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Effect of short-term creatine monohydrate supplementation on indirect markers of cellular damage in young soccer players
Science & Sports (2012) 27, 88—93
ORIGINAL ARTICLE
Effect of short-term creatine monohydrate supplementation on indirect markers of cellular damage in young soccer players Effet de la supplémentassion à court terme du monohydrate de créatine sur les marqueurs indirects de dommages cellulaires chez de jeunes footballeurs S. Atashak a, A. Jafari b,∗ a b
Department of Physical Education and Sports Sciences, Mahabad Branch, Islamic Azad University, Mahabad, Iran Faculty of Physical Education and Sports Sciences, University of Tabriz, 29 Bahman Street, Tabriz, Iran
Received 14 September 2010; accepted 1st June 2011 Available online 23 July 2011
KEYWORDS Creatine loading; Creatine kinase; Lactate dehydrogenase; Soccer
∗
Summary Purpose. — In recent years, creatine (Cr) supplementation has received great attention in both popular and scientific media. Nevertheless, there is anecdotal information on the side effects of this supplement. Hence, we hypothesized that Cr monohydrate (CrM) loading would induce increase in serum enzymes activity as indirect markers of cellular damage. Therefore, the present study was conducted to identify the effect of short-term Cr supplementation on serum lactate dehydrogenase (LDH), creatine phosphokinase (CK), and myocardial CK isoform (CKMB ) in young male soccer players. Methods. — Eighteen volunteer young male soccer players in a randomized and double-blind design were divided into two equal groups (Cr & Placebo groups). Creatine (CrM) group was ingested Cr supplementation (0.3 g/kg/d for 7 days), but placebo group (Pl) was not took any supplements. All subjects were participated in circuit weight training (eight exercises, three sets, 15 repetitions, and 60% one repetition maximum [1RM]). To identify enzymes activity (IU/L), venous blood samples were obtained before Cr loading and 24 h after last session of weight training, Moreover, VO2 max each of subjects were obtained with 20 m Shuttle Run test. Data were expressed as mean (±SD) and analyzed by paired and independent t-tests using SPSS15 at P ≤ 0.05.
Corresponding author. E-mail address: [email protected] (A. Jafari).
0765-1597/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2011.06.001
Creatine monohydrate supplementation on indirect markers of cellular dommage
89
Results. — The modest elevation of LDH activity in both groups were not significant following the short-term Cr supplementation, whereas, mean and changes range of CK (P = 0.0004, tCK = 5.13) and CKMB (P < 0.05, tCKMB = 2.10) activities in CrM group were significantly higher than in Pl group (P < 0.05). Conclusion. — The present results suggest that serum CK and CKMB activity as indirect markers of cellular damage increases by the oral short-term CrM supplementation in young male soccer players. Therefore, it can be concluded that CrM loading probably have significant adverse effects on serum indirect markers of cellular damage. Nevertheless, more research will be needed to determine effect of the short and long-term Cr supplementation on the other indirect markers of cellular damages in the future. © 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Supplémentation en créatine ; Créatine kinase ; Lactico-hydrogénase ; Footballeur
Résumé But. — Ces dernières années, la supplémentation de créatine a retenu la plus grande attention des médias populaires et scientifiques. Néanmoins, il n’y a pas d’information valide sur les effets secondaires de ce supplément. C’est pourquoi nous formulons l’hypothèse que le chargement de monohydrate de créatine (CrM) augmente l’activité d’enzymes de sérum comme des marqueurs indirects de dommages cellulaires. Donc, l’étude présente a été faite pour étudier l’effet à cour terme d’une supplémentation en créatine sur la lactico-déshydrogénase (LDH), la créatine phosphokinase (CK) et l’isoforme CK du myocarde (CKMB) sériques chez de jeunes footballeurs. Méthodes. — Dans une étude aléatoire en double aveugle, 18 jeunes footballeurs ont participé volontairement et ont été divisés en deux groupes égaux (le groupe de Créatine et le groupe de Placebo). Le premier groupe (CrM) a consommé le supplément de créatine (0,3 g/kg par jour pendant sept jours), mais le groupe de placebo (Pl) n’a pas pris des supplément. Tous les sujets ont participés à un programme d’entraînement (huit exercices, trois séries, 15 répétitions, et 60 % one repetition maximum [1RM]). Afin de détecter l’activité enzymatique (IU/L), les échantillons de sang veineux ont été prélevés avant le chargement en créatine et 24 heures après la dernière séance d’entraînement. De plus, VO2 max de chaque sujet a été obtenu avec le test navette (20 m). Les données ont été exprimées en moyens (± SD) et elles ont analysées par les t-tests dépendant et indépendant en utilisant SPSS15. Résultats. — L’augmentation moyenne d’activité de LDH dans les deux groupes n’était pas significative à la suite de la supplémentassion à court terme de créatine, tandis que, la moyenne et la limite de changement d’activité de CK (p = 0,0004 ; tCK = 5,13) et de CKMB (p < 0,05 ; tCKMB = 2,10) ont été significativement plus élevées dans le groupe de CrM par rapport au groupe de Pl (p < 0,05). Conclusion. — Les résultats présents suggèrent que l’activité des enzymes de CK et de CKMB comme des marqueurs indirects de dommages cellulaires apès la supplémentassions à court terme de CrM augmente chez