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ARTICLE Serum S100B level increases after running but not cycling exercise Cintia Mussi Alvim Stocchero, Jean Pierre Oses, Giovani Santos Cunha, Jocelito Bijoldo Martins, Liz Marina Brum, Eduardo Rigon Zimmer, Diogo Onofre Souza, Luis Valmor Portela, and Álvaro Reischak-Oliveira
Abstract: The objective of this study was to investigate the effect of running versus cycling exercises upon serum S100B levels and typical markers of skeletal muscle damage such as creatine kinase (CK), aspartate aminotransferase (AST) and myoglobin (Mb). Although recent work demonstrates that S100B is highly expressed and exerts functional properties in skeletal muscle, there is no previous study that tries to establish a relationship between muscle damage and serum S100B levels after exercise. We conducted a cross-sectional study on 13 male triathletes. They completed 2 submaximal exercise protocols at anaerobic threshold intensity. Running was performed on a treadmill with no inclination (RUN) and cycling (CYC) using a cycle-simulator. Three blood samples were taken before (PRE), immediately after (POST) and 1 h after exercise for CK, AST, Mb and S100B assessments. We found a significant increase in serum S100B levels and muscle damage markers in RUN POST compared with RUN PRE. Comparing groups, POST S100B, CK, AST and Mb serum levels were higher in RUN than CYC. Only in RUN, the area under the curve (AUC) of serum S100B is positively correlated with AUC of CK and Mb. Therefore, immediately after an intense exercise such as running, but not cycling, serum levels of S100B protein increase in parallel with levels of CK, AST and Mb. Additionally, the positive correlation between S100B and CK and Mb points to S100B as an acute biomarker of muscle damage after running exercise. Key words: biochemical markers, muscle damage, muscle contractions, S100B, myoglobin, creatine kinase. Résumé : Cette étude se propose d’examiner l’effet de la course comparativement au cyclisme sur la concentration sérique de S100B et les marqueurs typiques des lésions musculaires : créatine kinase (« CK »), aspartate aminotransférase (« AST ») et myoglobine (« Mb »). Même si des études récentes révèlent une expression élevée de S100B et des propriétés fonctionnelles dans le muscle squelettique, il n’y a pas encore d’études traitant de la relation entre les lésions musculaires et la concentration sérique postexercice de S100B. On réalise une étude transversale auprès de treize triathloniens. Ces derniers participent a` deux protocoles d’exercice sous-maximal au seuil anaérobie. La course s’effectue sur un tapis roulant non incliné (« RUN ») et le vélo (« CYC ») sur un cyclosimulateur. Avant (« PRE »), immédiatement après (« POST ») et 1 h plus tard, on prélève des échantillons de sang pour l’analyse des CK, AST, Mb et S100B. On observe une augmentation significative de la concentration sérique de S100B et des marqueurs de lésion musculaire dans la condition RUN POST comparativement a` la condition RUN PRE. Quand on compare les groupes, on observe en POST des valeurs sériques plus élevées des S100B, CK, AST et Mb en RUN comparativement a` CYC. La surface sous la courbe (« AUC ») de S100B sérique est positivement corrélée avec les AUC de CK et de Mb, et ce, uniquement en RUN. Par conséquent, après un exercice intense comme a` la course, mais pas a` vélo, le niveau sérique de S100B augmente parallèlement aux concentrations sériques des CK, AST et Mb. En outre, la corrélation positive observée entre S100B et CK et MB souligne le rôle de S100B comme biomarqueur immédiat de lésion musculaire a` la suite d’un exercice de course. [Traduit par la Rédaction] Mots-clés : marqueurs biochimiques, lésion musculaire, contractions musculaires, S100B, myoglobine, créatine kinase.
Introduction Creatine-kinase (CK), aspartate aminotransferase (AST) and myoglobin (Mb) are typical serum biomarkers of skeletal muscle damage studied in the field of competitive sports (Lavender and Nosaka 2008; Lippi et al. 2008; Singh et al. 2011). Furthermore, it has been shown that exercise with predominant eccentric contractions causes considerable damage to the sarcolemma, eliciting the leakage of a significant amount of muscle proteins into blood that may serve as biomarkers of damage (Lavender and Nosaka 2008). Skeletal muscle damage causes pain and functional impairment and has a negative
impact on the performance of athletes. Despite some controversies regarding tissue-specificity, the assessment of serum biomarkers provides a chance to reveal the dynamic of muscle abnormalities induced by training protocols. S100B is a calcium-binding protein, which has cerebral and extra-cerebral sources, and it can reach blood either by normal secretion or leakage that is due to damage (Vos et al. 2004; Chaves et al. 2010). In the field of competitive sports, serum quantification of S100B protein gained notoriety because its ability to reflect damage caused by direct impact in the brain (Otto et al. 2000;
Received 4 July 2013. Accepted 9 September 2013. C.M.A. Stocchero. Federal Institute of Education, Science and Technology of Rio Grande do Sul, Porto Alegre RS, Brazil; School of Physical Education, Federal University of Rio Grande do Sul, Porto Alegre RS, Brazil. J.P. Oses. Health and Behavior Graduate Program, Catholic University of Pelotas, Pelotas, Brazil. G.S. Cunha, J.B. Martins, and Á. Reischak-Oliveira. School of Physical Education, Federal University of Rio Grande do Sul, Porto Alegre RS, Brazil. L.M. Brum. Institute of Cardiology of Rio Grande do Sul, Porto Alegre RS, Brazil. E.R. Zimmer, D.O. Souza, and L.V. Portela. Department of Biochemistry, ICBS, Federal University of Rio Grande do Sul, 2600 Ramiro Barcelos street, 90035-003 Porto Alegre, RS, Brazil. Corresponding author: Luis Valmor Portela (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 340–344 (2014) dx.doi.org/10.1139/apnm-2013-0308
Published at www.nrcresearchpress.com/apnm on 27 September 2013.
