Serum paraoxonase activity and lipid hydroperoxide levels in adult football players after three days football tournament

Background: It has been suggested that physical activity is an important factor in the prevention and treatment of cardiovascular diseases. Low serum paraoxononase–1 (PON1) activity is with an associated risk of atherosclerotic disease. Objectives: In this study, we aimed to investigate serum PON1 activity and lipid hydroperoxide (LOOH) levels in adult football players after three days football tournament. Methods: Twenty-three adult male football players and 23 sedentary male subjects after three days football tournament were enrolled. Serum paraoxonase, arylesterase activities and LOOH levels were determined. Results: Serum paraoxonase and arylesterase activities were signiûcantly higher in football players than sedentary subjects (all, p<0.05), while LOOH levels were significantly lower (p< 0.05). Serum LOOH levels were inversely correlated with paraoxonase and arylesterase activities (r=-0.552, p<0.001; r=-0.812, p<0.001; respectively) in adult football players. Conclusion: Our data show, for the first time, that physical activity is associated with increased PON1 activity and decreased oxidative stress after three days football tournament. In addition, physical activity for a healthy life is important in increasing serum PON1 activity, and this may play a role in the prevention of atherosclerosis.


Introduction
Oxidative stress is defined as an increase in reactive oxygen species (ROS) production resulting from a deterioration of the balance between oxidants and antioxidants in the body 1 . ROS may be generated as observed during aerobic endurance stress. The major source of ROS is thought to be the mitochondria of active working muscles 2 . In this respect, the balance between free radical generation and antioxidant activity plays an important role in the pathogenesis of coronary heart disease.
It is generally accepted that sedentary lifestyle is related to an increased risk of coronary heart disease. In contrast, regular physical activity has been identified as a protective factor against the occurrence and progression of coronary heart disease 3 . On the other hand, it has been reported that acute exercise is considered to be a major source of free radical generation, oxidative stress, and lipid peroxidation 4 .
Paraoxonase-1 (PON1) is calciumdependent esterase in human serum that is synthesized in the liver 5 . Serum PON1 catalyzes the hydrolysis of many organophosphates and aromatic carboxylic acid esters 6 . PON1 is one of the antioxidant enzymes that are associated with high-density lipoprotein (HDL) to prevent oxidative modification of low density lipoprotein (LDL). Therefore, PON1 plays an important role in the protection against atherosclerosis by preventing oxidative modification of serum lipoproteins 7 . Furthermore, previous studies indicated that oxidative stress decreased PON1 activity and down-regulated the serum expression of PON1 8,9 . Moreover, it has been recently reported that PON1 activity can influence several factors-such as diet, lifestyle, and environmental factors 10,11 . Moreover, Manresa et al. 12 described the association between PON polymorphism, gender, and exercise. Furthermore, it has been suggested that low serum PON1 activity is significantly associated with an increased risk of atherosclerotic disease 13 .
The effects of physical exercise on PON activity have been investigated in a clinical 3 study. Recently, several studies found that serum PON1 activity was increased in adolescent wrestlers 14 and adolescent athletes 15 .
To the best of our knowledge, the serum PON1 activity and lipid hyroperoxide levels in adult football players have not yet been reported. Therefore, we investigated the serum PON1 activity and lipid hydroperoxide (LOOH) levels as a marker of oxidative stress in adult football players after three days football tournament.

Methods Subjects
A group of 46 healthy male subjects, 23 adult football players and 23 sedentary controls were included in this study. 23 adult football players were selected among Yuzuncu Yil University, Physical Education and Sports students.
Adult football players were asymptomatic with an unremarkable medical history and a normal physical examination. None of the adult football players had diabetes mellitus, hyperlipidemia, hypertension, coronary artery disease, and psychiatric, metabolic, hepatic or renal disease. Adult football players were non-smokers. None of the subjects reported the use of anti-inflammatory drugs or a regular antioxidant vitamin supplements including vitamin E or C. None of the subjects was following a special diet.
Control subjects were young men who had a sedentary lifestyle and did not regularly participate in physical activity. Control subjects underwent routine physical and laboratory evaluations to ensure that none had diabetes mellitus, hyperlipidemia, hypertension, coronary artery disease, smoking, usage of supplemental vitamins, psychiatric, metabolic, hepatic or renal disease.
All subjects were informed about the study and they gave their informed written consent prior to the beginning of the study. The protocol of the study was conducted in accordance with the Helsinki Declaration as revised in 2000 and was approved by the local ethics committe.

Blood samples
After three days football tournament, blood samples were collected at overnight fasting period, between 08:00 h and 10:00 h from an antecubital vein. Blood samples were collected into empty tubes and immediately stored at 4 0 C. The serum samples were then separated from the cells by centrifugation at 3,000 rpm for 10 min. The serum samples were stored in plastic tubes at -80 0 C and used for the analysis of the PON1 activity and LOOH levels.