les jeunes footballeurs. Donc, il peut être conclu que le chargement en CrM a probablement des effets significatifs défavorables sur les marqueurs sériques indirects de dommages cellulaires. Néanmoins, d’autres études seront nécessaires pour déterminer l’effet à long et court termes de la supplémentassions de créatine sur les autres marqueurs indirects de dommages cellulaires. © 2011 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Soccer is a multiple-sprint sport involves a mixture of repeated intense exercises followed by recovery periods or light activities [1]. Moreover, previous studies have shown that creatine (Cr) supplementation can significantly increases strength, power, sprint performance, and/or work performed during multiple sets of maximal effort muscle contractions in many professional and amateur athletes such as soccer players [2,3]. Since many athletes consume Cr supplements during intense training periods as well as before major competitions [3]. Cr monohydrate (CrM) (a-methyl guandino-acetic acid), a naturally occurring nitrogenous compound is both synthesized endogenously mainly in the
liver as well as in pancreas and kidney from three amino acids; glycine, arginine, methionine; and provided through normal dietary intake for body [4,5]. However, there is anecdotal information about of Cr supplementation effects on various aspects of athlete’s health [6—10]. Nevertheless, some previous studies did not show any obvious risk of ingesting Cr supplements at the recommended doses [8,9,11,12]. Whereas, other studies have demonstrated that Cr supplementation may increase muscle or liver damage [6,13—16]. Some studies imply that Cr supplementation increases cell membrane disruption and plasma enzymes levels such as creatine phosphokinase (CK) and lactate dehydrogenase (LDH) [6,13]. However, a definite mechanism has not been suggested for acute and chronic effects of Cr sup-
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S. Atashak, A. Jafari
plementation on cellular damages or undesirable changes in serum enzymes activity [14]. Since, previous results regarding the side effects of short or long-term Cr supplementation are conflicting [6,8,13,14], this study was conducted to determine effect of short-term CrM supplementation (loading phase) on serum LDH, Cr kinase, and myocardial CK isoform (CKMB ) in young male soccer players.
2. Methodology 2.1. Subjects Eighteen young male soccer players (aged 17.5 ± 0.3 years, height 173.33 ± 6.5 cm and weight 68.61 ± 11.8 kg) in a randomized and double-blind design were allocated in two equal CrM supplement and placebo groups. None of the subjects had ingested CrM, or any other dietary supplements before initiation of the study. In addition, they have no past history of kidney, heart and liver disease, diabetes or any physical damage and problems. The study design and experimental procedures approved by the Regional Research Ethics Committee of Tabriz University of Medical Sciences and conducted in the laboratory conditions (temperature 22—25◦ C; humidity 50—55%).
2.2. Supplementation protocol The subjects were randomly assigned to either the CrM (5 g CrM plus 10 g of flavored dextrose powder per packet equal to 0.3 g/day/kg or total of 20 g/day CrM produced by P.N.C Co) or placebo (PL — 10 g of flavored dextrose powder per packet) groups. Both treatments were effervescent powders, prepackaged to be identical in taste and appearance, and were dissolved in 250—300 mL of water. Each group ingested one packet four times per day at regular intervals (breakfast, lunch and dinner and at 10 pm) for 7 days [17—19].
2.3. Aerobic test In order to adjust and control the effects of maximal oxygen uptake on recovery time and fatigue in the circuit weight training, VO2 max was measured and recorded [20—22]. Therefore, one week before the supplementation protocol, all subjects participated in the maximal multistage 20-m shuttle run test [23]. The shuttle run test was carried out on flat terrain and between two parallel lines 20 m apart. The test ended when the participant failed to come within 2 m of the ends of the lines at the moment indicated by the tape. Therefore, the maximal oxygen uptake or VO2 max of each subject was predicted by the ended level and shuttle run test’s equation [24]. SpVO2 max. = −27.4 + 6.0 MAS. SpVO2 max is predicted VO2 max in mL/kg/min and MAS is maximal aerobic speed in km/hr i.e. maximum speed reached during the test.
Figure 1
Circuit training.
2.4. One repetition maximum (1RM) The true 1RM for the upper and lower extremities was estimated according to the National Strength and Conditioning Association guidelines and calculated by the Brzycki equation (1RM = Weight/(1.0278 − (0.0278 × Number of repetitions)). After a warm-up period, all subjects lifted submaximal loads until exhaustion (7—10 RM). The appropriate load was based on each subject’s weights used for circuit training in pre- competition season [25,26].
2.5. Training protocol All subjects were participated in a circuit weight training (eight exercises, three sets, 15 repetitions and 60% 1RM). Two days prior to initiation of training protocol, participants underwent a familiarization session of the training equipment that would be utilized during the study. The subjects performed a warm-up (15 min) before of each session. Eight stations of circuit training was included leg curls, bench press, leg extension, lat pull, sit & rich, push-up, biceps curl and chin-p. Rest periods between stations were considered from 60 to 90 seconds (Fig. 1).
