International Journal of Sport Nutrition and Exercise Metabolism, 2015, 25, 171  -178 http://dx.doi.org/10.1123/ijsnem.2014-0090 © 2015 Human Kinetics, Inc

www.IJSNEM-Journal.com ORIGINAL RESEARCH

Oxidative Stress Markers After a Race in Professional Cyclists Alfredo Córdova, Antoni Sureda, María L. Albina, Victoria Linares, Montse Bellés, and Domènec J. Sánchez The aim was to determine the levels and activities of the oxidative stress markers in erythrocytes, plasma, and urine after a flat cyclist stage. Eight voluntary male professional trained-cyclists participated in the study. Exercise significantly increased erythrocyte, leukocyte, platelet, and reticulocyte counts. The exercise induced significant increases in the erythrocyte activities of catalase (19.8%) and glutathione reductase (19.2%), while glutathione peroxidase activity decreased significantly (29.3%). Erythrocyte GSSG concentration was significantly increased after exercise (21.4%), whereas GSH was significantly diminished (20.4%). Erythrocyte malondialdehyde levels evidenced a significant decrease 3 h after finishing the stage (44.3%). Plasma malondialdehyde, GSH and GSSG levels significantly decreased after 3 hr recovery (26.8%, 48.6%, and 31.1%, respectively). The exercise significantly increased the F2-isoprostane concentration in urine from 359 ± 71 pg/mg creatinine to 686 ± 139 pg/mg creatinine. In conclusion, a flat cycling stage induced changes in oxidative stress markers in erythrocytes, plasma, and urine of professional cyclists. Urine F2-isoprostane is a more useful biomarker for assessing the effects of acute exercise than the traditional malondialdehyde measurement. Keywords: antioxidant enzymes; exercise; isoprostanes; lipid peroxidation; redox status Over the last few decades, intensive research in the field of oxidative stress reported that exhaustive and high endurance exercise exacerbates free radical and other reactive oxygen species (ROS) generation which can result in oxidative stress. The mitochondrial electron transport chain, neutrophils, xantine oxidase reaction, and hemoglobin oxidation have been identified as major sources of intracellular free radical generation during exercise (Sureda et al., 2005). Professional training that involves repeated bouts of exercise and high volume of physically practice sessions and competitive games may lead to a decline on performance associated to an increase in oxidative stress and inflammation (Margonis et al., 2007). Moreover, the regular practice of moderate exercise is beneficial to health because it induces a mild oxidative stress that stimulates the expression of certain antioxidant enzymes (Gomez-Cabrera et al., 2008). In fact, the typical reaction to ROS can be described by a bell-shaped curve: low concentrations induced by moderCórdova is with the Dept. of Biochemistry and Physiology, School of Physical Therapy, University of Valladolid, Soria, Spain. Sureda is with the Research Group on Community Nutrition and Oxidative Stress and CIBEROBN (Physiopathology of Obesity and Nutrition), University of Balearic Islands, Palma de Mallorca, Spain. Albina, Linares, Bellés, and Sánchez are with the Laboratory of Toxicology and Environmental Health, Rovira i Virgili University, Reus, Spain. Address author correspondence to Alfredo Córdova at [email protected].

ate exercise have a stimulating effect (signaling, receptor stimulation, enzymatic stimulation), while a massive level of ROS after exhaustive exercise can alter enzyme activity resulting in oxidative damage in cellular molecules (Radak et al., 2008). The production of intracellular reactive species is increased by two- to fourfold during skeletal muscle contractions. Many reactive species produced during exercise by contractile muscle but also by endothelial cells are membrane permeable, and, consequently, are able to diffuse out of the plasma membrane into plasma. Moreover, reactive species produced inside blood cells could potentially oxidize substances found in plasma. This is particularly relevant for nitric oxide, H2O2 and HOCl, which have relatively long half-life in aqueous media (Mutze et al., 2003). In fact, it was evidenced a direct relationship between neutrophil MPO activity and plasma MDA levels, indicating that neutrophils could be an important source of oxidants in plasma and thereby contributing to oxidative stress. Erythrocytes are also very susceptible to oxidative damage because of the high polyunsaturated free fatty acid content of their membranes and the high cellular concentrations of oxygen and hemoglobin, a potentially powerful promoter of oxidative processes, and because they are unable to repair damaged components by resynthesis (Clemens & Waller, 1987). It is well established that plasma and blood cells are able to produce significant amounts of reactive species and contain considerable quantities of oxidizable substances. F2-isoprostanes are a family of eicosanoids