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Stalnacke et al. 2003, 2004). However, we demonstrated that a non-impact exercise (swimming) also increased serum S100B levels in humans, providing the basis to assume that extra-cerebral sources could additionally release/leak protein into blood (Dietrich et al. 2003). For instance, experimental data show that under stress conditions, cardiomyocytes and adipocytes can leak S100B into medium (Mazzini et al. 2005, 2009; Gonçalves et al. 2010). Furthermore, S100B protein is strategically located on the skeletal muscle membranes, sarcoplasmic reticulum, and transverse tubules acting in association with other proteins linked with calcium homeostasis (Arcuri et al. 2002). The demonstration that S100B is highly expressed and exerts functional properties in skeletal muscle provides the premise upon which to investigate S100B abnormalities in response to exercise-induced muscle damage (Arcuri et al. 2002; Donato et al. 2009; Tubaro et al. 2010; Riuzzi et al. 2011). Moreover, the assessments of serum concentrations of a candidate biomarker along with typical muscle biomarkers may help to determine the cell/tissue origin site and the behavior profile post-injury (Hasselblatt et al. 2004). Here we performed a cross-sectional study to investigate the effects of 2 exercise protocols on serum levels of typical skeletal muscle biomarkers CK, AST and Mb, and S100B protein. To achieve this main objective we used running versus cycling protocols performed at the same relative intensity.
Materials and methods Participants and ethics Thirteen recreational male triathletes (mean ± SD: age, 33.9 ± 6.0 years; height, 177 ± 0.1 cm; mass, 74.7 ± 6.8 kg; body fat, 11.1% ± 4.7%; maximal oxygen uptake (V˙O2max), 62.7 ± 6.6 mL·kg−1·min−1) participated in this study. We have estimated that this sample size would provide sufficient statistical power (B = 0.20) to detect an association of at least 0.7 among muscle damage markers and S100B protein. Reference values for sample size determination were obtained from a previous study investigating S100B and CK association (Hasselblatt et al. 2004). The athletes’ training program included running an average 41.5 ± 14.3 km·week−1, cycling 235 ± 104.8 km·week−1 and swimming 8.44 ± 4.7 km·week−1. The participants were fully informed about the procedures and all possible risks involved in this study. Each volunteer provided written informed consent before participating. The study was approved by the Research Ethics Committee of Federal University of Rio Grande do Sul, Brazil (no. 2007708). Experimental design and exercise protocols All participants of the study were required to complete 4 visits to the laboratory to evaluate their V˙O2max on the treadmill and bicycle ergometers, document their training, and participate in the experimental protocols. All participants reported to be free of acute or chronic illnesses and were not taking prescribed medication. After completion of the maximal tests, the individual ventilatory threshold for running and cycling were determined according to previously established criteria (Dekerle et al. 2003). To participate in the experimental protocols, the athletes were asked to refrain from vigorous exercise and also to abstain from running the day before the exercise protocol. The experimental protocols consisted of running on a treadmill at ventilatory threshold intensity for 40 min (RUN) and cycling (CYC) at the same duration and relative intensity in their own bicycles in a cycle simulator (Cateye CS1000, Osaka, Japan). All testing procedures took place in a laboratory with temperature ranging from 18 to 21 °C and relative humidity ranging from 50% to 70%. During the V˙O2max and experimental protocols the following parameters were obtained: oxygen and carbon dioxide fractions and ventilation (MEDGRAPHICS, Cardiopulmonary Diagnostic System, Saint Paul, Minn., USA), heart rate (Polar Electro, Kempele, Finland) and rated perceived exertion (RPE).