2.6. Data collection All subjects were asked to be present in laboratory one day before starting of loading protocol in morning (9—10 am) to obtain blood samples. Blood samples were obtained from subject’s antecubital vein using venepuncture (5 mL; made by SUHA Co) before and 24 h after the loading protocol. Finally, serum was separated by a centrifuge (made by SAHAND Co) and serum CK, CKMB and LDH activities determined by commercial kits (Sigma Chemical Co) with automatic analyzers (RA-1000; made by American TECHNICOM Co).
2.7. Statistical analysis Data distribution normality was tested using the Kolmogorov Smirnov test. Normally distributed data were expressed
Creatine monohydrate supplementation on indirect markers of cellular dommage Table 1 Physical characteristics of the creatine and placebo groups. Variable
Creatine (n = 9)
Placebo (n = 9)
Age (years) Weight (kg) Height (cm) BMI (kg/m2 ) Body fat (%) VO2 max (ml/kg/min)
17.41 ± 0.26 66.90 ± 7.89 174.60 ± 4.87 22.40 ± 2.84 15.48 ± 2.83 42.58 ± 2.92
17.55 ± 0.35 70.33 ± 15.22 172.08 ± 7.91 21.91 ± 1.97 16.64 ± 5.29 40.22 ± 2.84
Values are mean ± standard deviation.
as means (± SD) and analyzed by dependent (paired) and independent t-tests. A paired t-test was performed to detect differences of pre-post means of each group separately. Then pre-post changes range differences between the groups were analyzed by independent t-test. Moreover, correlations between the parameters were examined with the Pearson’s correlation test (r). All statistical analyses were performed using the SPSS statistical software package (SPSS version 15.0 for Windows, SPSS Inc., Chicago, IL, USA). The significance level was set at P ≤ 0.05.
3. Results There were no differences among groups at the beginning of the research for age, body weight, height, percent body fat or lean mass and VO2 max (Table 1). All subjects reported adherence to the experimental protocol and complete ingestion of the supplement. The pre-post serum enzymes concentrations are presented in Table 2. The results show that short-term Cr supplementation has no significant effect (P > 0.05) on LDH enzyme activity. However, serum CK and CKMB activities were significantly increased following the short-term supplementation (P < 0.05 (Table 2). The pre-post changes of LDH activity (P = 0.609, tLDH = 0.52) indicated no significant differences between Cr and placebo groups, whereas mean and changes range of CK (P = 0.0004, tCK = 5.13) and CKMB (P < 0.05, tCKMB = 2.10) in CrM group were significantly higher than in Pl group (Table 2).
4. Discussion Despite a large number of publications on the ergogenic effects of Cr [5,9,12,17], there are limited, anecdotal reports on the possible adverse effects of this supplement
Table 2
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[6—9,12]. Hence, this study was done to clarify the effects of CrM loading on some serum indirect markers of cellular damage in young soccer players who participated in one week circuit weight training. Indirect markers of cellular damage were chemically inferred through serum CK, CKMB and LDH activities. The present results show that short-term Cr supplementation does not have significant effect on serum LDH activity in young soccer players (Table 2). This finding confirms the results of Bassit et al., Kreider et al., Rosene et al., Santos et al. and Schröder et al. [8,13,27—29], whereas it contrasts to the finding of Rawsson et al. [30]. The discrepancy between the present result and Rawsson finding may be due to the greater baseline of the enzyme activity at the beginning of the present study (Pre LDH 400 IU/L). Moreover, it was clarified that the peak activity in serum LDH observes 8 h after exercise, while serum enzymes activity in present study determined 24 hours after the last session of weight training. In addition, the positive significant correlation between post and changes ranges of LDH activity (r = 0.846, P < 0.001) indicates that the modest increase of LDH activity may result in the oral short-term Cr supplementation. However, it should be pointed out that the post value of LDH and the other serum enzymes (CK & CKMB ) in this study were slightly above the clinical norms for untrained individuals and within the normal range for athletes [27]. In consistent with previous studies [13,30—32], the increased serum CK and CKMB activities in CrM group were significantly higher than Pl group following the short-term Cr supplementation. Furthermore, the positive significant relationship between pre-post changes and post values in these enzymes confirms that all alterations have occurred following Cr loading. Nevertheless, this result is contrast to some previous findings of Cancela et al., Rawson et al., Schröder et al. and Kreider et al. [13,30—33]. The discrepancy between our result and some previous studies may be related to differences in methodology design (human versus animal model studies), supplementation (short-term versus long-term supplementation with low or supraphysiological doses) and exercise training protocols (intensity, duration and frequency of various exercises). Souza et al., for example, have demonstrated that four weeks of supraphysiological doses of Cr supplementation resulted increase the levels of direct and indirect markers of tissue damage (Histochemical, and extracellular enzymes levels change) in untrained rats (but not in trained). Hence, they suggested that the exercise could block the side effects of high-dose Cr supplementation [6]. While Kreider
Changes of serum enzymes activity in the placebo and creatine groups following supplementation. Stages
LDH activities (IU/L)
CK activities (IU/L)
CKMB activities (IU/L)
Before Supplementation After Supplementation
393.33 ± 42.42 407.56 ± 44.66
141.22 ± 42.88 165.33 ± 65.29a
19.67 ± 1.5 19.88 ± 3.86a
Creatine Before Supplementation group After Supplementation
400 ± 51.38 435.56 ± 79.39
126 ± 27.18 345.67 ± 106.03a,b
19.89 ± 2.03 22 ± 5.43a
Placebo group
LDH: lactate dehydrogenase; CK: creatine phosphokinase; CKMB : myocardial CK. a If P < 0.05 between placebo and creatine groups. b If P < 0.05 between pre-post supplementation.