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172  Córdova et al.

end products of the tissue phospholipids oxidation by free radicals, including ROS (Sánchez-Moreno et al., 2004). Urinary F2-isoprostane levels have been reported to be reliable biomarkers of lipid peroxidation and a valuable tool for assessing oxidative stress (Il’yasova, et al., 2012). Intense exercise has been evidenced to increase the cellular oxidative status and markers of oxidative stress such as increases in lipid peroxidation, protein oxidation or increased glutathione disulfide concentration (Santos-Silva et al., 2001; Tauler et al., 2002). To avoid the potentially deleterious effects of the ROS, erythrocytes contain an elaborate antioxidant defense system that involves antioxidant enzymes that eliminate these ROS. Erythrocytic antioxidant enzyme activities have shown adaptations to oxidative stress induced by exhaustive exercise (Sureda et al., 2005). Antioxidants in both hydrophilic and lipophilic compartments of plasma are actively involved as a defense system against ROS, which are continuously generated (Yeum et al., 2004). Since the pattern of change of blood oxidative markers is a good reflex of oxidative stress owing to exercise, and both aerobic and anaerobic exercise causes oxidative stress, the purpose of the current study was to determine the levels and activities of the oxidative stress markers in erythrocytes, plasma, and urine after a flat professional cyclist stage.

Materials and Methods Subjects and Study Design Eight male subjects participated in this study. They were professional trained-cyclists participating in the professional race “Volta Ciclista a Catalunya” (Spain). The physical characteristics of cyclists were: age 25.7 ± 3.3 years old, height 1.79 ± 0.06 m, weight 70.0 ± 5.8 Kg, body fat percentage 9.4 ±0.5% and VO2max 78.2 ± 4.3 ml· kg-1·min-1. The evaluated exercise was the third stage, a flat cyclist race of 157.8 km. The two prior stages were the following: one time race of 20 km (Time trial), and one flat cyclist race without any mountain pass of 186.8 km. The cyclists took the same time of 230 min at 41.8 Km/h to complete this stage because all finished the stage with the main group and, with a mean heart rate of 127 ± 8 pulses/min. Cyclists were informed about the research protocol and volunteered to participate in the study. The study was designed in accordance with the recommendations for clinical research of the Declaration of Helsinki of the World Medical Association. The protocol was approved by the ethics committee of the Rovira and Virgili University (Reus, Spain). Further, all subjects signed a written informed consent. All subjects completed a medical questionnaire comprising a cardiopulmonary and electrocardiographic examination. A mandatory biological control according International Cycling Union (UCI) (hematological and biochemistry parameters) were also performed one week before study entrance. None of the

subjects smoked, drank alcohol, or were taking medications known to alter the hormonal response. Concomitant pathology was discarded by clinical rapport and medical examination. All subjects followed similar diet supervised by the medical doctor of the team.

Experimental Procedure Venous blood samples were obtained from the antecubital vein of cyclists in suitable vacutainers with EDTA as anticoagulant. Venous blood samples were obtained before the clycling stage after overnight fasting (basal sample) and 3 hr after finishing the stage and plasma and erythrocytes were purified. Preanalytical management of the samples was performed following the official rules of the World Anti-Doping Agency (WADA) (WADA, 2011) and UCI (2012) concerning blood sample collection and sample transport. Blood samples were stored at 4 °C in a temperature-controlled portable electric refrigerator and in less of 30 min were processed in the laboratory to ensure the sample stability. Blood samples were centrifuged at 900g at 4 °C for 30 min. The plasma was recovered, and the erythrocyte phase at the bottom was washed twice with 0.9% saline, centrifuged as above, and reserved to determine reduced glutathione (GSH), oxidized glutathione (GSSG), malondialdehyde (MDA) levels as a marker of lipid peroxidation and the antioxidant enzyme activities. Plasma samples were used to determine GSH, GSSG and MDA. Antioxidant enzyme activities and TBARS were determined freshly whereas and aliquot of both erythrocytes and plasma were precipitated with cold 70% trichloroacetic acid (TCA) (final concentration 10%), centrifuged and stored –20 °C until GSH and GSSG determination. All erythrocyte biochemical results were expressed per g Hb. Urine samples were collected in clean, dry containers either from the first morning void in rest conditions and 3 hr after finishing the race. Urine samples were briefly centrifuged at 1000g for 5 min at 4 °C to remove sediment. Frozen samples were analyzed in less than a week since collection.

