Metabolic muscle damage and oxidative stress ...

Eur J Appl Physiol (2011) 111:1341–1350 DOI 10.1007/s00421-010-1762-6

ORIGINAL ARTICLE

Metabolic muscle damage and oxidative stress markers in an America’s Cup yachting crew Carlos Barrios • Michal Hadala • Inmaculada Almansa • Francisco Bosch-Morell Jose´ M. Palanca • Francisco J. Romero

•

Accepted: 27 November 2010 / Published online: 10 December 2010 Ó Springer-Verlag 2010

Abstract Activities of enzymes involved in muscle damage [creatine kinase (CK) and aspartate aminotransferase (AST)] and levels of malondialdehyde (MDA) as a marker of oxidative stress were monitored in the plasma of 27 members of an America’s Cup yachting crew. The preventive benefits of allopurinol on muscle damage were also tested. In racing period A, the crew was divided into two groups according to their tasks on board. Blood samples from all 27 sailors were obtained before the start of a 5-day fleet race, after the last race, and after the ten match races. In period B, crew members were divided at random into two groups. One group (13 participants) received 300 mg/day of allopurinol 3 h before racing. The other ten members received placebo. Blood samples were collected just before and after the second round of the Louis Vuitton Cup. All participants showed increased CK and AST activities after the racing period A. The increase in CK activity was highest in sailors involved in strenuous physical work. At the end of period A, plasma MDA levels were higher in all participants as compared with non-participant athletes. In period B, a significant decrease in CK

Communicated by Martin Flueck. C. Barrios (&) M. Hadala Orthopaedics and Trauma Unit, Department of Surgery, Valencia University Medical School, Avda Blasco Iban˜ez 17, 46010 Valencia, Spain e-mail: [email protected] J. M. Palanca Anaesthesiology Unit, Department of Surgery, Valencia University Medical School, Valencia, Spain I. Almansa F. Bosch-Morell F. J. Romero Department of Physiology, Pharmacology and Toxicology, Universidad Cardenal Herrera-CEU, Valencia, Spain

activity, but not in AST, appeared among participants receiving allopurinol. Plasma MDA decreased in sailors treated with allopurinol, but this reduction did not reach statistical significance. America’s Cup is a sailing sport with high physical demands, as shown by the increase in muscle-damage markers. Treatment with allopurinol appeared to decrease the levels of muscle damage markers. Keywords Muscle damage Creatine kinase Aspartate aminotransferase Oxidative stress Malondialdehyde America’s Cup

Introduction Exhausting exercise has important consequences on muscle physiology that in turn induces muscle damage by both structural, i.e., microfibrillar rupture, and enzymatic mechanisms (Clarkson et al. 1992; Noakes 1987). Microruptures of muscle fibres following strong and repetitive contractions cause an outflow of creatine kinase (CK) (Davies et al. 1982) into the bloodstream. In light or moderate exercise, CK is maintained in normal ranges in serum; thus, increased CK levels have been found to indicate skeletal muscle fibre damage after heavy physical exercise (Brancaccio et al. 2007, 2010). Aspartate aminotransferase (AST), an enzyme localised primarily not only in the liver but also in skeletal and myocardial muscle, has been mainly used as a hepatotoxic biomarker. However, AST activity was also significantly increased immediately after muscular exertion, remaining at high levels for 24 h (Lippi et al. 2008; Nie et al. 2010). This increase is also related to the duration of exercise (Kim et al. 2009). In chronic muscle disease, AST activity can also be increased, but it is rarely high in subjects with