92 et al. reported that high extracellular enzymes activity in athletes following Cr supplementation probably might be a consequence of participating in the high intensity exercises [27]. Moreover, Souza et al. speculated that exercise training could protect against kidney and liver alterations, by increasing Cr consumption by skeletal muscles during exercise training, resulting in a decreased Cr accumulation in the kidneys and liver [6]. This group hypothesized that the Cr accumulation into the tissue, which has low metabolic capacity to convert Cr into creatinine and is enzymatically capable of accomplishing the metilation processes, contributing to the formation and accumulation of citotoxic substances, such as formaldehyde and methylamine [6]. Because some studies have demonstrated that short-term, high-dose oral Cr supplementation can increase the production of methylamine and formaldehyde (Deaminated methylamine) as potential cytotoxic compounds, may potentially cause protein cross-linkage and increase blood malondialdehyde (an indicator of lipid peroxidation of phospholipids’ membrane or cellular damage) and subsequently serious unwanted side effects [6,14,15]. On the other hand, it can be speculated that deamination of methylamine in circulation may induce increase of cellular damages in different tissues (especially renal glomerula). Nevertheless, in humans, most of the studies investigated the potential for toxicity of Cr supplementation has not found evidence of adverse effects when ingested at low doses in long-term supplementation [11,27,29]. According to the present results and previous findings, it can be concluded that short-term CrM supplementation (during loading phase) may cause undesirable increase in serum enzymes activity, as indirect markers of cellular damage in young soccer players (but within normal range of athletes). However, since the exact mechanism in which Cr supplementation may affect in cellular damage have not fully been elucidated and the ratio of CKMB /CK as cardiovascular risk factor is less than 0.2 hence, it is not possible to affirm that Cr supplementation lead to tissue damage. On the other hand, further studies are necessary to clarify the possible side effects of short and long-term Cr supplementation in different organs. Therefore, it should be cautious about the Cr ingestion until the actual adverse effects of this supplement on the young athletes are completely identified.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments This study was carried out in the faculty of Physical Education & Sports Sciences in university of Tabriz. This paper is based on a thesis submitted by S. Atashak to the Ministry of Education, in partial fulfillment of the requirements for the MSc degree in physiology from university of Tabriz, Tabriz, Iran. We thank Loghman Radpey and Faezeh Sohrabi for their suggestion that help to improve the manuscript. The authors are indebted to Dr. R.S. Shabani for French translation of the abstract.
S. Atashak, A. Jafari Authors’ contributions: Afshar Jafari coordinated the study and prepared the manuscript. Sirvan Atashak carried out the design and participated in all the experiments and the manuscript preparation. All authors have read and approved the content of the manuscript.
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Creatine monohydrate supplementation on indirect markers of cellular dommage [17] Gotshalk LA, Kraemer WJ, Mendonca MAG, Vingren JL, Kenny AM, Spiering BA, et al. Creatine supplementation improves muscular performance in older women. Eur J Appl Physiol 2008;102(2):223—31. [18] Gualano B, Ugrinowitsch C, Novaes RB, Artioli GG, Shimizu MH, Seguro AC, et al. Effects of creatine supplementation on renal function: a randomized, double-blind, placebocontrolled clinical trial. Eur J Appl Physiol 2008;103(1): 33—40. [19] Smith AE, Walter AA, Herda TJ, Ryan ED, Moon JR, Cramer JT, et al. Effects of creatine loading on electromyographic fatigue threshold during cycle ergometry in college-aged women. J Int Soc Sports Nutr 2007;4:20. [20] Williams AJ. Effect of oxygen on recovery from maximal exercise in COPD. Thorax 2005;60(3):257—8. [21] Stevenson NJ, Calverley PMA. Effect of oxygen on recovery from maximal exercise in patients with chronic obstructive pulmonary disease. Thorax 2004;59(8):668—72. [22] Short KR, Sedlock DA. Excess postexercise oxygen consumption and recovery rate in trained and untrained subjects. J Appl Physiol 1997;83(1):153—9. [23] Leger LA, Lambert J. A maximal multistage 20-m shuttle run test to predict VO2 max. Eur J Appl Physiol Occup Physiol 1982;49(1):1—12. [24] Ramsbottom R, Brewer J, Williams C, progressive shuttle run test to estimate maximal oxygen A, Br F uptake. Sports Med 1988;22(4):141—4. [25] Brzycki M. A practical approach to strength training. 3rd ed. Indianapolis: Masters Press; 1995.