Hematological parameters The number of erythrocytes, reticulocytes, hemoglobin concentration (Hb), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), leukocytes, and platelets, before and after the cycling stage, were determined in blood samples with a Sysmex XE-2100 hematology analyzer (Kobe, Japan). Quality control check for the hematology analyzer was routinely performed following the standard procedures supplied by the manufacturer.

Oxidative stress markers GSH and GSSG were determined with a fluorimetic method based on the reaction with o-phthalaldehyde (OPT) as a fluorescent reagent (Hissin & Hilf, 1976). MDA concentration was analyzed by a colorimetric assay kit specific for MDA determination (Sobioda, Grenoble,

Oxidative Stress in Cyclists   173

France) following the manufacturer’s instructions. The detection limit was 0.11 mmol of MDA per liter of plasma and the overall intra-assay coefficient of variation has been calculated to be 1.8–3.3% and the interassay coefficient of variation 3.3–4.4%. Antioxidant enzyme activities—Superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX)- were determined as we previously described (Sureda et al., 2005).

Urinary F2-isoprostane determination A competitive immunoassay was used for the quantitation of urinary isoprostane levels (8-isoPGF2a) (Cayman Chemical, Charlotte, USA) following the manufacturer instructions. The assay has a detection limit of 2.7 pg/ mL. The intra-assay and the interassay reproducibility were lower than 8% in both cases.

Statistical Analysis Statistical analysis of the results was performed with the SPSS version 13.0. The Kolmogorov-Smirnov test was used to assess the normality of distribution of investigated parameters. All parameters in our study were distributed normally. Student’s t-test for paired data were used to identify differences between pre- and postexercise. Results are presented as mean values ± SD. For all data, the level of significance was set at p < .05.

Results Hematological parameters determined before and 3 hr after finishing the cyclist race are presented in Table 1. Exhaustive exercise caused a significant increase in erythrocytes, leukocytes, platelets, and reticulocytes (p < .05). A significant decrease in the MCV and MCH parameters

were reported (p < .05). No differences between before and after exercise were observed in the Hb, HCT and MCHC values. Table 2 shows the antioxidant enzyme activities -SOD, CAT, GR, GPX-, as well as the GSH, GSSG and TBARS concentration in erythrocytes before and 3 hr after finishing the cyclist stage. The exercise induced significant increases in the activities of CAT and GR, while GPx activity decreased significantly (p < .05). No significant differences were observed in SOD activity. GSSG concentration was significantly increased after exercise, whereas GSH was significantly diminished (p < .05). However, the increase in GSSG/GSH ratio was not statistically significant. Lipid peroxidation, determined as MDA levels, evidenced a significant decrease 3 hr after finishing the cyclist stage (p < .05). The plasma oxidative stress markers, before and after exercise, are shown in Table 3. Plasma MDA as well as GSH and GSSG levels showed a significant decrease in the recovery period (p < .05). Although there was a marked decrease in plasma GSH and GSSG, the GSSG/ GSH ratio remained unaltered after exercise. Urine isoprostanes are presented in Figure 1. The cycling stage significantly increased the isoprostanes concentration in urine samples from 359 ± 71–686 ± 139 pg/mg creatinine (p < .05).

