i need summary for article 2 pages as APA formatting

Morning-to-evening difference of biomarkers of muscle injury and

antioxidant status in young trained soccer players

Omar Hammouda a,b , Henda Chahed

c , Hamdi Chtourou

a , Salyma Ferchichi

c ,

Abdelhedi Miled c and Nizar Souissi

a,d *

a Research Laboratory ‘‘Sports performance optimization’’ National Center of Medicine and Science in Sports (CNMSS), Tunis, Tunisia;

b Research Unit, High Institute of Sport and

Physical Education, Sfax, Tunisia; c Biochemistry laboratory, CHU Farhat Hached, Sousse,

Tunisia; d High Institute of Sport and Physical Education, Ksar-Saı̈d, Manouba University,

Tunisia

(Received 26 April 2011; final version received 15 June 2011)

The aim of this study was (i) to evaluate whether homocysteine (Hcy), total antioxidant status (TAS), and biological markers of muscle injury would be affected by time-of-day (TOD) in well-trained soccer players. In a counter- balanced order, 20 soccer players participated on two different occasions between 07:00 and 08:30 h and between 17:00 and 18:30 h. Fasting blood samples were collected from a forearm vein during each session. The results showed that the values of white blood cells (WBC), neutrophils (NE), lymphocytes (LY), and monocytes (MO) are higher in the evening than the morning. Although there was no TOD effect on blood lactate (Lac) levels, significant difference was observed for urea (URE), creatinine (CRE), and blood glucose (GLC) indicating higher evening levels. Moreover, the results also showed diurnal variations of core temperature, resting Hcy levels, and all biological markers of muscle injury [i.e., aspartate aminotransferase (ASAT), creatine kinase (CK), lactate dehydrogenase (LDH)].These parameters were lowest in the morning and tended to rise throughout the day. Furthermore, biomarkers of antioxidant status [i.e., TAS, uric acid (UA), and total bilirubin (TBIL)] displayed a significant effect of TOD with higher morning levels. In conclusion, the present study confirms the diurnal variations of Hcy, selected biological markers of cellular damage, and antioxidant status in young trained soccer players. Our finding suggests the fact that muscle damage and inflammation could be more important in the evening and that antioxidant status is more efficient in the morning.

Keywords: time-of-day; muscle damage; total antioxidant status; homocysteine; soccer

Introduction

Many physiological and behavioral functions follow a circadian rhythm. Recently, it has been concluded that markers of muscle damage [e.g. creatine kinase (CK), lactate dehydrogenase (LDH), alanine (ALAT), aspartate aminotransferase (ASAT), and phosphatase alkaline (PAL)], blood lactate (Lac), and white blood cells (WBC)

*Corresponding author. Email: [email protected]

Biological Rhythm Research

Vol. 43, No. 4, August 2012, 431–438

ISSN 0929-1016 print/ISSN 1744-4179 online

� 2012 Taylor & Francis http://dx.doi.org/10.1080/09291016.2011.599638

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display a transient increase during high-intensity maximal tasks (Nieman and Pedersen 1999; Brancaccio et al. 2008, 2010). These parameters are indicative of muscle fatigue and overtraining (Rietjens et al. 2005; Main et al. 2010). Moreover, the serum level of skeletal muscle enzymes is a marker of the functional status of muscle tissue and varies widely in both the pathological and physiological conditions. An increase in these enzymes may be an index of cellular necrosis and tissue damage following acute or chronic muscle injuries (Szumilak et al. 1998). In fact, monitoring of serum enzymes in athletes might have a role in studying muscle response to training, and this method could give more indications on muscle adaptation to physical work (Brancaccio et al. 2008).

It is well established that biochemical markers of cellular damage (Kanabrocki et al. 1990; Rivera-Coll et al. 1993; Gutenbrunner 2000), as well as WBC and their subpopulations (Haus et al. 1983), are time-of-day (TOD)-dependent with acrophases generally observed in the evening. The acrophases for the quantities studied coincide with times near those of the oral temperature acrophase (Rivera-Coll et al. 1993). In addition, plasma homocysteine (Hcy) levels, a prognostic marker for cardiovascular morbidity and mortality, were also shown to vary in a daily manner in humans with an evening peak and a morning nadir (Bönsch et al. 2007).