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[26] Eston R, Evans HJL. The validity of submaximal ratings of perceived exertion to predict one repetition maximum. J Sport Sci Med 2009;8(4):567—73. [27] Kreider RB, Melton C, Rasmussen CJ, Greenwood M, Lancaster S, Cantler EC, et al. Long-term creatine supplementation does not significantly affect clinical markers of health in athletes. Mol Cell Biochem 2003;244(1):95— 104. [28] Santos RVT, Bassit RA, Caperuto EC, Rosa LFBPC. The effect of creatine supplementation upon inflammatory and muscle soreness markers after a 30 km race. Life Sci 2004;75(16): 1917—24. [29] Bassit RA, Pinheiro CH, Vitzel KF, Sproesser AJ, Silveira LR, Curi R. Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity. Eur J Appl Physiol 2010;108(5): 945—55. [30] Rawson ES, Gunn B, Clarkson PM. The effects of creatine supplementation on exercise-induced muscle damage. J Strength Cond Res 2001;15(2):178—84. [31] Cancela P, Ohanian C, Cuitino E, Hackney AC. Creatine supplementation does not affect clinical health markers in soccer players. Br J Sports Med 2007. [32] Cancela P, Ohanian C, Cuitino E, Hackney AC. Creatine supplementation does not affect clinical health markers in football players. Brit J Sport Med 2008;42(9):731—5. [33] Kreider RB. Effects of creatine supplementation on performance and training adaptations. Mol Cell Biochem 2003;244(1):89—94.
ORIGINAL ARTICLE
Effect of short-term creatine monohydrate supplementation on indirect markers of cellular damage in young soccer players Effet de la supplémentassion à court terme du monohydrate de créatine sur les marqueurs indirects de dommages cellulaires chez de jeunes footballeurs S. Atashak a, A. Jafari b,∗ a b
Department of Physical Education and Sports Sciences, Mahabad Branch, Islamic Azad University, Mahabad, Iran Faculty of Physical Education and Sports Sciences, University of Tabriz, 29 Bahman Street, Tabriz, Iran
Received 14 September 2010; accepted 1st June 2011 Available online 23 July 2011
KEYWORDS Creatine loading; Creatine kinase; Lactate dehydrogenase; Soccer
∗
Summary Purpose. — In recent years, creatine (Cr) supplementation has received great attention in both popular and scientific media. Nevertheless, there is anecdotal information on the side effects of this supplement. Hence, we hypothesized that Cr monohydrate (CrM) loading would induce increase in serum enzymes activity as indirect markers of cellular damage. Therefore, the present study was conducted to identify the effect of short-term Cr supplementation on serum lactate dehydrogenase (LDH), creatine phosphokinase (CK), and myocardial CK isoform (CKMB ) in young male soccer players. Methods. — Eighteen volunteer young male soccer players in a randomized and double-blind design were divided into two equal groups (Cr & Placebo groups). Creatine (CrM) group was ingested Cr supplementation (0.3 g/kg/d for 7 days), but placebo group (Pl) was not took any supplements. All subjects were participated in circuit weight training (eight exercises, three sets, 15 repetitions, and 60% one repetition maximum [1RM]). To identify enzymes activity (IU/L), venous blood samples were obtained before Cr loading and 24 h after last session of weight training, Moreover, VO2 max each of subjects were obtained with 20 m Shuttle Run test. Data were expressed as mean (±SD) and analyzed by paired and independent t-tests using SPSS15 at P ≤ 0.05.
Corresponding author. E-mail address: [email protected] (A. Jafari).