Discussion A flat cycling stage induced an acute phase immune response and alterations in the erythrocyte and plasma redox state resulting in antioxidant adaptations in professional cyclists (Cases et al., 2006; Sureda et al., 2007). As we analyzed the third stage, it is plausible to think that the two previous stages could have some effects in the obtained results from the third studied stage. However, in a previous study we evidenced that glutathione homeo-

Table 1  Hematological Parameters in Cyclists Before and After a Cyclist Stage Before

After

4.49 ± 0.19

4.55 ± 0.18*

Hb (g·dl-1)

13.7 ± 0.7

13.7 ± 0.7

HCT (%)

40.7 ± 2.0

40.6 ± 1.8

MCV (fl)

90.5 ± 2.1

89.3 ± 2.2*

MCH (pg)

30.5 ± 0.3

30.2 ± 0.4*

Erythrocytes

(106·μl-1)

MCHC (%)

33.7 ± 0.6

33.8 ± 0.9

Leukocytes (103·μl-1)

6.04 ± 1.15

10.2 ± 0.6*

203 ± 23

218 ± 28*

6.64 ± 1.85

16.0 ± 7.9*

Platelets (103·μl-1) Reticulocytes (103·μl-1)

Results are expressed as mean values ± SD. Red blood cells; Hb = hemoglobin; HCT = hematocrit; MCV = mean cell volume; MCH = mean cell hemoglobin; MCHC: mean cell hemoglobin concentration; WBC = white blood cells. *Statistical differences before and after exercise at p < .05.RBC:

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Table 2  Markers of Oxidative Stress in Erythrocyte of Cyclists Before and After a Cyclist Stage Before

After

3.24 ± 1.44

2.58 ± 0.97*

GSSG (μmol·g-1 Hb)

1.54 ± 0.32

1.87 ± 0.64*

GSSG/GSH

0.56 ± 0.31

0.78 ± 0.44

1.31 ± 0.55

0.73 ± 0.60*

172 ± 29

206 ± 38*

GPx (μmol·min-1·g-1 Hb)

22.2 ± 7.5

15.7 ± 5.1*

GR (μmol·min-1 ·g-1 Hb)

2.71 ± 0.70

3.23 ± 0.83*

1983 ± 433

2072 ± 529

GSH

(μmol·g-1

MDA CAT

SOD

Hb)

(nmol·g-1

Hb)

(mmol·min-1

(U·g-1

·g-1

Hb)

Hb)

Note. Results are expressed as mean values ± SD. GSH = reduced glutathione; GSSG = oxidized glutathione; TBARS = thiobarbituric acid reactive substances; CAT =catalase; GPx = glutathione peroxidase; GR = glutathione reductase; SOD =superoxide dismutase. *Statistical differences before and after exercise at p < .05.

Table 3  Markers of Oxidative Stress in Plasma of Cyclists Before and After a Cyclist Stage Before

After

GSH (nmol·ml-1)

32.1 ± 7.2

16.5 ± 5.9*

GSSG (nmol·ml-1)

33.1 ± 7.2

22.8 ± 5.2*

GSSG/GSH

1.03 ± 0.35

1.48 ± 0.85

1.27 ± 0.30

0.93 ± 0.19*

MDA

(nmol·ml-1)

Note. Results are expressed as mean values ± SD. GSH = reduced glutathione; GSSG =oxidized glutathione; MDA = malondialdehyde. *Statistical differences before and after exercise at p < .05.

stasis and blood cell counts were normalized the morning after finishing a 171 Km mountain cycling stage (Tauler et al., 2002; Aguiló et al., 2005). Moreover, 4-day road cycling competition reported no significant changes in the erythrocyte glutathione pool of professional cyclists (Serrano et al., 2010). These results suggest that in professional and well trained cyclist the recovery period between three stages is enough to normalize the redox state. Leukocyte concentrations increased after the physical work in accordance with previous studies (Tauler et al., 2002; Villa et al., 2003). The underlying mechanisms are multifactorial and include neuroendocrinological and metabolic factors such as catecholamines and cortisol liberation and/or the inflammatory process generated by the exercise (Cordova et al., 2006). Greater increases of leukocyte levels where reported in marathon and triathlon when compared with long distance cycling suggesting greater inflammatory effects of these endurance sports (Hanke et al., 2010). The exercise stimulates the production of new erythrocytes which are smaller and therefore have less hemoglobin as it was evidenced by the lower values of MCV and MCH after exercise (San-Sabrafen et al., 2006). The change in erythrocytes counts after the