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no disease and normal CK activity (Nathwani et al. 2005). In athletes, the implications of increased serum AST should be considered in combination with the activity of CK (Lippi et al. 2008; Nie et al. 2010). The intimate relationship and interdependence of CK and AST activities still deserves further research. Apart from the release of cytosolic enzymes, highintensity exercise also increases the generation of free radicals in muscle and other tissues, which causes oxidation of protein, lipids, DNA, and other substrates. Excessive formation of free radicals has therefore been found to be a marker of muscle damage following exhaustive exercise (Gomez-Cabrera et al. 2003; Sastre et al. 1992). Although there is no single biomarker that can be considered the gold standard for lipid peroxidation (Veskoukis et al. 2008), malondialdehyde (MDA) is a good indirect indicator for estimation of oxidative stress. MDA increases significantly after marathon running, the ironman triathlon, and other intensive sports activities such as cycling stageraces (Gomez-Cabrera et al. 2003, 2006; Knez et al. 2007). Activation of xanthine oxidase (XO), a free radicalgenerating enzyme involved in the ischaemia–reperfusion syndrome, has been suggested to be aetiologically related to muscular oxidative damage after strenuous exercise (Hellsten et al. 1996; Vinã et al. 2000a, b). Although the mechanism has not been completely elucidated, serum hypoxanthine and xanthine concentrations increase dramatically in humans after intense exercise (Sahlin et al. 1991). This was confirmed in experimental animals when a tenfold increase of XO activity in the plasma was detected in rats after repeated high-intensity running to exhaustion (Radak et al. 1995). Allopurinol, an inhibitor of XO, protects against cell damage caused by exhaustive exercise in experimental animals (Vinã et al. 2000a, b), which highlights the role of XO in oxidative stress generation during exercise. Inhibition of XO has been shown to prevent plasma lipid peroxidation in marathon runners as measured by MDA levels (Gomez-Cabrera et al. 2006). The preventive effect of allopurinol on associated muscle damage due to exhaustive exercise has also been demonstrated in Tour de France cyclists (Gomez-Cabrera et al. 2003). Within sailing sports, the America’s Cup Challenge is considered to have high physiological and conditioning demands because of the exhausting training and its particular racing characteristics (Allen 2005; Allen and De Jong 2006; Hadala and Barrios 2009a, b; Hadala et al. 2010; Lambert 2001; Neville et al. 2006, 2009). The 32nd America’s Cup yachting race included 13 consecutive competitions and a final event, the Louis Vuitton Cup. Each competition consisted of five to seven consecutive days of races at the same venue, with one to two races per day that

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lasted 1–2 h per race. Each team spent approximately three additional hours sailing before the races began, during which the sailors would review their equipment and sails and adapt to the weather conditions. The long sessions of heavy physical work while training and racing might have important repercussions on the oxidative stress of the sailors. According to the 32nd America’s Cup rules, the crew was composed of 17 sailors with different positions and responsibilities within the boat. Each position had a specific role that required different physical work (Neville et al. 2009; Hadala and Barrios 2009b). For physical conditioning purposes, positions on the boat can be divided into three categories depending on the physical work intensity involved: high physical work intensity (grinders, mastmen, and bowmen); mild physical work intensity (pitman, trimmer, traveller, and runner); and low physical work intensity (navigator, tactician, and helmsman). Grinders, mastmen, and bowmen are exposed to high degrees of physical and psychological stress (Neville et al. 2006, 2009; Hadala and Barrios 2009a, b, Hadala et al. 2010). There is no information concerning muscle damage and oxidative stress markers in America’s Cup sailors. In this study, we address this issue by monitoring the activity of muscle enzymes involved in tissue damage (CK and AST) and levels of MDA, a marker of lipid peroxidation arising from oxidative stress, in members of an America’s Cup yachting crew during a racing period. The preventive benefit of allopurinol (an XO inhibitor) on free radicalinduced muscle damage was also tested.