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Serum sampling Three blood samples were taken during each protocol: pre exercise (PRE; rest), postexercise (POST; within 15 min), and 1 h after exercise (1h). At each sampling, blood was collected into a plain vaccutainer (BD, USA) and allowed to coagulate for 10 min, before centrifugation (3000g) for 7 min at room temperature. Serum aliquots were immediately frozen at −70 °C. For all blood collection, a 5-mL whole blood sample was taken from the antecubital region from each subject. Serum biochemical analysis Measurements of S100B were performed using an immunoluminescent assay kit (Diasorin, Italy), which uses an antibody labeled with isoluminol. S100B standard curve was linear up to 20 ng/mL and the coefficient of variation (CV) of duplicates in all range levels of standards and samples were within 5%. S100B levels are expressed as ng/mL (Portela et al. 2002). Serum total CK and AST activity were detected using an automatic analyzer (Cobas Integra 400; Roche Diagnostics). Mb concentration was analyzed by electrochemiluminescence immunoassays (Elecsys 2010; Roche Diagnostics). The intra-assay CV was 1.3% for CK, 1.2% for AST and 2.6% for Mb. Statistical analysis To analyze the data distribution, we used the Shapiro–Wilk normality test. We used two-way ANOVA with repeated-measures followed by Bonferroni post hoc test to analyze differences between groups and time points. Area under the curve (AUC) was calculated by definite integral (兰f(x)dx, [a,b]) for each subject where a and b define PRE and 1h, respectively. Differences in AUC were compared using Student’s t test. Outliers were excluded based on z score distribution according to Shiffler (1988), where values higher than ±3 z scores were considered to be outliers. Correlation analyzes between AUC of serum muscle biomarkers and S100B were analyzed using Pearson’s correlation. Data are presented as means ± SE. Statistical significance was accepted at p < 0.05.
Results All participants completed 40 min in each submaximal test. There was no significant difference among relative intensity (RUN = 72 ± 0.05% and CYC = 69% ± 0.06% V˙O2max), respiratory exchange ratio (RUN = 0.93 ± 0.09 and CYC = 0.93 ± 0.03) and RPE (data not shown) on both experimental situations. Serum CK levels were increased after RUN and CYC (time effect: F[2,24] = 39.30, p = 0.0001); however, there was no effect of exercise type (exercise effect: F[1,12] = 1.240, p = 0.2872). In addition, we found an interaction between time and exercise type (time × exercise effect: F[2,24] = 16,62, p = 0.0001). Multiple comparisons correction showed that RUN CK levels at POST and 1h were higher than PRE. However, in CYC only POST was higher than PRE (Fig. 1A). Also, AUC of CK was significantly higher in RUN compared with CYC (p = 0.0114) (Fig. 1B). Serum Mb levels were increased after exercise (time effect: F[2,24] = 24.19, p = 0.0001), and there was a significant effect of exercise type (exercise effect: F[1,12] = 23.69, p = 0.0004). Furthermore, we showed an interaction between time and exercise type (time × exercise effect: F[2,24] = 7.643, p = 0.0027). Multiple comparisons correction showed that RUN Mb levels at POST and 1h were significantly higher than PRE. In CYC, Mb 1h was higher than PRE and POST (Fig. 1C). In addition, AUC of Mb was significantly higher in RUN compared with CYC (p = 0.0078) (Fig. 1D). After exercise, serum AST levels were also increased (time effect: F[2,24] = 26.70, p = 0.0001); however, there was no effect of exercise type (exercise effect: F[1,12] = 1.766, p = 0.2086). In addition, we found an interaction between time and exercise type (time × exercise effect: F[2,24] = 7.569, p = 0.0028). Multiple comparisons correction showed that RUN AST levels POST and 1h were higher Published by NRC Research Press
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Fig. 1. Serum levels of skeletal muscle damage markers (Creatine-kinase (CK), aspartate aminotransferase (AST), and myoglobin (Mb)) in running (RUN) and cycling (CYC) protocols. (A) Before (PRE), immediately after (POST), and 1 h after (1h) CK serum levels; (B) CK area under the curve (AUC); (C) PRE, POST and 1h Mb serum values; (D) Mb AUC; (E) PRE, POST and 1h AST serum levels; (F) AST AUC. Data are presented as means ± SE; lines with circles and lines with triangles represent each replicate. The statistical significance adopted was p < 0.05 (*, RUN vs CYC; †, compared with PRE; ‡, POST vs 1 h).
than PRE (Fig. 1E). Additionally, POST CYC AST level was significantly higher than PRE and 1h. Also, AUC of AST was significantly higher in RUN compared with CYC (p = 0.0052) (Fig. 1F). Serum S100B levels were increased after exercise (time effect: F[2,24] = 4.172, p = 0.0279), but there was no influence of exercise type (exercise effect: F[1,12] = 3.8, p = 0.0415). Additionally, there was an interaction between time and exercise type (time × exercise effect: F[2,24] = 3.915, p = 0.0338). Multiple comparisons correction showed increased S100B in RUN POST when compared with PRE. However, there were no significant changes in S100B levels during CYC protocol (Fig. 2A). Additionally, AUC of S100B was significantly higher in RUN than CYC (p = 0.0352) (Fig. 2B). AUC of S100B showed a significant correlation with AUC of CK (r = 0.6237, p = 0.030) (Fig. 3A) and Mb (r = 0.6118, p = 0.0345) (Fig. 3B) in RUN but not in CYC (r = −0.0430, p = 0.8890; and r = −0.008, p = 0.9792, respectively). Correlation analysis between AUC of S100B and AST showed a trend to reach statistical significance in RUN protocol (r = 0.5637, p = 0.0563), whereas no ten-
dency to significance in CYC was observed (r = 0.1164, p = 0.7048) (Fig. 3C).
