

Blood samples were collected at rest and fasted for 12 h. The collection was performed 48 h after the last swimming train, or after the last physical education

White blood cells and neutrophil activation

The total and the differential WBC count, and the concentration of some of their degranulation products, presented by the two groups, are shown in Table 1. We found similar values for total WBC count; however, the differential WBC count showed significantly higher values of lymphocytes in swimmers (P<0.001). Concerning elastase, lactoferrin and GM-CSF, we found that the mean values are higher in swimmers, though only elastase presented a statistically significant rise (P<0.05).

RBC damage

Table 2 presents

Discussion

Leukocytosis is a striking and consistent change associated with exercise [34] and is mainly due to a rise in neutrophils. The magnitude of this rise is determined by exercise intensity and duration. High WBC values are sustained for several hours, returning afterwards to preexercise levels or to even lower values. To exclude this immediate effect of exercise, we collected blood 48 h after the last physical exercise bout in both groups.

We found (Table 1) that total WBC, granulocytes and

Acknowledgements

This study was in part supported by grants from the “Fundação Gomes Teixeira”, the Oporto University and Praxis XXI no. 2/2.1/SAU/1240/95 (JNICT). The authors are greatful to Dr. Laura Pereira for the technical support.

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