Measurement of paraoxonase and arylesterase activities
Paraoxonase and arylesterase activities were measured using commercially available kits (Relassay, Gaziantep, Turkey). PON1 activity was determined using two different substrates. 16 First, the rate of hydrolysis of paraoxon was measured by monitoring the increase in absorbance at 412 nm and 25 o C due to the formation of p-nitrophenol. PON1 activity assays were carried out in the absence of basal activity and the presence of NaCl (salt-stimulated activity). The serum sample to be tested was added to a cuvette containing 1.0 mmol paraoxon, 1.0 mmol CaCl 2 and 50 mmol glycine/NaOH buffer at pH 10.5. The amount of p-nitrophenol generated was calculated from the molar extinction coefûcient at pH 10.5, which was 18 290/mol per centimetre. PON1 activity was expressed as U/L and defined as 1 mmol p-nitrophenol generated per minute under wellestablished conditions. Second, the PON1 activity was measured in an arylesterase assay using phenyl acetate as substrate. In 10 mL of prediluted (1:3) serum samples were added to 1.5 mL reaction mixture containing 1.0 mmol phenylacetate, 9.0 mmol Tris-HCl buffer, and 0.9 mmol CaCl 2 at pH 8.0 and 25 o C Enzymatic activity was calculated from the molar extinction coefûcient 1310/mol per centimetre. Arylesterase activity is expressed as kU/ L and defined as 1 mmol phenol generated per minute under the conditions. 17 Blanks without enzyme were used to correct for spontaneous hydrolysis of both substrates.

Measurement of lipid hydroperoxide levels
Serum LOOH levels were measured with ferrous ion oxidation-xylenol orange assay. The principle of the assay depends on the oxidation of ferrous ion to ferric ion via various oxidants and the produced ferric ion is measured with xylenol orange. LOOHs are reduced by triphenyl phosphine (TPP),which is a specific reductant for lipids. The difference between with and without TPP pretreatment gives LOOH levels. 18 Other parameters The levels of triglyceride (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) were determined using commercially available assay kits (Abbott®) with an autoanalyzer (Aeroset®, Abbott®).

Statistical analysis
The results were expressed as the means and their standard deviation (mean±SD). Nonparametric continuous variables were compared by the Mann-Whitney U-test. Parametric variables were compared using Student's test. Correlation analyses were performed using Pearson's correlation test. The results were considered to be statistically significant when p value was less than 0.05. The data were analyzed using the SPSS® for Windows computing program (Version 11,0).

Results
Demographic characteristics of groups are presented in table 1. There were no significant differences between the groups in terms of age and body mass index (p>0.05) (table 1).
Serum TG, TC and LDL-C levels were significantly lower in adult football players compared to in sedentary subjects (all, p<0.05), while the HDL-C levels were significantly higher (p<0.05) (table 1).

Discussion
In the present study, we measured serum PON1 activity and LOOH levels as a marker of oxidative stress in adult football players after three days football tournament. We observed that adult football players had increased paraoxonase and arylesterase activity along with decreased LOOH levels. In addition, we found a negative correlation between paraoxonase, arylesterase activity and LOOH in adult football players. To the best of our knowledge, this study is the first to show that physical exercise is associated with decreased LOOH along with increased PON1 activity.
ROS are physiological products of aerobic metabolism. They contribute to the stimulation of anti-oxidative processes 19 . It has been shown that regular physical activity is an important factor in the prevention and treatment of cardiovascular diseases 20 . Regular physical exercise is a protective factor against cardiovascular diseases and enhances antioxidant systems, whereas acute exercise appears to be a major source of increased oxidative stress 20 . It has been reported that exercise training appears to enhance antioxidant levels 21 and reduce lipid peroxidation levels 4 , improving serum oxidant status in humans 22 .
The useful effect of exercise on health has been well known for centuries. Regular exercise is linked to reduced risk of developing obesity, hypertension, coronary artery disease and diabetes, and helps prevent premature death 23 . It has been shown that regular physical activity has beneficial effects on lipoprotein metabolism, including a decrease in plasma triglyceride levels and an increase in HDL-cholesterol levels, which bears antioxidant properties 24 . In the present study, we observed that adult football players had decreased serum TG, TC and LDL-C levels compared to in sedentary subjects, while the HDL-C levels were significantly higher.
Paraoxonases are a new addition to the antioxidant enzyme family. Several lines of evidence suggest that PON1 is responsible for the antioxidant properties of HDL on LDL particle 25,26 . Reduced PON1 activity has been reported in various diseases such as diabetes mellitus 27 , atherosclerotic heart disease 28 and hypercholesterolemia 29 and in patients who are under increased oxidative stress 30 . PON1 is also one of the antioxidants that act as an enzymatic defense against lipid hydroperoxides 31 . In addition, oxidative stress has been reported to affect the expression and activity of PON1 8,9 .
Several studies have investigated PON1 activity in many different conditions 3 32 . In addition, regular aerobic exercise in well-trained amateur athletes has been associated with an increase in PON1 activity 33 . To the best of our knowledge, the serum PON1 activity and lipid hyroperoxide levels in adult football players have not yet been reported.
Although PON1 activity and concentration are determined genetically, several factors such as diet, lifestyle, and environmental factors, can influence PON1 activity and/or concentration. Degraded cooking oil has been reported to decrease serum PON1 levels in humans 34 .
The mechanism of the observed increase in serum PON1 activity in football players remains unclear. This increase could be related to decrease lipid peroxidation because oxidized lipids are reported to inhibit PON1 activity. Furthermore, PON1 is an HDLassociated enzyme, and PON1 activity is positively correlated with HDL-C levels 35 . Indeed, in this study, adult football player's subjects had higher HDL-C levels than controls. However, in contrast to a previous study 36 , there was no correlation between PON1 activity and HDL-C.

Conclusion
Our data show, for the first time, that physical activity is associated with increased PON1 activity and decreased oxidative stress after three days football tournament. These results indicate that physical activity for a healthy life is important for increasing serum PON1 activity and that this may play a role in the prevention of atherosclerosis. Further studies with larger populations are needed to explore this relationship.