Moreover, considerable attention has been recently focused on the chronobio- logical aspects of the antioxidant system. The respiratory and motor activities, which directly determine the production of free radicals, are diurnally variable like the total antioxidant capacity (Borisenkov et al. 2007), the activities of some antioxidant enzymes (Hardeland et al. 2003), and the rate of lipid peroxidation (Kanabrocki et al. 2002; Cardona 2004). From these studies, the antioxidant system is more efficient in the early morning than in the evening.

Additionally, Kanabrocki et al. (1990, 2004) concluded that the peak values of uric acid (UA) are situated around 02:00 h. Although origins of these fluctuations are unclear, it has been identified that the physiological levels of melatonin contribute to total antioxidant capacity of human serum (Benot et al. 1999), this hormone having a powerful antioxidant role (Tan et al. 1993). However, these studies used healthy individuals with average age generally �30 years (Kanabrocki et al. 1990, 2004; Rivera-Coll et al. 1993). Furthermore, competitive sport imposes substantial energy, mechanical, mental, and emotional burdens on the human. This reflects, among other things, on a number of biochemical and hematological properties, which display significant differences between athletes and non-athletes in blood samples collected at rest (Nikolaidis et al. 2003; Mayr et al. 2006).

To the best of our knowledge, the effect of TOD on plasma levels of biomarkers of muscle injury and antioxidant status in young trained subjects have not been investigated. Therefore, the purpose of this study was to examine whether the selected biological markers of muscle injury and antioxidant status would be affected by TOD in trained soccer player.

Methods

Subjects

Twenty male soccer players (age: 17.6 + 0.6 years; weight: 71.3 + 4.8 kg; height: 181.3 + 5.4 cm) volunteered to participate in this study. Subjects are usually trained for at least 4 days per week for average of 2 h a day. After receiving a thorough

432 O. Hammouda et al.

explanation of the possible risks and discomforts associated with the experimental procedures, they gave a written informed consent. Answers to the Horne and Östberg (1976) questionnaire categorized subjects as either ‘‘moderately morning’’ (n ¼ 8) or ‘‘intermediate’’ (n ¼ 12) chronotypes. None of the subjects was taking any vitamin or antioxidant supplements. The experimental design of the study was approved by the University’s Ethic Committee and meets the ethical standards of the Declaration of Helsinki.

Experimental design

Subjects performed two sessions in a randomized and balanced order over 2 days with only one session a day, allowing a recovery period �36 h. One session was conducted in the morning (07:00–08:30 h) and the other in the evening (17:00–18:30 h). These time points were chosen as they span the portion of the day during which people typically participate in physical activity and training (Bernard et al. 1998).

Upon arrival for their first session, each subject’s body mass (Tanita, Tokyo, Japan) and height were recorded. During each session, after resting for 5 min, 10 mL of blood samples were collected.

To ensure control of confounding factors, subjects were asked to keep as closely as possible their usual sleeping habits with a minimum of 7 h sleep taken on the night preceding each session. Before the morning session, subjects were instructed to wake up at 06:00 h. They were fasting and allowed to drink only one glass of water to avoid the effects of postprandial thermogenesis (Touitou et al. 2004). For evening test session, they were requested to ingest their last meal at least 5 h before the evening test session as recommended by Bougard et al. (2009). The diet was identical for all participants before each test session. The overall daily energy intake goal was set at 10.5 MJ (2500 kcal) per capita per day. Throughout the experimental period, subjects were requested to maintain their habitual physical activity and to avoid strenuous activity during the 24 h before each test session.

Blood sampling and analysis

Fasting blood samples were collected from a forearm vein after 5 min of seated rest. Samples were placed in an ice bath and centrifuged immediately. Aliquots of the resulting plasma were stored at 7808C until analyzed. Serum creatinine (CRE), ASAT, ALAT, PAL, CK, LDH, and g-glutamyl transferase (GT) activities were determined by the enzymic rate method, whereas blood glucose (GLC), UA, and Lac were determined by a quantitative enzymatic technique, using a Randox kit (Randox, Antrim, UK). Hcy was measured by an immunologic technique. All these measures were done in an automated analyzer CX 9 PRO (Beckman Coulter, Brea, CA, USA).