0765-1597/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.scispo.2011.06.001
Creatine monohydrate supplementation on indirect markers of cellular dommage
89
Results. — The modest elevation of LDH activity in both groups were not significant following the short-term Cr supplementation, whereas, mean and changes range of CK (P = 0.0004, tCK = 5.13) and CKMB (P < 0.05, tCKMB = 2.10) activities in CrM group were significantly higher than in Pl group (P < 0.05). Conclusion. — The present results suggest that serum CK and CKMB activity as indirect markers of cellular damage increases by the oral short-term CrM supplementation in young male soccer players. Therefore, it can be concluded that CrM loading probably have significant adverse effects on serum indirect markers of cellular damage. Nevertheless, more research will be needed to determine effect of the short and long-term Cr supplementation on the other indirect markers of cellular damages in the future. © 2011 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Supplémentation en créatine ; Créatine kinase ; Lactico-hydrogénase ; Footballeur
Résumé But. — Ces dernières années, la supplémentation de créatine a retenu la plus grande attention des médias populaires et scientifiques. Néanmoins, il n’y a pas d’information valide sur les effets secondaires de ce supplément. C’est pourquoi nous formulons l’hypothèse que le chargement de monohydrate de créatine (CrM) augmente l’activité d’enzymes de sérum comme des marqueurs indirects de dommages cellulaires. Donc, l’étude présente a été faite pour étudier l’effet à cour terme d’une supplémentation en créatine sur la lactico-déshydrogénase (LDH), la créatine phosphokinase (CK) et l’isoforme CK du myocarde (CKMB) sériques chez de jeunes footballeurs. Méthodes. — Dans une étude aléatoire en double aveugle, 18 jeunes footballeurs ont participé volontairement et ont été divisés en deux groupes égaux (le groupe de Créatine et le groupe de Placebo). Le premier groupe (CrM) a consommé le supplément de créatine (0,3 g/kg par jour pendant sept jours), mais le groupe de placebo (Pl) n’a pas pris des supplément. Tous les sujets ont participés à un programme d’entraînement (huit exercices, trois séries, 15 répétitions, et 60 % one repetition maximum [1RM]). Afin de détecter l’activité enzymatique (IU/L), les échantillons de sang veineux ont été prélevés avant le chargement en créatine et 24 heures après la dernière séance d’entraînement. De plus, VO2 max de chaque sujet a été obtenu avec le test navette (20 m). Les données ont été exprimées en moyens (± SD) et elles ont analysées par les t-tests dépendant et indépendant en utilisant SPSS15. Résultats. — L’augmentation moyenne d’activité de LDH dans les deux groupes n’était pas significative à la suite de la supplémentassion à court terme de créatine, tandis que, la moyenne et la limite de changement d’activité de CK (p = 0,0004 ; tCK = 5,13) et de CKMB (p < 0,05 ; tCKMB = 2,10) ont été significativement plus élevées dans le groupe de CrM par rapport au groupe de Pl (p < 0,05). Conclusion. — Les résultats présents suggèrent que l’activité des enzymes de CK et de CKMB comme des marqueurs indirects de dommages cellulaires apès la supplémentassions à court terme de CrM augmente chez les jeunes footballeurs. Donc, il peut être conclu que le chargement en CrM a probablement des effets significatifs défavorables sur les marqueurs sériques indirects de dommages cellulaires. Néanmoins, d’autres études seront nécessaires pour déterminer l’effet à long et court termes de la supplémentassions de créatine sur les autres marqueurs indirects de dommages cellulaires. © 2011 Elsevier Masson SAS. Tous droits réservés.
1. Introduction Soccer is a multiple-sprint sport involves a mixture of repeated intense exercises followed by recovery periods or light activities [1]. Moreover, previous studies have shown that creatine (Cr) supplementation can significantly increases strength, power, sprint performance, and/or work performed during multiple sets of maximal effort muscle contractions in many professional and amateur athletes such as soccer players [2,3]. Since many athletes consume Cr supplements during intense training periods as well as before major competitions [3]. Cr monohydrate (CrM) (a-methyl guandino-acetic acid), a naturally occurring nitrogenous compound is both synthesized endogenously mainly in the
liver as well as in pancreas and kidney from three amino acids; glycine, arginine, methionine; and provided through normal dietary intake for body [4,5]. However, there is anecdotal information about of Cr supplementation effects on various aspects of athlete’s health [6—10]. Nevertheless, some previous studies did not show any obvious risk of ingesting Cr supplements at the recommended doses [8,9,11,12]. Whereas, other studies have demonstrated that Cr supplementation may increase muscle or liver damage [6,13—16]. Some studies imply that Cr supplementation increases cell membrane disruption and plasma enzymes levels such as creatine phosphokinase (CK) and lactate dehydrogenase (LDH) [6,13]. However, a definite mechanism has not been suggested for acute and chronic effects of Cr sup-
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plementation on cellular damages or undesirable changes in serum enzymes activity [14]. Since, previous results regarding the side effects of short or long-term Cr supplementation are conflicting [6,8,13,14], this study was conducted to determine effect of short-term CrM supplementation (loading phase) on serum LDH, Cr kinase, and myocardial CK isoform (CKMB ) in young male soccer players.
2. Methodology 2.1. Subjects Eighteen young male soccer players (aged 17.5 ± 0.3 years, height 173.33 ± 6.5 cm and weight 68.61 ± 11.8 kg) in a randomized and double-blind design were allocated in two equal CrM supplement and placebo groups. None of the subjects had ingested CrM, or any other dietary supplements before initiation of the study. In addition, they have no past history of kidney, heart and liver disease, diabetes or any physical damage and problems. The study design and experimental procedures approved by the Regional Research Ethics Committee of Tabriz University of Medical Sciences and conducted in the laboratory conditions (temperature 22—25◦ C; humidity 50—55%).
2.2. Supplementation protocol The subjects were randomly assigned to either the CrM (5 g CrM plus 10 g of flavored dextrose powder per packet equal to 0.3 g/day/kg or total of 20 g/day CrM produced by P.N.C Co) or placebo (PL — 10 g of flavored dextrose powder per packet) groups. Both treatments were effervescent powders, prepackaged to be identical in taste and appearance, and were dissolved in 250—300 mL of water. Each group ingested one packet four times per day at regular intervals (breakfast, lunch and dinner and at 10 pm) for 7 days [17—19].