Figure 1 — F2-isoprostane (8-isoPGF2α) concentration in urine of cyclists before and after a flat cyclist stage. Results are expressed as mean values ± SD. Superscript* indicates statistical differences before and after exercise at p < .05

stage and the lack of changes in hematocrit and hemoglobin indicates that the cycling stage did not induced significant hemolysis and hemoconcentration. In accordance with the present results, several studies reported an increase in platelet counts after resistance exercise probably due to a release of platelets from the spleen, bone marrow, and lungs (Ahmadizad & El-Sayed, 2003; McKenzie et al., 1999). Moreover, it was reported that in young well-trained athletes, supramaximal exercise causes release of immature reticulocytes (Morici et al., 2005). This progenitor mobilization has been suggested to occur in response to tissue hypoxia, or signals originating in skeletal muscle at a very high workload. In this way, the professional cyclists are training and in competition from more than 4 months. Moreover, activation of angiogenesis was suggested by the rise in vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) supporting a possible role of tissue hypoxia in modulating the response (Grochot-Przeczek et al., 2013). Although,

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several hematological parameters were increased after acute exercise it was reported that long-term training and competition periods such as 3-week professional competition (Giro d’Italia stage race) resulted in lower values of hemoglobin, red blood cells and hematocrit than baseline (Banfi et al., 2011; Corsetti et al., 2012). Thiol redox cycles play central roles in maintaining the redox state in cells. Blood glutathione homeostasis is essential for the normal cell metabolism and it has been suggested to participate in exercise-induced oxidative stress (Radak et al., 2103). The effort of the cycling race produced oxidative stress in erythrocytes, which was evidenced by the reduction of the GSH levels and the increase in GSSG levels. The present results agree with those previously reported by various authors who reported similar results after exhaustive exercise (Fatouros et al., 2010; Sastre et al., 1992). An explanation for GSH decreases and GSSG increases during exercise would reside in an increase in the metahemoglobin formation. The hemoglobin oxidation produces superoxide anion (O2.-), which is dismutated by into hydrogen peroxide (H2O2). In erythrocytes, H2O2 is decomposed by GPx and/or CAT action, producing O2 and water. The use of GSH by GPx to detoxify superoxide anion could be responsible at least in part of the oxidation of GSH to GSSG. Although the GSSG/GSH ratio was increased there were not statistically differences before and after the race in accordance with previous results (Tauler et al., 2005). The lack of significant differences in the GSSG/ GSH ratio could be a consequence of the well-training status of the cyclists and/or to the stage that was not hard enough to induce more evident changes in the GSSG/ GSH ratio in these well-trained professional cyclists. In fact, the stage was a flat stage without any significant mountain difficulty. The GSH and GSSG levels were significantly diminished in plasma, in comparison with the values previous to the exercise. It was evidenced that increases in plasma GSH and GSSG after exercise might be explained by an efflux from the muscle of liver cells when internal levels increase (Lew et al., 1985). The decrease in the plasma levels of GSH and GSSG suggests that the muscle was not damaged by the exercise. It was reported that efflux of GHS from the liver to plasma could be used to deliver GSH to skeletal muscle during strenuous physical exercise activity. The muscle capitation of GSH could explain decrease in GSH and GSSG. This may be a mechanism to protect skeletal muscle from oxidative damage under conditions of increased physical activity. The present results showed a significant rise in erythrocytic GR and CAT 3 hr after the cycling stage, while a significant decrease in GPx activity was reported. Notwithstanding, SOD did not vary significantly after competition. This pattern of change activity agrees with previous results registered by various investigators (Smith 1995; Tauler et al., 1999). It is important to note that erythrocytes cannot synthesize proteins, so the increase in enzyme maximal activities in erythrocytes could be attributed to covalent modification of proteins, to other protein interactions or to interactions with reactive