Total antioxidant status (TAS) was measured using a kit purchased from Randox Laboratories (Crumlin, Northern Ireland). In this assay, metmyoglobin reacts with H2O2 to form the radical species ferrylmyoglobin. A chromogen (2,2

0-azinodi- [ethylbenzthiazoline sulfonate], ABTS) is incubated with the ferrylmyoglobin to produce the radical cation species ABTS

þ . This has a relatively stable blue-green

color which is measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree which is proportional to their concentration.

Biological Rhythm Research 433

Hematological parameters were determined in a multichannel automated blood cell analyzer Beckman Coulter Gen System-2 (Coulter T540). We measured simul- taneously red blood cells (RBC), WBC, and principal derivative subpopulations such as monocytes (MO), neutrophils (NE), and lymphocytes (LY).

Statistical analyses

All statistical tests were processed using STATISTICA Software (StatSoft, France). All values are expressed as mean + SD. Biological parameters data were analyzed using a paired Student t test. A probability level of 0.05 was selected as the criterion for statistical significance.

Results

Hematological parameters

Table 1 shows the means of the hematological parameters at 07:00 h and 17:00 h. Statistical analyses showed higher resting evening values of WBC, NE, LY, and MO. However, RBC count was significantly higher in the morning sessions.

Biochemical parameters

Means of the Lac, CRE, GLC, and urea (URE) are presented in Table 2. Although there was no TOD effect on Lac levels, significant difference was observed for GLC and CRE indicating higher evening levels.

Means of ALAT, ASAT, CK, LDH, PAL, GT, Hcy, and core temperature (Table 3) showed a similar profile. These parameters were lowest in the morning and tended to rise throughout the day. However, means of TAS, total bilirubin (TBIL), and UA levels shown in Table 4 displayed a significant effect TOD with higher morning levels.

Discussion

The purpose of this study was to confirm the diurnal variations of some biological markers of muscle injury and antioxidant status in young trained soccer player. Our results showed that resting levels of Hcy, biomarkers of cellular damage, and leucocytes were higher in the evening; however, markers of antioxidant status (i.e. TAS, UA, and TBIL) were higher in the morning. As these biomarkers of muscle injury are indicative of muscle fatigue and overtraining (Rietjens et al. 2005; Main

Table 1. Diurnal variations of resting hematological parameters (mean + SD).

Parameters Morning Evening

WBC (10 3 /mL) 6.32 + 0.83 7.19 + 0.74a

NE (10 3 /mL) 3.55 + 0.98 4.27 + 1.04b

LY (10 3 /mL) 2.15 + 0.37 2.77 + 0.4c

MO (10 3 /mL) 0.66 + 0.29 0.8 + 0.28c

RBC (10 6 /mL) 5.4 + 0.35c 4.94 + 0.27

Note: a Significant difference between TOD of blood sampling (p 5 0.05); b significant difference between

TOD of blood sampling (p 5 0.001); c significant difference between TOD of blood sampling (p 5 0.01).

434 O. Hammouda et al.

et al. 2010), it is imperative to study the TOD effect on the selected parameters in trained subjects. Moreover, the levels of skeletal muscle enzymes are widely different between trained and untrained subjects (Nikolaidis et al. 2003; Mayr et al. 2006). Additionally, exercise training increases the resistance against oxidative stress and enhances activity of antioxidant enzymes, providing enhanced protection (for review see Radak et al. 2008).

Our findings showed that WBC and their subpopulations (MO, LY, and NE) are significantly higher in the evening than in the morning. Accordingly, most previous research have identified that peak times for these variables are situated between 18:00 h and 24:00 h (Haus et al. 1983; Kanabrocki et al. 1990) in clinically healthy subjects. The greater levels of WBC in the evening could be because of the higher concentrations of catecholamines at this time point (Kanabrocki et al. 1990). Indeed, a pronounced circadian variation in catecholamine activity has been observed at rest

Table 2. Diurnal variations of selected biochemical parameters (mean + SD).