2.3. Aerobic test In order to adjust and control the effects of maximal oxygen uptake on recovery time and fatigue in the circuit weight training, VO2 max was measured and recorded [20—22]. Therefore, one week before the supplementation protocol, all subjects participated in the maximal multistage 20-m shuttle run test [23]. The shuttle run test was carried out on flat terrain and between two parallel lines 20 m apart. The test ended when the participant failed to come within 2 m of the ends of the lines at the moment indicated by the tape. Therefore, the maximal oxygen uptake or VO2 max of each subject was predicted by the ended level and shuttle run test’s equation [24]. SpVO2 max. = −27.4 + 6.0 MAS. SpVO2 max is predicted VO2 max in mL/kg/min and MAS is maximal aerobic speed in km/hr i.e. maximum speed reached during the test.
Figure 1
Circuit training.
2.4. One repetition maximum (1RM) The true 1RM for the upper and lower extremities was estimated according to the National Strength and Conditioning Association guidelines and calculated by the Brzycki equation (1RM = Weight/(1.0278 − (0.0278 × Number of repetitions)). After a warm-up period, all subjects lifted submaximal loads until exhaustion (7—10 RM). The appropriate load was based on each subject’s weights used for circuit training in pre- competition season [25,26].
2.5. Training protocol All subjects were participated in a circuit weight training (eight exercises, three sets, 15 repetitions and 60% 1RM). Two days prior to initiation of training protocol, participants underwent a familiarization session of the training equipment that would be utilized during the study. The subjects performed a warm-up (15 min) before of each session. Eight stations of circuit training was included leg curls, bench press, leg extension, lat pull, sit & rich, push-up, biceps curl and chin-p. Rest periods between stations were considered from 60 to 90 seconds (Fig. 1).
2.6. Data collection All subjects were asked to be present in laboratory one day before starting of loading protocol in morning (9—10 am) to obtain blood samples. Blood samples were obtained from subject’s antecubital vein using venepuncture (5 mL; made by SUHA Co) before and 24 h after the loading protocol. Finally, serum was separated by a centrifuge (made by SAHAND Co) and serum CK, CKMB and LDH activities determined by commercial kits (Sigma Chemical Co) with automatic analyzers (RA-1000; made by American TECHNICOM Co).
2.7. Statistical analysis Data distribution normality was tested using the Kolmogorov Smirnov test. Normally distributed data were expressed
Creatine monohydrate supplementation on indirect markers of cellular dommage Table 1 Physical characteristics of the creatine and placebo groups. Variable
Creatine (n = 9)
Placebo (n = 9)
Age (years) Weight (kg) Height (cm) BMI (kg/m2 ) Body fat (%) VO2 max (ml/kg/min)
17.41 ± 0.26 66.90 ± 7.89 174.60 ± 4.87 22.40 ± 2.84 15.48 ± 2.83 42.58 ± 2.92
17.55 ± 0.35 70.33 ± 15.22 172.08 ± 7.91 21.91 ± 1.97 16.64 ± 5.29 40.22 ± 2.84
Values are mean ± standard deviation.
as means (± SD) and analyzed by dependent (paired) and independent t-tests. A paired t-test was performed to detect differences of pre-post means of each group separately. Then pre-post changes range differences between the groups were analyzed by independent t-test. Moreover, correlations between the parameters were examined with the Pearson’s correlation test (r). All statistical analyses were performed using the SPSS statistical software package (SPSS version 15.0 for Windows, SPSS Inc., Chicago, IL, USA). The significance level was set at P ≤ 0.05.
3. Results There were no differences among groups at the beginning of the research for age, body weight, height, percent body fat or lean mass and VO2 max (Table 1). All subjects reported adherence to the experimental protocol and complete ingestion of the supplement. The pre-post serum enzymes concentrations are presented in Table 2. The results show that short-term Cr supplementation has no significant effect (P > 0.05) on LDH enzyme activity. However, serum CK and CKMB activities were significantly increased following the short-term supplementation (P < 0.05 (Table 2). The pre-post changes of LDH activity (P = 0.609, tLDH = 0.52) indicated no significant differences between Cr and placebo groups, whereas mean and changes range of CK (P = 0.0004, tCK = 5.13) and CKMB (P < 0.05, tCKMB = 2.10) in CrM group were significantly higher than in Pl group (Table 2).