Methods Subjects All 27 members of an America’s Cup yachting crew including sailors from six different countries participated in the study. Most of the sailors had previous professional experience consisting of participation in the Olympic Games, World Cups, Volvo Ocean Races, or previous America’s Cups. There were 13 members (high-intensity work group HIW) who acted as grinders (n = 9), mastmen (n = 2), and bowmen (n = 2). Eleven sailors were included in the mild-intensity work group (MIW) (pitman, n = 3; trimmer, n = 5; traveller, n = 2; and runner, n = 1). The low physical intensity work group (LIW) consisted of a navigator, a tactician, and a helmsman. For comparisons, the LIW group was considered in combination with the MIW group because of the small number of members. All participants signed a written informed consent form. The experimental protocol conformed to the standards set by the Declaration of Helsinki and received


approval from the ethics board of the Valencia University Medical School. Study design Venous blood samples were taken from all 27 members of the America’s Cup yachting crew during two official racing periods. In period A, samples were obtained the morning before the start of a 5-day fleet race, after the last race (6 races in total), and after the ten match races (8 days) corresponding to the first round of the 2007 Louis Vuitton Cup. Each race took an average of 90 min. Post-race venous blood samples were taken 3 h after the last regatta (i.e., when the activity of cytosolic enzymes in plasma was expected to be maximal if cellular damage occurred). Seven members of the crew (5 of the HIW and 2 of the MIW) never took part in the races because they were not selected for the team who will be sailing the races. This was a decision of the team manager and was not due to a previous sports injury. These seven sailors, acting as a spares, were used as the reference group. Therefore, the participant group consisted of 20 subjects. In period B, samples were collected just after the second round of the Louis Vuitton Cup. In period B, crew members were divided into two groups according to random numbers. Sailors were blind regarding the group assignment. Four sailors finally declined to take part in this phase of the study. Thus, a first group of 13 participants (8 HIW, 4 MIW and 1 LIW) was given a daily dose of 300 mg of allopurinol (an inhibitor of xanthine oxidase) 3 h before racing. The other ten-member group received a placebo (2 HIW, 4 MIW and 4 LIW). The pharmacokinetics of allopurinol indicates that the administered dose is sufficient to effectively inhibit XO and to treat hyperuricaemia in clinical practice (Heunks et al. 1999). Allopurinol is not included in the list of drugs prohibited by the International Olympic Committee.

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Plasma lipid peroxide levels (an indicator of oxidative stress) were determined by measuring MDA according to a modification of the high-pressure liquid chromatography method of Richard et al. (1992). Briefly, 0.1 mL of sample and 0.75 mL of working solution (thiobarbituric acid 0.37% and perchloric acid 6.4%, 2:1 v/v) were mixed and heated to 95°C for 1 h. After cooling (10 min in an ice water bath), the flocculent precipitate was removed by centrifugation at 12,000 rpm for 10 min. The supernatant (0.2 mL) was neutralised to pH 6–7 and filtered with a syringe filter prior to injection on a Cromasil C18 5 lm column (150 9 4.6 mm). The mobile phase consisted of 50 mM phosphate buffer (pH 6.0):methanol (58:42, v/v). Isocratic separation was performed with 1.0 mL/min flow (HPLC System, Waters) and detection at 532 nm (UV/VIS HPLC-Detector 2475, Waters). Calibration curves were run daily. Statistical data analysis Results were analysed using first standard descriptive statistics. The mean values and standard deviations (SD) of each item were calculated for the entire sample, for participants and non-sailing groups, for the two physical work subgroups according to on-board physical tasks (HIW and mixed MIW/LIW), and for the experimentally treated and placebo group. There was a normal distribution of data according to the Kolmogorov–Smirnov test. Statistical analysis involved comparisons between groups using the parametric unpaired t test. Comparisons within groups (participants, the two physical work groups, non-sailing athletes, experimental and placebo groups) at the different stages of the competition were assessed by the parametric two-tailed t test. Correlations between variables were made through the Pearson coefficient correlation. Significance was accepted at p \ 0.05. All statistical calculations were performed using SPSS 16 software for Mac (SPSS Inc., Champaign, IL, USA).

Laboratory determinations The blood samples were left to coagulate at room temperature and were centrifuged at 3,000g for 10 min to separate the serum, which was stored at -20°C for later analysis. As indices of muscle damage, serum CK, and AST activities were spectrophotometrically measured using commercially available reagents on an automatic random access analyser (Autolab 18, Boehringer, Mannheim, Germany). The intra-assay coefficient of variation was 4.5% for CK and 4.2% for AST. The catalytic concentrations of CK and AST were expressed as U/L at 37°C. The age- and gender-specific upper limits of the normal reference for these biomarkers were 171 U/L for CK and 35 U/L for AST.