Parameters Morning Evening

Lac (mmol/L) 0.96 + 0.28 1.01 + 0.35 CRE (mmol/L) 71.3 + 6 81.8 + 9.69a

GLC (mmol/L) 4.33 + 0.3 4.76 + 0.38b

URE (mmol/L) 68.60 + 7.18 85.20 + 13.79b

Note: a Significant difference between TOD of blood sampling (p 5 0.01); bsignificant difference between

TOD of blood sampling (p 5 0.001).

Table 3. Diurnal variations of resting biochemical analyses of muscle injury and core temperature (mean + SD).

Parameters Morning Evening

ALAT (IU/L) 20.6 + 3.27 23.7 + 4.97a

ASAT (IU/L) 24.8 + 4.26 31 + 4.69a

CK (IU/L) 150.7 + 42.35 179.8 + 71.27b

GT (IU/L) 12.2 + 3.79 14.2 + 4.24a

LDH (IU/L) 347.1 + 67.42 402.1 + 75.04a

PAL (IU/L) 113.5 + 25.7 146.9 + 38.95a

Hcy (mmol/L) 17 + 2.75 18.57 + 2.51b

Temperature 36.16 + 0.24 36.87 + 0.23b

Note: a Significant difference between TOD of blood sampling (p 5 0.01); bsignificant difference between

TOD of blood sampling (p 5 0.001).

Table 4. Diurnal variations of resting biochemical analyses of antioxidant status (mean + SD).

Parameters Morning Evening

TBIL (mmol/L) 15.82 + 3.47a 14.54 + 3.27 UA (mmol/L) 275.7 + 40.5b 228.2 + 36.22 SAT (mmol/L) 1.22 + 0.22a 1.11 + 0.18

Note: a Significant difference between TOD of blood sampling (p 5 0.05); b significant difference between

TOD of blood sampling (p 5 0.01).

Biological Rhythm Research 435

(Åkerstedt 1979) with a peak in the early afternoon and a trough occurring during the night, independent of sleep patterns.

In addition, we found that plasma levels of CK, LDH, ASAT, ALAT, PAL, and GT, known as markers of muscle injury during training and exercise (Nathwani et al. 2005; Brancaccio et al. 2010), are higher in the evening than in the morning. Similar profile has been observed in the previous reports (Kanabrocki et al. 1990; Rivera- Coll et al. 1993; Gutenbrunner 2000). These diurnal variations of leucocytes and muscle enzymes are linked to the circadian rhythm of core temperature (Haus et al. 1983; Rivera-Coll et al. 1993). In fact, Dalton et al. (1997) supposed that the evening elevation in body temperature would increase the activity of the enzymes such as phosphofructokinase and LDH. In agreement, our results showed that core temperature improved from morning to evening.

Moreover, plasma levels of Hcy, URE, CRE, and GLC were higher in the evening than in the morning. These results are in agreement with Bönsch et al. (2007) and Bremner et al. (2000) for Hcy and Kanabrocki et al. (1990) for URE, CRE, and GLC. Animal studies support a relationship between high blood levels of Hcy and oxidative stress, signal-transduction pathways leading to inflammation and apoptosis being activated (Wilson and Lentz 2005). Thus, it would be expected that the higher levels of Hcy and biomarkers of muscle injury in the evening confirm, at least in part, the TOD effect on oxidative stress as shown by Kanabrocki et al. (2002).

On the other hand, we showed that TAS and UA levels were greater in the morning than in the evening. UA is a powerful antioxidant (Hooper et al. 2000) and has a protective role of vitamins E and C (Ma et al. 1994). However, to date, only few studies have investigated the diurnal variations of TAS (Benot et al. 1999; Borisenkov et al. 2007) and UA (Kanabrocki et al. 1990, 2004) in human. These investigations found that acrophases of TAS and UA were observed in the early morning. The respiratory and motor activities, which directly determine the production of free radicals, are diurnally variable and seem to influence the diurnal variation of the antioxidant status (Hardeland et al. 2003; Borisenkov et al. 2007) which could affect the rate of lipid peroxidation (Kanabrocki et al. 2002; Cardona 2004). Indeed, higher levels of muscle damage and oxidative stress have been observed in the evening (Rivera-Coll et al. 1993; Kanabrocki et al. 2002). Furthermore, it is well known that the reciprocal circadian relationship between serum nitric oxide (NO