4. Discussion Despite a large number of publications on the ergogenic effects of Cr [5,9,12,17], there are limited, anecdotal reports on the possible adverse effects of this supplement
Table 2
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[6—9,12]. Hence, this study was done to clarify the effects of CrM loading on some serum indirect markers of cellular damage in young soccer players who participated in one week circuit weight training. Indirect markers of cellular damage were chemically inferred through serum CK, CKMB and LDH activities. The present results show that short-term Cr supplementation does not have significant effect on serum LDH activity in young soccer players (Table 2). This finding confirms the results of Bassit et al., Kreider et al., Rosene et al., Santos et al. and Schröder et al. [8,13,27—29], whereas it contrasts to the finding of Rawsson et al. [30]. The discrepancy between the present result and Rawsson finding may be due to the greater baseline of the enzyme activity at the beginning of the present study (Pre LDH 400 IU/L). Moreover, it was clarified that the peak activity in serum LDH observes 8 h after exercise, while serum enzymes activity in present study determined 24 hours after the last session of weight training. In addition, the positive significant correlation between post and changes ranges of LDH activity (r = 0.846, P < 0.001) indicates that the modest increase of LDH activity may result in the oral short-term Cr supplementation. However, it should be pointed out that the post value of LDH and the other serum enzymes (CK & CKMB ) in this study were slightly above the clinical norms for untrained individuals and within the normal range for athletes [27]. In consistent with previous studies [13,30—32], the increased serum CK and CKMB activities in CrM group were significantly higher than Pl group following the short-term Cr supplementation. Furthermore, the positive significant relationship between pre-post changes and post values in these enzymes confirms that all alterations have occurred following Cr loading. Nevertheless, this result is contrast to some previous findings of Cancela et al., Rawson et al., Schröder et al. and Kreider et al. [13,30—33]. The discrepancy between our result and some previous studies may be related to differences in methodology design (human versus animal model studies), supplementation (short-term versus long-term supplementation with low or supraphysiological doses) and exercise training protocols (intensity, duration and frequency of various exercises). Souza et al., for example, have demonstrated that four weeks of supraphysiological doses of Cr supplementation resulted increase the levels of direct and indirect markers of tissue damage (Histochemical, and extracellular enzymes levels change) in untrained rats (but not in trained). Hence, they suggested that the exercise could block the side effects of high-dose Cr supplementation [6]. While Kreider
Changes of serum enzymes activity in the placebo and creatine groups following supplementation. Stages
LDH activities (IU/L)
CK activities (IU/L)
CKMB activities (IU/L)
Before Supplementation After Supplementation
393.33 ± 42.42 407.56 ± 44.66
141.22 ± 42.88 165.33 ± 65.29a
19.67 ± 1.5 19.88 ± 3.86a
Creatine Before Supplementation group After Supplementation
400 ± 51.38 435.56 ± 79.39
126 ± 27.18 345.67 ± 106.03a,b
19.89 ± 2.03 22 ± 5.43a
Placebo group
LDH: lactate dehydrogenase; CK: creatine phosphokinase; CKMB : myocardial CK. a If P < 0.05 between placebo and creatine groups. b If P < 0.05 between pre-post supplementation.
92 et al. reported that high extracellular enzymes activity in athletes following Cr supplementation probably might be a consequence of participating in the high intensity exercises [27]. Moreover, Souza et al. speculated that exercise training could protect against kidney and liver alterations, by increasing Cr consumption by skeletal muscles during exercise training, resulting in a decreased Cr accumulation in the kidneys and liver [6]. This group hypothesized that the Cr accumulation into the tissue, which has low metabolic capacity to convert Cr into creatinine and is enzymatically capable of accomplishing the metilation processes, contributing to the formation and accumulation of citotoxic substances, such as formaldehyde and methylamine [6]. Because some studies have demonstrated that short-term, high-dose oral Cr supplementation can increase the production of methylamine and formaldehyde (Deaminated methylamine) as potential cytotoxic compounds, may potentially cause protein cross-linkage and increase blood malondialdehyde (an indicator of lipid peroxidation of phospholipids’ membrane or cellular damage) and subsequently serious unwanted side effects [6,14,15]. On the other hand, it can be speculated that deamination of methylamine in circulation may induce increase of cellular damages in different tissues (especially renal glomerula). Nevertheless, in humans, most of the studies investigated the potential for toxicity of Cr supplementation has not found evidence of adverse effects when ingested at low doses in long-term supplementation [11,27,29]. According to the present results and previous findings, it can be concluded that short-term CrM supplementation (during loading phase) may cause undesirable increase in serum enzymes activity, as indirect markers of cellular damage in young soccer players (but within normal range of athletes). However, since the exact mechanism in which Cr supplementation may affect in cellular damage have not fully been elucidated and the ratio of CKMB /CK as cardiovascular risk factor is less than 0.2 hence, it is not possible to affirm that Cr supplementation lead to tissue damage. On the other hand, further studies are necessary to clarify the possible side effects of short and long-term Cr supplementation in different organs. Therefore, it should be cautious about the Cr ingestion until the actual adverse effects of this supplement on the young athletes are completely identified.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments This study was carried out in the faculty of Physical Education & Sports Sciences in university of Tabriz. This paper is based on a thesis submitted by S. Atashak to the Ministry of Education, in partial fulfillment of the requirements for the MSc degree in physiology from university of Tabriz, Tabriz, Iran. We thank Loghman Radpey and Faezeh Sohrabi for their suggestion that help to improve the manuscript. The authors are indebted to Dr. R.S. Shabani for French translation of the abstract.
S. Atashak, A. Jafari Authors’ contributions: Afshar Jafari coordinated the study and prepared the manuscript. Sirvan Atashak carried out the design and participated in all the experiments and the manuscript preparation. All authors have read and approved the content of the manuscript.
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