Results Anthropometric characteristics of the whole 27-member crew are given in Table 1. When the sailors were divided into two groups according to the intensity of the physical work required for their tasks on board, statistically significant differences were found in some of the anthropometric parameters. Compared with the group with moderate or low physical work intensity, sailors performing physical activities of high intensity (mastmen, grinders, and bowmen) were younger (mean 27.3 vs. 35.8 years, p = 0.0005) and heavier (mean 96.9 vs. 85.1 kg, p = 0.001). This group also showed higher BMI values (28.4 vs. 25.9,

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Table 1 Anthropometric profile of the whole crew and of the two groups established according to the intensity of the physical work on board Variables

Age

All crew members (n = 27)

Work on board High intensity (n = 13) Mean (SD)

Medium–low intensity (n = 14) Mean (SD)

p

Mean (SD)

Range

31.7 (6.87)

21–44

27.38 (4.72)

35.86 (6.06)

0.005

Weight (kg)

90.82 (13.84)

63–118

96.97 (16.54)

85.11 (7.6)

0.001

Height (cm) BMI (kg/m2)

183 (0.07) 27.2 (3.02)

168–198 21.43–34.36

184 (0.07) 28.48 (3.53)

181 (0.07) 25.93 (1.72)

ns 0.001

Muscle (kg)

51.34 (8.47)

34.2–64.9

55 (9.77)

47.94 (5.45)

0.003

Muscle (%)

53.13 (8.87)

16.2–61.8

51.89 (11.46)

54.59 (4.92)

ns

Body fat (kg)

16.44 (5.52)

5.8–32.4

17.29 (6.85)

15.59 (3.88)

ns

Body fat (%)

17.22 (3.67)

9.2–25.2

16.75 (4.34)

17.66 (3.03)

ns

LBM (kg)

76.75 (10.46)

57.6–98.2

82.59 (11.83)

71.32 (4.88)

0.0001

p values correspond to the comparisons between the two physical work groups ns not significant

p = 0.001), greater muscle mass (55 vs. 47.9 kg, p = 0.003), and increased lean body mass (82.5 vs. 71.3 kg, p = 0.001). Body fat components were similar in both groups. Muscle damage markers Non-intervention phase (racing period A) All participants showed an increase in plasma CK and AST activities after fleet and match races as compared to nonparticipants (Fig. 1a, b). The elevation was highest after the fleet races, where the average CK increased from 224.4 to 363.7 U/L (p \ 0.01), and the average AST increased from 25.7 to 29.7 U/L (p \ 0.05). The CK and AST activity showed maximal peaks for sailors performing heavy physical work (Fig. 2a, b). CK mean values after the fleet races reached 505.5 U/L for sailors performing heavy physical work versus 261.4 U/L (p \ 0.01) for sailors with moderate–low physical work. After match races, these CK values were 451.8 and 254.4 U/L, respectively (p \ 0.01). In both fleet and match races, mean AST values increased significantly in the HIW group but not in the other group [basal 26.2 U/L, fleet race 34.4 U/L (p \ 0.01), match race 30.3 U/L (p \ 0.05)]. There were significant correlations between CK and AST values in all three conditions (Table 2). This correlation existed for both groups of sailors according to physical work intensity. Figure 3a shows the linear regression plot corresponding to CK and AST values after the fleet race in both groups. Changes in muscle damage markers showed no correlation with MDA changes (Fig. 3b). When Spearman bivariate correlation tests were applied between muscle damage markers and anthropometric

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Fig. 1 Creatine kinase (CK) and Aspartate aminotransferase (AST) activities in plasma after fleet and match races (Racing period A). Values are presented as the mean (SD). *p \ 0.01 versus basal prerace values. **p \ 0.05 versus basal pre-race values. vp \ 0.05 versus values of the participant group

variables among participants, the only statistically significant correlation was found between CK at basal conditions and muscle mass percentage (Table 3).