Discussion Here we showed that immediately after a submaximal exercise protocol such as running, but not cycling, serum S100B level increased in parallel with levels of CK, AST and Mb, suggesting the leakage of S100B from skeletal muscles was due to eccentric contraction and reversible damage. Also, the lack of significant differences in serum S100B concentrations observed in our CYC protocol corroborates previous results by Schulte et al. (2011) in which subjects were monitored up to 240 min postcycling trials to exhaustion with or without vibration. Considering that in our study both exercise protocols were performed at the same relative intensity with the same duration, the differences in serum S100B levels can be attributed to type of exercise. Moreover, different types of exercise involve distinct strategies of muscle contraction, leading to different levels of Published by NRC Research Press
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Fig. 2. Serum levels of S100B in running (RUN) and cycling (CYC) protocols. (A) Before (PRE), immediately after (POST), and 1 h after (1h) S100B serum levels; (B) S100B area under the curve (AUC). Data are presented as means ± SE; lines with circles and lines with triangles represent each replicate. The statistical significance adopted was p < 0.05 (*, RUN vs CYC; †, compared with PRE).
Fig. 3. Correlations between areas under the curve (AUCs) of serum S100B and typical biomarkers of muscle damage. (A) Positive correlation between creatine-kinase (CK) and S100B levels; (B) positive correlation between myoglobin (Mb) and S100B levels; and (C) correlation between aspartate aminotransferase (AST) and S100B levels. Data are presented as 1 sample per point. The statistical significance adopted was p < 0.05.
muscle injury (Lieber and Friden 2002). Thus, the RUN protocol presents a strong eccentric component and a high mechanical stress imposed on muscles, whereas CYC involves a predominant concentric component, which induces comparatively less muscle damage than RUN. Besides, in our study there is no interindividual variability since the same athletes were engaged in both exercise protocols. In light of this, we assumed that the increase in serum typical biomarkers and S100B levels reveals the higher skeletal muscle damage present shortly after RUN. Actually, Koller et al. (1998) showed that alpine ultramarathon athletes had higher skeletal muscle protein leakage (CK and myosin heavy chain) than alpine long-distance cycling. Mechanistically, it seems that S100B provides a danger signaling from injured muscles and thus, participates in the promyogenic or mitogenic signaling cascades, depending on several factors, including S100B concentration (Riuzzi et al. 2011, 2012). Recent findings have demonstrated that following a mechanical crush, S100B is released from skeletal muscle, and that upon chemical injury to skeletal muscle of mice in vivo, the S100B release peaks at 1 day post-injury (Riuzzi et al. 2011, 2012). These results add to the notion that serum S100B may come from skeletal muscle injured sites and that muscle-derived S100B is a potential myokine (Pedersen and Febbraio 2012) involved in skeletal muscle trophism and (or) regeneration (Riuzzi et al. 2011, 2012). However, to our knowledge few studies have addressed the connection between the typical muscle damage markers and S100B in humans submitted to well-controlled exercise protocols. Based on the functional concepts discussed above, we conjectured that S100B might serve as biomarker of muscle injury in vivo. Although it is well accepted that serum S100B level increases in humans engaged in intense physical exercise, there are controversies regarding the sources of such increment and the dichotomy between trophic release and leakage by damage cells (Dietrich et al. 2003; Hasselblatt et al. 2004). Considering that human stud-
ies encompass limitations regarding mechanistic interpretations, we compared the profile of typical biomarkers of skeletal muscle damage with S100B to test our hypothesis. Remarkably, we found that AUC of S100B is positively correlated with AUC of CK and Mb in RUN protocol, but not in CYC. Similarly, Hasselblatt et al. (2004) reported in marathon runners increased serum S100B and CK levels with different time-to-peak (3 and 20 h, respectively) and a strong positive correlation between AUC of these biomarkers. Considering that is well established that serum CK activity in runners are derived from skeletal muscle, the authors concluded that the robust correlation between S100B and CK strongly suggests extracranial source of S100B. They also propose that S100B and CK determination improves the tissue specificity of S100B as biomarker of injury (Hasselblatt et al. 2004). Considering that we determined the biomarkers profile in a short time frame (up to 1 h after RUN or CYC), our study was not able to predict the exact time-to-peak of S100B release. Although our results reinforce the perception that serum S100B might come from injured myofibers after running, a long-term follow up is necessary to overcome these limitations.
Conclusion The results of this investigation show that immediately after an intense exercise protocol such as running, but not cycling, serum levels of S100B protein increase in parallel with levels of CK, AST and Mb. Likewise, the positive correlation between AUC of S100B and AUC of markers of muscle damage (CK and Mb) points to S100B as an acute biomarker of muscle damage after running exercise.