oxygen species or molecules generated during exercise, producing changes in the redox status of the protein. The observed lack of change of SOD activity in cyclists could indicate the compensated decomposition of H2O2 through increased activity of CAT during the stage. CAT levels increase after competition, thus increasing the potential for H2O2 decomposition in erythrocytes. The increase in CAT activity was mimicked “in vitro” in a previous study by incubating haemolysed erythrocytes in the xanthine/ xanthine oxidase system (Tauler et al., 2005). The in vitro activation of CAT by superoxide anion might explain the levels of CAT activity observed in the erythrocytes after the race. The superoxide anion produced during the exercise overwhelmed the SOD capacity of the erythrocyte and the superoxide anion activated CAT. The different kinetics of catalase and glutathione peroxidase indicates an almost exclusive role for catalase in the removal of H2O2 in normal human erythrocytes during a cycling stage (Mueller et al., 1997). In fact, in the present results GPx activity decreased after exercise indicating a deactivation process and also reinforcing the main role of CAT in detoxifying H2O2 (Pigeolet & Remacle, 1991). The increase in GR activity after exercise could be a response to the increased GSSG levels to recover the GSH. It was previously described an in vitro study a decrease in glutathione reductase activity in the presence of hydrogen peroxide. Consequently, the increased activity of catalase after the cycling stage might have produced an adequate decomposition of hydrogen peroxide, thus preventing an inhibition of glutathione reductase activity, and allowing this activity to increase after the cycling stage. Indeed, it has been observed that under oxidizing conditions glutathione reductase may form molecular aggregates with a decrease in its activity (Worthington & Rosemeyer, 1976). MDA concentration was surprisingly reduced both in erythrocytes and plasma 3 hr after the cycling stage. Some studies have found increases or not significant changes in MDA levels (Alessio et al., 2000; Dixon et al., 2006; Jimenez et al., 2000). The exact reason for this is unknown but it may be due to methodical variation between studies or the experimental procedure. It should also be noted that MDA is not a stable end product of lipid peroxidation and it can undergo further chemical reactions and it is also metabolized in vivo. This assay has been criticized for its lack of specificity and tendency to react with other nonrelated aldehydes, carbohydrates, and prostaglandins which can reduce the detected MDA after a recovery period (Gutteridge & Halliwell, 1990). The decreased concentration may also be due to the rapid clearance activation of MDA from plasma an erythrocytes (Groussard et al., 2003) which can make it difficult to obtain accurate in vivo measurements of MDA. F2-isoprostanes are a family of eicosanoids of nonenzymatic origin produced by the random oxidation of tissue phospholipids by oxygen radicals. It was reported that isoprostanes are reliable markers of oxidative stress in vivo with a sensitive lower limit of detection in the picogram range (Nikolaidis et al., 2012). Moreover, F2-isoprostanes, are chemically stable compounds and

176  Córdova et al.

their urinary excretion levels are not sensitive to dietary intake (Il’yasova at al., 2012). Elevated F2-isoprostanes levels were determined in plasma of subjects with known oxidative stress, including patients suffering cystic fibrosis and Alzheimer’s diseases (Collins et al., 1999; Pratico et al., 1998). The observed increase in F2-isoprostanes after the cycling stage supports our hypothesis that the cyclists experienced increased oxidative stress. However, the origin of F2-isoprostanes detected in plasma and urine remains controversial, as a multitude of tissues, including skeletal muscle and plasma, can produce F2-isoprostanes (Nikolaidis et al., 2011). The main limitation of the current study is that we did not obtain a blood sample at the beginning of the race in basal conditions and consequently, we cannot absolutely discard any effect of the previous two stages. Working with professional and well-trained subjects may minimize the changes in the analyzed parameters and make difficult to extrapolate to the general population. In summary, the current study found that a cycling stage is associated with changes in markers of oxidative stress in erythrocytes, plasma, and urine of professional cyclists. The fact that F2-isoprostane levels increased after exercise but not the plasma MDA levels indicate that urine F2-isoprostane is a more sensitive biomarker for assessing the effects of acute exercise. These data provide grounds for further research in this field aimed at investigating the effects of oxidative stress instauration following exercise on performance.

Novelty Statement A cycling stage increases markers of oxidative stress in professional cyclist. Urine F2-isoprostane is a more useful biomarker than plasma or erythrocyte MDA.

Practical Application Statement Urine F2-isoprostane is a no invasive and useful biomarker to monitor exercise-induced oxidative stress. Acknowledgments The study was designed by AC and DJS; data were collected and analyzed by MLA, MVL and MB; data interpretation and manuscript preparation were undertaken by all the authors. All authors approved the final version of the paper. The authors thank Laboratory of the Sant Pau and Santa Tecla Hospital of Tarragona their help in the identification of various blood parameters. A. Sureda was supported by the Spanish Ministry of Health and Consumption CIBERobn (CB12/03/30038). The authors state that they do not have any conflict of interest.

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