1345 Table 2 Correlations between CK and AST levels during racing period A Parameters

AST basal

AST fleet race

AST match race

CK basal Pearson correlation Significance (two-tailed) n

0.680** 0.000 25

0.527** 0.007 25

0.421* 0.040 24

CK fleet race Pearson correlation

0.608**

0.815**

0.803**

Significance (two-tailed)

0.001

0.000

0.000

n

25

25

24

CK match race Pearson correlation

0.582**

0.814**

0.898**

Significance (two-tailed)

0.003

0.000

0.000

n

24

24

24

* p \ 0.05, ** p \ 0.01

athletes (mean values 1.58 vs. 1.18 lM/mL, p \ 0.05). This difference was again dependent on whether the sailors exercised at high intensity. In the basal condition and after the match race, sailors of the HIW group showed statistically higher peaks values compared with non-participants [basal 1.89 vs. 1.10 lM/mL (p \ 0.05); match race 2.08 vs. 1.23 lM/mL (p \ 0.05)] (Fig. 5). Fig. 2 Muscle damage enzymatic activity in relation to the intensity of physical activity according to boat positions (Racing period A). HIW, high-intensity work; M-LIW, moderate- and low-intensity work. Values are presented as the mean (SD). *p \ 0.01 versus basal pre-race values. **p \ 0.05 versus basal pre-race values. vp \ 0.05 versus values of the HIW group

Intervention phase (racing period B) A significant decrease in CK activity was found among participants who received allopurinol (from 445.8 to 333.6 U/L, p \ 0.05) compared with those taking the placebo (from 275.8 to 237.3 U/L, p [ 0.05, ns) (Fig. 4a). However, AST mean values showed almost no change (treated group, from 31.0 U/L before the race to 31.1 U/L after the race; placebo group, from 24.2 U/L before the race to 24.5 U/L after the race) (Fig. 4b). Oxidative stress Non-intervention phase (racing period A) At the end of the ten match races of the first round of the Louis Vuitton Cup (period A), we found higher levels of MDA in all participants as compared with non-participant

Intervention phase (racing period B) After the intervention period, there was a decrease in MDA (1.55 ± 0.44 vs. 1.03 ± 0.56 lM/mL) in sailors treated with allopurinol during the seven races of the second round of the Louis Vuitton Cup (period B). The reduction in MDA did not reach statistical significance. No changes were found in the placebo group (Fig. 6).

Discussion This work describes for the first time the monitoring of muscle enzymes involved in muscle damage in members of an America’s Cup yachting crew during a short racing period. An important increase in CK and AST levels was found after the races, which reflects the high physiological requirements of this particular sailing modality and also implies muscle damage. In addition, muscle damage prevention through XO inhibition by allopurinol was proven to be effective in these athletes, as those receiving a daily 300 mg dose showed a significant decrease in muscle damage markers after a heavy 5-day race. Serum CK levels are used widely as an index of skeletal muscle fibre damage in sports and exercise physiology

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Fig. 3 Linear regression chart showing the correlation between CK and AST values after fleet race (p \ 0.01 for both work intensity levels). CK and MDA values showed no correlation