Acknowledgements This work was supported in part by grants from Brazilian agencies FAPERGS, CNPq, CAPES and INCT-Excitotoxicity and Neuroprotection. Published by NRC Research Press
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ARTICLE Serum S100B level increases after running but not cycling exercise Cintia Mussi Alvim Stocchero, Jean Pierre Oses, Giovani Santos Cunha, Jocelito Bijoldo Martins, Liz Marina Brum, Eduardo Rigon Zimmer, Diogo Onofre Souza, Luis Valmor Portela, and Álvaro Reischak-Oliveira
Abstract: The objective of this study was to investigate the effect of running versus cycling exercises upon serum S100B levels and typical markers of skeletal muscle damage such as creatine kinase (CK), aspartate aminotransferase (AST) and myoglobin (Mb). Although recent work demonstrates that S100B is highly expressed and exerts functional properties in skeletal muscle, there is no previous study that tries to establish a relationship between muscle damage and serum S100B levels after exercise. We conducted a cross-sectional study on 13 male triathletes. They completed 2 submaximal exercise protocols at anaerobic threshold intensity. Running was performed on a treadmill with no inclination (RUN) and cycling (CYC) using a cycle-simulator. Three blood samples were taken before (PRE), immediately after (POST) and 1 h after exercise for CK, AST, Mb and S100B assessments. We found a significant increase in serum S100B levels and muscle damage markers in RUN POST compared with RUN PRE. Comparing groups, POST S100B, CK, AST and Mb serum levels were higher in RUN than CYC. Only in RUN, the area under the curve (AUC) of serum S100B is positively correlated with AUC of CK and Mb. Therefore, immediately after an intense exercise such as running, but not cycling, serum levels of S100B protein increase in parallel with levels of CK, AST and Mb. Additionally, the positive correlation between S100B and CK and Mb points to S100B as an acute biomarker of muscle damage after running exercise. Key words: biochemical markers, muscle damage, muscle contractions, S100B, myoglobin, creatine kinase. Résumé : Cette étude se propose d’examiner l’effet de la course comparativement au cyclisme sur la concentration sérique de S100B et les marqueurs typiques des lésions musculaires : créatine kinase (« CK »), aspartate aminotransférase (« AST ») et myoglobine (« Mb »). Même si des études récentes révèlent une expression élevée de S100B et des propriétés fonctionnelles dans le muscle squelettique, il n’y a pas encore d’études traitant de la relation entre les lésions musculaires et la concentration sérique postexercice de S100B. On réalise une étude transversale auprès de treize triathloniens. Ces derniers participent a` deux protocoles d’exercice sous-maximal au seuil anaérobie. La course s’effectue sur un tapis roulant non incliné (« RUN ») et le vélo (« CYC ») sur un cyclosimulateur. Avant (« PRE »), immédiatement après (« POST ») et 1 h plus tard, on prélève des échantillons de sang pour l’analyse des CK, AST, Mb et S100B. On observe une augmentation significative de la concentration sérique de S100B et des marqueurs de lésion musculaire dans la condition RUN POST comparativement a` la condition RUN PRE. Quand on compare les groupes, on observe en POST des valeurs sériques plus élevées des S100B, CK, AST et Mb en RUN comparativement a` CYC. La surface sous la courbe (« AUC ») de S100B sérique est positivement corrélée avec les AUC de CK et de Mb, et ce, uniquement en RUN. Par conséquent, après un exercice intense comme a` la course, mais pas a` vélo, le niveau sérique de S100B augmente parallèlement aux concentrations sériques des CK, AST et Mb. En outre, la corrélation positive observée entre S100B et CK et MB souligne le rôle de S100B comme biomarqueur immédiat de lésion musculaire a` la suite d’un exercice de course. [Traduit par la Rédaction] Mots-clés : marqueurs biochimiques, lésion musculaire, contractions musculaires, S100B, myoglobine, créatine kinase.
Introduction Creatine-kinase (CK), aspartate aminotransferase (AST) and myoglobin (Mb) are typical serum biomarkers of skeletal muscle damage studied in the field of competitive sports (Lavender and Nosaka 2008; Lippi et al. 2008; Singh et al. 2011). Furthermore, it has been shown that exercise with predominant eccentric contractions causes considerable damage to the sarcolemma, eliciting the leakage of a significant amount of muscle proteins into blood that may serve as biomarkers of damage (Lavender and Nosaka 2008). Skeletal muscle damage causes pain and functional impairment and has a negative
impact on the performance of athletes. Despite some controversies regarding tissue-specificity, the assessment of serum biomarkers provides a chance to reveal the dynamic of muscle abnormalities induced by training protocols. S100B is a calcium-binding protein, which has cerebral and extra-cerebral sources, and it can reach blood either by normal secretion or leakage that is due to damage (Vos et al. 2004; Chaves et al. 2010). In the field of competitive sports, serum quantification of S100B protein gained notoriety because its ability to reflect damage caused by direct impact in the brain (Otto et al. 2000;
Received 4 July 2013. Accepted 9 September 2013. C.M.A. Stocchero. Federal Institute of Education, Science and Technology of Rio Grande do Sul, Porto Alegre RS, Brazil; School of Physical Education, Federal University of Rio Grande do Sul, Porto Alegre RS, Brazil. J.P. Oses. Health and Behavior Graduate Program, Catholic University of Pelotas, Pelotas, Brazil. G.S. Cunha, J.B. Martins, and Á. Reischak-Oliveira. School of Physical Education, Federal University of Rio Grande do Sul, Porto Alegre RS, Brazil. L.M. Brum. Institute of Cardiology of Rio Grande do Sul, Porto Alegre RS, Brazil. E.R. Zimmer, D.O. Souza, and L.V. Portela. Department of Biochemistry, ICBS, Federal University of Rio Grande do Sul, 2600 Ramiro Barcelos street, 90035-003 Porto Alegre, RS, Brazil. Corresponding author: Luis Valmor Portela (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 340–344 (2014) dx.doi.org/10.1139/apnm-2013-0308
Published at www.nrcresearchpress.com/apnm on 27 September 2013.