. ) level and serum CO2 and UA levels reflects the generation

of (NO . )/peroxynitrite (ONOO

– )/CO2-dependent radicals and their inactivation by

reaction with UA (Kanabrocki et al. 2004). It has been identified that the physiological levels of melatonin contribute to total antioxidant capacity of human serum (Benot et al. 1999) and could affect the UA rhythm. Indeed, the acrophase of this indole was observed between 2 h and 4 h, and it displays a powerful antioxidant role (Tan et al. 1993). Concerning plasma TBIL, as previously shown (Kanabrocki et al. 1990; Larsson et al. 2009), we found a significant diurnal variation with higher morning values. These diurnal variations of TBIL are not well understood, but they could be attributed to heme degradation (Raghuram et al. 2007).

In conclusion, the present study confirms the diurnal variations of the selected markers of muscle damage and antioxidant status in young trained soccer players. Although the biological markers selected in this study are not very specific, our finding suggests the fact that inflammation could be more important in the evening and that antioxidant status is more efficient in the morning. Respecting the TOD of

436 O. Hammouda et al.

measure of the biological markers of muscle damage and antioxidant status may protect the physicians from misinterpreting of abnormal values when evaluating the training level of their athletes.

Acknowledgments

The authors wish to express their sincere gratitude to all the participants for their maximal effort and cooperation.

References

Åkerstedt T. 1979. Altered sleep/wake patterns and circadian rhythms. Acta Physiol Scand Suppl. 469:1–48.

Benot S, Goberna R, Reiter RJ, Garcia-Maurino S, Osuna C, Guerrero JM. 1999. Physiological levels of melatonin contribute to the antioxidant capacity of human serum. J Pineal Res. 27:59–64.

Bernard T, Giacomoni M, Gavarry O, Seymat M, Falgairette G. 1998. Time-of-day effects in maximal anaerobic leg exercise. Eur J Appl Physiol Occup Physiol. 77:133–138.

Bönsch D, Hothorn T, Krieglstein C, Koch M, Nehmer C, Lenz B, Reulbach U, Kornhuber J, Bleich S. 2007. Daily variations of homocysteine concentration may influence methylation of DNA in normal healthy individuals. Chronobiol Int. 24:315–326.

Borisenkov MF, Erunova LA, Lyuseva EM, Pozdeeva NV. 2007. Diurnal changes in the total antioxidant activity of human saliva. Hum Physiol. 33:375–376.

Bougard C, Moussay S, Gauthier A, Espié S, Davenne D. 2009. Effects of waking time and breakfast intake prior to evaluation of psychomotor performance in the early morning. Chronobiol Int. 26:324–336.

Brancaccio P, Giuseppe L, Nicola M. 2010. Biochemical markers of muscular damage. Clin Chem Lab Med. 48:757–767.

Brancaccio P, Maffulli N, Buonauro R, Limongelli FM. 2008. Serum enzyme monitoring in sports medicine. Clin Sports Med. 27:1–18.

Bremner WF, Holmes EW, Kanabrocki EL, Hermida RC, Ayala D, Garbincius J, Third JL, Ryan MD, Johnson M, Foley S, et al. 2000. Circadian rhythm of serum total homocysteine in men. Am J Cardiol. 86:1153–1156.

Cardona F. 2004. Periodic dip of lipid peroxidation in humans: a redox signal to synchronize peripheral circadian clocks? Med Hypotheses. 63:841.

Dalton B, McNaughton L, Davoren B. 1997. Circadian rhythms have no effect on cycling performance. Int J Sports Med. 18:538–542.

Gutenbrunner C. 2000. Circadian variations of the serum creatine kinase level: a masking effect? Chronobiol Int. 17:583–590.

Hardeland R, Coto-Montes A, Poeggeler B. 2003. Circadian rhythms, oxidative stress, and antioxidative defense mechanisms. Chronobiol Int. 20:921.

Haus E, Lakatua D, Jacqueline S, Sackett-Lundeen L. 1983. Chronobiology in hematology and immunology. Am J Anat. 168:467–517.

Hooper DC, Scott GS, Zborek A. 2000. Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood–CNS barrier permeability changes and tissue damage in a mouse model of multiple sclerosis. FASEB J. 14:691–698.