(Brancaccio et al. 2007; Mougios 2007). Increased release of CK and other myocellular enzymes has been specifically related to eccentric exercises rather than to concentric exercises (Brancaccio et al. 2007; Nosaka et al. 2002; Stupka et al. 2001). Elevated serum CK concentrations have been reported in sports such as football (Ehlers et al. 2002; Hoffman et al. 2002), marathons and other running activities (Kim et al. 2009; Lippi et al. 2008; Malm et al. 2004; Nie et al. 2010), triathlons (Neubauer et al. 2008), and weight training (Bolgiano 1994; Tokmakidis et al. 2003), where strenuous eccentric exercises are common. America’s Cup sailors in boat positions requiring heavy physical activity (grinders, mastmen, and bowmen) develop close chain muscle work with a combination of eccentric and concentric contractions. As shown in our study, increased CK levels in these sailors are therefore in accordance with the specific type of muscle work developed during races. There are no previous data on CK reference values and exercise-related variations in sailing sports, and thus comparisons with our America’s Cup sailor sample cannot be made. The subgroup of sailors involved in high-intensity exercise showed resting mean values twice those of those athletes not participating in the competition for different reasons. This last group had CK levels within the range reported for non-active individuals (Mougios 2007; Brancaccio et al. 2007). The upper limit in our study was obtained from a bowman of African origin who had 3,500 U/L at baseline prior to racing period A and 7,705 U/L just after the last match race of that period. Because of these extremely disparate values, this subject was not included in the calculations. Some authors consider CK levels of 5–10 times the normal range as the definition of exertional rhabdomyolysis (Bolgiano 1994; Patel et al. 2009). However, this athlete never had unusual post-exertion muscle soreness or systemic consequences. Maybe a silent metabolic myopathy could be responsible for the high CK values and should be worth investigating more in detail.

Table 3 Correlations between CK basal activity and MDA levels after match races and some anthropometric parameters of participant sailors Parameters

Weight (kg)

BMI

Muscle (%)

Body fat (kg)

Lean body mass (kg)

CK basal Pearson correlation

0.053

0.089

0.440*

-0.050

0.135

Significance

ns

ns

0.041

ns

ns

25

24

22

24

25

Pearson correlation

0.454*

0.472*

-0.306

0.509*

0.478*

Significance

0.026

0.023

ns

0.013

0.018

n

24

23

21

23

24

n MDA match race

* Correlation is significant at the p \ 0.05 level (two-tailed)

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Fig. 5 Malondialdehyde levels during racing period A. Values are presented as the mean (SD). HIW, high-intensity work group. MLIW, moderate- and low-intensity work group. *p \ 0.05 between groups

Fig. 4 CK activity and AST levels in sailors treated with allopurinol and in those receiving a placebo (racing period B). *p \ 0.05 versus pre-treatment values

A criticism for this work might concern the timing of blood sample collection. Some authors have found that the serum concentration of CK peaks 1–4 days after exercise and remains elevated for several days (Clarkson 2006; Clarkson et al. 2006; Paschalis et al. 2005). Furthermore, athletes participating in daily training have higher resting values than non-athletes (Evans et al. 1986; Nikolaidis et al. 2003; Mougios 2007). Thus, CK values obtained in these sailors may not accurately reflect the severity of muscle damage. In our study design, post-racing blood samples were taken 3 h after the last match. At this time, the activity of cytosolic enzymes in the plasma is expected to be maximal if cellular damage occurs. Furthermore, given that serum CK remains elevated for several days post-exercise and that the America’s Cup crew is engaged in daily heavy training, the values used in this analysis should be considered as the cumulative effect of a set of match races. In fact, the CK serum levels obtained in the first blood probe were slightly higher than the reference data given by the laboratory and can be explained by this cumulative effect.

Fig. 6 Malondialdehyde levels in serum before and after the intervention phase during the Louis Vuitton Cup (racing period B). Values are presented as the mean (SD)

Another source of CK serum increase consists of the skeletal muscle cell injury caused by oxidative stress derived from the liberation of reactive oxygen species (ROS) (Duarte et al. 1993; Clark 1997; Veskoukis et al. 2008). One of the possible sources for ROS is thought to be XO, an enzyme located in the capillary endothelium (Duarte et al. 1993). The role of the XO pathway in muscle oxidative stress has been confirmed by inhibition of this enzyme with allopurinol. It has been found that this drug prevents muscle damage after exhaustive exercise in mice, rats and humans (Duarte et al. 1993; Vinã et al. 2000a, b).