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Stalnacke et al. 2003, 2004). However, we demonstrated that a non-impact exercise (swimming) also increased serum S100B levels in humans, providing the basis to assume that extra-cerebral sources could additionally release/leak protein into blood (Dietrich et al. 2003). For instance, experimental data show that under stress conditions, cardiomyocytes and adipocytes can leak S100B into medium (Mazzini et al. 2005, 2009; Gonçalves et al. 2010). Furthermore, S100B protein is strategically located on the skeletal muscle membranes, sarcoplasmic reticulum, and transverse tubules acting in association with other proteins linked with calcium homeostasis (Arcuri et al. 2002). The demonstration that S100B is highly expressed and exerts functional properties in skeletal muscle provides the premise upon which to investigate S100B abnormalities in response to exercise-induced muscle damage (Arcuri et al. 2002; Donato et al. 2009; Tubaro et al. 2010; Riuzzi et al. 2011). Moreover, the assessments of serum concentrations of a candidate biomarker along with typical muscle biomarkers may help to determine the cell/tissue origin site and the behavior profile post-injury (Hasselblatt et al. 2004). Here we performed a cross-sectional study to investigate the effects of 2 exercise protocols on serum levels of typical skeletal muscle biomarkers CK, AST and Mb, and S100B protein. To achieve this main objective we used running versus cycling protocols performed at the same relative intensity.
Materials and methods Participants and ethics Thirteen recreational male triathletes (mean ± SD: age, 33.9 ± 6.0 years; height, 177 ± 0.1 cm; mass, 74.7 ± 6.8 kg; body fat, 11.1% ± 4.7%; maximal oxygen uptake (V˙O2max), 62.7 ± 6.6 mL·kg−1·min−1) participated in this study. We have estimated that this sample size would provide sufficient statistical power (B = 0.20) to detect an association of at least 0.7 among muscle damage markers and S100B protein. Reference values for sample size determination were obtained from a previous study investigating S100B and CK association (Hasselblatt et al. 2004). The athletes’ training program included running an average 41.5 ± 14.3 km·week−1, cycling 235 ± 104.8 km·week−1 and swimming 8.44 ± 4.7 km·week−1. The participants were fully informed about the procedures and all possible risks involved in this study. Each volunteer provided written informed consent before participating. The study was approved by the Research Ethics Committee of Federal University of Rio Grande do Sul, Brazil (no. 2007708). Experimental design and exercise protocols All participants of the study were required to complete 4 visits to the laboratory to evaluate their V˙O2max on the treadmill and bicycle ergometers, document their training, and participate in the experimental protocols. All participants reported to be free of acute or chronic illnesses and were not taking prescribed medication. After completion of the maximal tests, the individual ventilatory threshold for running and cycling were determined according to previously established criteria (Dekerle et al. 2003). To participate in the experimental protocols, the athletes were asked to refrain from vigorous exercise and also to abstain from running the day before the exercise protocol. The experimental protocols consisted of running on a treadmill at ventilatory threshold intensity for 40 min (RUN) and cycling (CYC) at the same duration and relative intensity in their own bicycles in a cycle simulator (Cateye CS1000, Osaka, Japan). All testing procedures took place in a laboratory with temperature ranging from 18 to 21 °C and relative humidity ranging from 50% to 70%. During the V˙O2max and experimental protocols the following parameters were obtained: oxygen and carbon dioxide fractions and ventilation (MEDGRAPHICS, Cardiopulmonary Diagnostic System, Saint Paul, Minn., USA), heart rate (Polar Electro, Kempele, Finland) and rated perceived exertion (RPE).