Horne JA, Östberg O. 1976. A self-assessment questionnaire to determine morningness– eveningness in human circadian rhythms. Int J Chronobiol. 4:97–110.

Kanabrocki EL, Dennis M, Ramon C, Gwen S, Fraser Bremner W, May D, Diana E, Jane LHC, Parvez S, Bernard A, et al. 2002. Circadian variation in oxidative stress markers in healthy and type II diabetic. Chronobiol Int. 19:423–439.

Kanabrocki EL, Ryan MD, Hermida RC, Ayala DE, Scott GS, Murray D, Bremner WF, Third JL, Johnson MC, Foley S, et al. 2004. Altered circadian relationship between serum nitric oxide, carbon dioxide, and uric acid in multiple sclerosis. Chronobiol Int. 21:739– 758.

Kanabrocki EL, Sothern RB, Scheving LE, Vesely DL, Tsai TH, Shelstad J, Cournoyer C, Greco J, Mermall H, Ferlin H, et al. 1990. Reference values for circadian rhythms of 98 variables in clinically healthy men in fifth decade of life. Chronobiol Int. 7:445–461.

Biological Rhythm Research 437

Larsson A, Hassan M, Ridefelt P, Axelsson J. 2009. Circadian variability of bilirubin in healthy men during normal sleep and after an acute shift of sleep. Chronobiol Int. 26:1613–1621.

Ma YS, Stone WL, Leclair IO. 1994. The effects of vitamin C and urate on the oxidation kinetics of human low-density lipoprotein. Proc Soc Exp Biol Med. 206:53–59.

Main LC, Dawson B, Heel K, Grove R, Landers GJ, Goodman C. 2010. Relationship between inflammatory cytokines and self-report measures of training overload. Res Sports Med. 18:127–139.

Mayr A, Kuipers H, Falk M, Santer P, Wierer B. 2006. Comparison of hematologic data in world elite junior speed skaters and in non-athletic juniors. Int J Sports Med. 27:283–288.

Nathwani RA, Pais S, Reynolds TB, Kaplowitz N. 2005. Serum alanine aminotransferase in skeletal muscle diseases. Hepatology. 41:380–382.

Nieman DC, Pedersen BK. 1999. Exercise and immune function. Recent developments. Sports Med. 27:73–80.

Nikolaidis MG, Protosygellou MD, Petridou A, Tsalis G, Tsigilis N, Mougios V. 2003. Hematologic and biochemical profile of juvenile and adult athletes of both sexes: implications for clinical evaluation. Int J Sports Med. 24:506–511.

Radak Z, Chung HY, Goto S. 2008. Systemic adaptation to oxidative challenge induced by regular exercise. Free Radic Biol Med. 44:153–159.

Raghuram S, Stayrook KR, Huang P, Rogers PM, Nosie AK, McClure DB, Burris LL, Khorasanizadeh S, Burris TP, Rastinejad F. 2007. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta. Nat Struct Mol Biol. 14:1207–1213.

Rietjens GJ, Kuipers H, Adam JJ, Saris WH, Van Breda E, Van Hamont D, Keizer HA. 2005. Physiological, biochemical and psychological markers of strenuous training-induced fatigue. Int J Sports Med. 26:16–26.

Rivera-Coll A, Fuentes-Arderiu X, Diez-Noguera A. 1993. Circadian rhythms of serum concentration of 12 enzymes of clinical interest. Chronobiol Int. 10:190–200.

Szumilak D, Sulowicz W, Walatek B. 1998. Rhabdomyolysis: clinical features, causes, complications and treatment. Przegl Lek. 55:274–279.

Tan DX, Pöeggeler B, Reiter RJ, Chen LD, Chen S, Manchester LC, Barlow-Walden LR. 1993. The pineal hormone melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in vivo. Cancer Lett. 70:65–71.

Touitou Y, Portaluppi F, Smolensky MH, Rensing L. 2004. Ethical principles and standards for the conduct of human and animal biological rhythm research. Chronobiol Int. 21:161– 170.

Wilson KM, Lentz SR. 2005. Mechanisms of the atherogenic effects of elevated homocysteine in experimental models. Semin Vasc Med. 5:163–171.

438 O. Hammouda et al.

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