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In elite sportsmen, the first test of muscle damage prevention through XO inhibition by allopurinol was conducted at the Tour de France, the most prestigious cycling race (Gomez-Cabrera et al. 2003). A group of participants receiving a placebo (no XO inhibition) showed a significant increase in CK and AST activities in plasma after the team time trial stage, in which riders performed at their peak level of exertion. Such an increase was not found among participants who received 300 mg allopurinol. A similar protective effect of allopurinol against muscle damage following exhaustive exercise was found in our study of an America’s Cup yachting crew. A significant decrease in the activity of CK, but not of AST, was found among sailors who received allopurinol, especially those performing heavy tasks on board. The potential antioxidant role of allopurinol was also studied in cyclists during the Tour de France (GomezCabrera et al. 2003). An evident increase in MDA was found in all participants at the end of the 21-day race; however, the increase was significantly greater in the placebo group, without XO inhibition, than in the allopurinol group. The same research group has recently proven that the inhibition of XO prevents plasma lipid peroxidation in marathon runners (Gomez-Cabrera et al. 2006). MDA was significantly increased after marathon running. However, this increase was absent following allopurinol treatment. In our study, the group of sailors at high intensity had MDA levels significantly higher than those of the non-participant group. However, the increase in MDA was not significant after the last match race. Similarly, the decrease in MDA levels after allopurinol treatment was also found to not be statistically significant. It is possible that the sailing race period of 5 days was not sufficient for detection of relevant changes in oxidative stress in these athletes. Cyclists took the same 300 mg dose of allopurinol but during the whole 3-week racing period. Consequently, our data do not entirely confirm that XO inhibition by allopurinol prevents the oxidative damage induced by intensive exercise. The effects of XO inhibition on physical performance deserve further research. In Tour de France cyclists (Gomez-Cabrera et al. 2003), as in our study on America’s Cup sailors, an effect on performance level could not be noticed. However, recent studies have raised several concerns about the recommendation of taking antioxidant supplements before exercise (Gomez-Cabrera et al. 2005, 2006, 2008). For non-elite marathon runners, it has been reported that ROS produced in exercise act as signals that regulate molecular events important in cell adaptations to exercise (Gomez-Cabrera et al. 2006). Thus, in addition to causing oxidative damage, free radicals also up-regulate enzymes that are important for antioxidant defence. Antioxidant administration prevents such adaptation; thus, the recommendation to take antioxidant supplements before

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exercise should be revised as the supplements may prevent useful adaptations induced by exercise. Other studies performed by the same research group (Gomez-Cabrera et al. 2005, 2008) addressed the upregulation of antioxidant genes by training in rats. In the gastrocnemius muscle, exercise caused an activation of MAP kinases, which in turn activated the NF-kappaB pathway and consequently the expression of enzymes associated with defence against ROS (superoxide dismutase) and adaptation to exercise. All of these changes were abolished when ROS production was inhibited by allopurinol. Thus, a decrease of ROS formation prevents the activation of signalling pathways that cause useful adaptations in cells. The authors pointed out that interference of free radical metabolism by antioxidants might hamper useful adaptations to training. A recent experimental study more deeply focused on the effect of allopurinol on oxidative stress and physical performance after rats swam until exhaustion (Veskoukis et al. 2008). As expected, allopurinol inhibited XO activity. However, compared with their non-treated counterparts, rats receiving allopurinol showed a 35% decrease in physical performance, as indicated by the shorter swimming time to exhaustion. This feature has not been detected yet in humans and was not found in our study. Data from these three recent papers open an important issue for further discussion and additional research. In conclusion, the America’s Cup is a sailing sport with high physical demands, as shown by the increase in muscle damage markers after a relatively short racing period. Furthermore, the xanthine oxidase pathway seems to be implicated in the tissue damage that occurs after this exhaustive exercise, as the most sensitive muscle damage marker–CK decreases in sailors treated with allopurinol, an XO-inhibiting drug. Acknowledgments This work was supported in part by funds from Ministerio de Ciencia e Innovacioń (SAF 2007-66801), CoopernicusSantander Program of the Universidad CEU-Cardenal Herrera.

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