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Serum sampling Three blood samples were taken during each protocol: pre exercise (PRE; rest), postexercise (POST; within 15 min), and 1 h after exercise (1h). At each sampling, blood was collected into a plain vaccutainer (BD, USA) and allowed to coagulate for 10 min, before centrifugation (3000g) for 7 min at room temperature. Serum aliquots were immediately frozen at −70 °C. For all blood collection, a 5-mL whole blood sample was taken from the antecubital region from each subject. Serum biochemical analysis Measurements of S100B were performed using an immunoluminescent assay kit (Diasorin, Italy), which uses an antibody labeled with isoluminol. S100B standard curve was linear up to 20 ng/mL and the coefficient of variation (CV) of duplicates in all range levels of standards and samples were within 5%. S100B levels are expressed as ng/mL (Portela et al. 2002). Serum total CK and AST activity were detected using an automatic analyzer (Cobas Integra 400; Roche Diagnostics). Mb concentration was analyzed by electrochemiluminescence immunoassays (Elecsys 2010; Roche Diagnostics). The intra-assay CV was 1.3% for CK, 1.2% for AST and 2.6% for Mb. Statistical analysis To analyze the data distribution, we used the Shapiro–Wilk normality test. We used two-way ANOVA with repeated-measures followed by Bonferroni post hoc test to analyze differences between groups and time points. Area under the curve (AUC) was calculated by definite integral (兰f(x)dx, [a,b]) for each subject where a and b define PRE and 1h, respectively. Differences in AUC were compared using Student’s t test. Outliers were excluded based on z score distribution according to Shiffler (1988), where values higher than ±3 z scores were considered to be outliers. Correlation analyzes between AUC of serum muscle biomarkers and S100B were analyzed using Pearson’s correlation. Data are presented as means ± SE. Statistical significance was accepted at p < 0.05.
Results All participants completed 40 min in each submaximal test. There was no significant difference among relative intensity (RUN = 72 ± 0.05% and CYC = 69% ± 0.06% V˙O2max), respiratory exchange ratio (RUN = 0.93 ± 0.09 and CYC = 0.93 ± 0.03) and RPE (data not shown) on both experimental situations. Serum CK levels were increased after RUN and CYC (time effect: F[2,24] = 39.30, p = 0.0001); however, there was no effect of exercise type (exercise effect: F[1,12] = 1.240, p = 0.2872). In addition, we found an interaction between time and exercise type (time × exercise effect: F[2,24] = 16,62, p = 0.0001). Multiple comparisons correction showed that RUN CK levels at POST and 1h were higher than PRE. However, in CYC only POST was higher than PRE (Fig. 1A). Also, AUC of CK was significantly higher in RUN compared with CYC (p = 0.0114) (Fig. 1B). Serum Mb levels were increased after exercise (time effect: F[2,24] = 24.19, p = 0.0001), and there was a significant effect of exercise type (exercise effect: F[1,12] = 23.69, p = 0.0004). Furthermore, we showed an interaction between time and exercise type (time × exercise effect: F[2,24] = 7.643, p = 0.0027). Multiple comparisons correction showed that RUN Mb levels at POST and 1h were significantly higher than PRE. In CYC, Mb 1h was higher than PRE and POST (Fig. 1C). In addition, AUC of Mb was significantly higher in RUN compared with CYC (p = 0.0078) (Fig. 1D). After exercise, serum AST levels were also increased (time effect: F[2,24] = 26.70, p = 0.0001); however, there was no effect of exercise type (exercise effect: F[1,12] = 1.766, p = 0.2086). In addition, we found an interaction between time and exercise type (time × exercise effect: F[2,24] = 7.569, p = 0.0028). Multiple comparisons correction showed that RUN AST levels POST and 1h were higher Published by NRC Research Press
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Fig. 1. Serum levels of skeletal muscle damage markers (Creatine-kinase (CK), aspartate aminotransferase (AST), and myoglobin (Mb)) in running (RUN) and cycling (CYC) protocols. (A) Before (PRE), immediately after (POST), and 1 h after (1h) CK serum levels; (B) CK area under the curve (AUC); (C) PRE, POST and 1h Mb serum values; (D) Mb AUC; (E) PRE, POST and 1h AST serum levels; (F) AST AUC. Data are presented as means ± SE; lines with circles and lines with triangles represent each replicate. The statistical significance adopted was p < 0.05 (*, RUN vs CYC; †, compared with PRE; ‡, POST vs 1 h).
than PRE (Fig. 1E). Additionally, POST CYC AST level was significantly higher than PRE and 1h. Also, AUC of AST was significantly higher in RUN compared with CYC (p = 0.0052) (Fig. 1F). Serum S100B levels were increased after exercise (time effect: F[2,24] = 4.172, p = 0.0279), but there was no influence of exercise type (exercise effect: F[1,12] = 3.8, p = 0.0415). Additionally, there was an interaction between time and exercise type (time × exercise effect: F[2,24] = 3.915, p = 0.0338). Multiple comparisons correction showed increased S100B in RUN POST when compared with PRE. However, there were no significant changes in S100B levels during CYC protocol (Fig. 2A). Additionally, AUC of S100B was significantly higher in RUN than CYC (p = 0.0352) (Fig. 2B). AUC of S100B showed a significant correlation with AUC of CK (r = 0.6237, p = 0.030) (Fig. 3A) and Mb (r = 0.6118, p = 0.0345) (Fig. 3B) in RUN but not in CYC (r = −0.0430, p = 0.8890; and r = −0.008, p = 0.9792, respectively). Correlation analysis between AUC of S100B and AST showed a trend to reach statistical significance in RUN protocol (r = 0.5637, p = 0.0563), whereas no ten-
dency to significance in CYC was observed (r = 0.1164, p = 0.7048) (Fig. 3C).
Discussion Here we showed that immediately after a submaximal exercise protocol such as running, but not cycling, serum S100B level increased in parallel with levels of CK, AST and Mb, suggesting the leakage of S100B from skeletal muscles was due to eccentric contraction and reversible damage. Also, the lack of significant differences in serum S100B concentrations observed in our CYC protocol corroborates previous results by Schulte et al. (2011) in which subjects were monitored up to 240 min postcycling trials to exhaustion with or without vibration. Considering that in our study both exercise protocols were performed at the same relative intensity with the same duration, the differences in serum S100B levels can be attributed to type of exercise. Moreover, different types of exercise involve distinct strategies of muscle contraction, leading to different levels of Published by NRC Research Press
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Fig. 2. Serum levels of S100B in running (RUN) and cycling (CYC) protocols. (A) Before (PRE), immediately after (POST), and 1 h after (1h) S100B serum levels; (B) S100B area under the curve (AUC). Data are presented as means ± SE; lines with circles and lines with triangles represent each replicate. The statistical significance adopted was p < 0.05 (*, RUN vs CYC; †, compared with PRE).
Fig. 3. Correlations between areas under the curve (AUCs) of serum S100B and typical biomarkers of muscle damage. (A) Positive correlation between creatine-kinase (CK) and S100B levels; (B) positive correlation between myoglobin (Mb) and S100B levels; and (C) correlation between aspartate aminotransferase (AST) and S100B levels. Data are presented as 1 sample per point. The statistical significance adopted was p < 0.05.
muscle injury (Lieber and Friden 2002). Thus, the RUN protocol presents a strong eccentric component and a high mechanical stress imposed on muscles, whereas CYC involves a predominant concentric component, which induces comparatively less muscle damage than RUN. Besides, in our study there is no interindividual variability since the same athletes were engaged in both exercise protocols. In light of this, we assumed that the increase in serum typical biomarkers and S100B levels reveals the higher skeletal muscle damage present shortly after RUN. Actually, Koller et al. (1998) showed that alpine ultramarathon athletes had higher skeletal muscle protein leakage (CK and myosin heavy chain) than alpine long-distance cycling. Mechanistically, it seems that S100B provides a danger signaling from injured muscles and thus, participates in the promyogenic or mitogenic signaling cascades, depending on several factors, including S100B concentration (Riuzzi et al. 2011, 2012). Recent findings have demonstrated that following a mechanical crush, S100B is released from skeletal muscle, and that upon chemical injury to skeletal muscle of mice in vivo, the S100B release peaks at 1 day post-injury (Riuzzi et al. 2011, 2012). These results add to the notion that serum S100B may come from skeletal muscle injured sites and that muscle-derived S100B is a potential myokine (Pedersen and Febbraio 2012) involved in skeletal muscle trophism and (or) regeneration (Riuzzi et al. 2011, 2012). However, to our knowledge few studies have addressed the connection between the typical muscle damage markers and S100B in humans submitted to well-controlled exercise protocols. Based on the functional concepts discussed above, we conjectured that S100B might serve as biomarker of muscle injury in vivo. Although it is well accepted that serum S100B level increases in humans engaged in intense physical exercise, there are controversies regarding the sources of such increment and the dichotomy between trophic release and leakage by damage cells (Dietrich et al. 2003; Hasselblatt et al. 2004). Considering that human stud-
ies encompass limitations regarding mechanistic interpretations, we compared the profile of typical biomarkers of skeletal muscle damage with S100B to test our hypothesis. Remarkably, we found that AUC of S100B is positively correlated with AUC of CK and Mb in RUN protocol, but not in CYC. Similarly, Hasselblatt et al. (2004) reported in marathon runners increased serum S100B and CK levels with different time-to-peak (3 and 20 h, respectively) and a strong positive correlation between AUC of these biomarkers. Considering that is well established that serum CK activity in runners are derived from skeletal muscle, the authors concluded that the robust correlation between S100B and CK strongly suggests extracranial source of S100B. They also propose that S100B and CK determination improves the tissue specificity of S100B as biomarker of injury (Hasselblatt et al. 2004). Considering that we determined the biomarkers profile in a short time frame (up to 1 h after RUN or CYC), our study was not able to predict the exact time-to-peak of S100B release. Although our results reinforce the perception that serum S100B might come from injured myofibers after running, a long-term follow up is necessary to overcome these limitations.
Conclusion The results of this investigation show that immediately after an intense exercise protocol such as running, but not cycling, serum levels of S100B protein increase in parallel with levels of CK, AST and Mb. Likewise, the positive correlation between AUC of S100B and AUC of markers of muscle damage (CK and Mb) points to S100B as an acute biomarker of muscle damage after running exercise.
Acknowledgements This work was supported in part by grants from Brazilian agencies FAPERGS, CNPq, CAPES and INCT-Excitotoxicity and Neuroprotection. Published by NRC Research Press
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Copyright of Applied Physiology, Nutrition & Metabolism is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
