Research Article

Received: 17 March 2008, Revised: 21 October 2008, Accepted: 9 November 2008, Published online 27 February 2009 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/bio.1104

Copyright © 2009 John Wiley & Sons, Ltd. Luminescence 2009; 24: 224–229

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John Wiley & Sons, Ltd.Effects of 2.5-hour sumo training on serum opsonic activitySumo and opsonic activityArata Kojimaa,b, Takashi Umedaa, Kazuo Saitob, Yoshikazu Ookuboa,b, Junya Satoa, Shigeyuki Nakajia*, Masashi Matsuzakaa, Makoto Yaegakia, Motoki Ohnishia, Maki Miyazawaa and Ippei Takahashia

ABSTRACT: Sumo is a traditional Japanese sport, but the effect of actual daily training on neutrophil function is unknown. Weevaluated the effect of sumo training on serum opsonic activity (SOA), which is one of the main neutrophil-related functions.Seventeen male university sumo wrestlers participated in the study. Changes in anthropometric parameters, concentrationsof serum immunoglobulins (IgG, IgA and IgM), complements (C3 and C4), myogenic enzymes (lactate dehydrogenase, asparateaminotransferase, alanine aminotransferase and creatine kinase), white blood cell/neutrophil counts and SOA were measuredimmediately before and after actual daily training for 2.5 h. Compared with the pre-values, immunoglobulins and comple-ments, myogenic enzymes and white blood cell/neutrophil counts significantly increased (p< 0.01 for all). As for SOA, the valuesof the peak height and the area under the curve significantly increased after the training when assessed using lucigenin as achemiluminigenic probe (p < 0.01 for all), but showed no significant change when luminol was used as the chemiluminigenicprobe. In conclusion, daily actual sumo training for 2.5 h increases SOA, thus possibly activating the reactive oxygen speciesproduction of neutrophils. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: complements; immunoglobulins; myogenic enzymes; reactive oxygen species; serum opsonic activity

Introduction

It has been reported that exercise of high intensity and frequencyincreases the tendency of athletes to catch a cold (1–3) andinduces the ‘over-training syndrome’. One reason for this isthat the nonspecific immune system, particularly neutrophilfunctions, including their reactive oxygen species (ROS) produc-tion capability, is suppressed by such intense training exercise(1).

Sumo is a fighting sport; two wrestlers fight on a raised 4.5 mdiameter hard clay circle (dohyo) dressed in only a belt-like loincloth (mawashi). Sumo wrestling is an original Japanese combatsport with a history and tradition rooted in the Shinto religion ofmany hundreds of years. Sumo wrestlers undergo an extremelygrueling and physically demanding effort during what is usuallya very short bout: first, there is the enormous physical impactwhen the two combatants, over 100 or even 200 kg each, collidetogether in the center of the ring; then there is the damagedone when one (or sometimes both) of them crashes to theground either in or out of the ring (Fig. 1). Both professional andamateur sumo is also associated with very exhausting, demand-ing, unique and established training methods, which are basedon centuries of tradition.

To date several studies on sumo wrestlers have been performed.However, most of them have focused on the obesity and obesityrelated-conditions such as hyperlipidemia (4, 5). Only the previ-ous studies from our group have surveyed the physiologicalchanges brought about by sumo training as follows: Umedaet al. reported that sumo training caused muscular damage andan increase in the neutrophil count as a response (6). In the presentstudy, although neutrophil oxidative burst activity increased,neutrophil phagocytic activity decreased after training.

Serum opsonic activity (SOA) is an activity which activatesthe ROS production capacity through the enhancement of theadhesion function of neutrophils against foreign pathogens.Neutrophils have a bactericidal action through phagocytosisof various foreign bodies using ROS and play an importantrole in the host defense system (7–10). On the other hand, the

Figure 1. Sumo wrestling.

* Correspondence to: S. Nakaji, Department of Social Medicine, HirosakiUniversity Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori036-8562, Japan. E-mail: nakaji@cc.hirosaki-u.ac.jp

a Department of Social Medicine, Hirosaki University Graduate School ofMedicine, 5 Zaifu-cho, Hirosaki, 036-8562 Aomori, Japan

b Department of Physical Education, Nippon Sport Science University, 1-1Fukasawa, Setagaya, 158-8508 Tokyo, Japan

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overproduction of ROS may cause oxidative injury to normaltissue (11, 12). Many studies have examined the relationshipbetween neutrophil ROS production capability and exercise orshort- and long-term training (13–27). However, only our studieshave researched the changes in SOA following exercise ortraining (13, 28–30) and the results of these are summarized inTable 1. In general, this data has shown that exercise increasesSOA.

Among the measurements of three major neutrophil immunefunctions, the measurement of SOA is the most practical,because SOA can be measured at any time using frozen serumbut ROS and PA must be measured rapidly using live neutrophil.

In this study, we evaluated the effect of a 2.5 h sumo trainingsession on SOA in order to clarify the relationship betweenchanges in SOA and exercise loading in sumo wrestlers. The reasonfor this is that there are no studies measuring the changes inSOS, although it has been measured for several other sports.

Subjects and methods

Subjects

Seventeen male university sumo wrestlers were enrolled into thestudy, with an average age (± standard deviation) of 20.2 ± 1.5years. The body mass, height and fat-free mass of the subjects(± standard deviation) were 112.3 ± 27.7 kg, 176.3 ± 6.2 cm and86.7 ± 13.8 kg, respectively (Table 2).

This study was conducted after obtaining the approval ofthe Ethics Committee of Hirosaki University School of Medicine.In addition, before the study began, the study‘s objectivesand requirements were explained to the subjects and writteninformed consent was obtained from each volunteer.

Methods

Measuring points and items measured. We measured thechanges in anthropometric parameters, concentrations ofserum immunoglobulins, complements and myogenic enzymes,white blood cell/neutrophil counts and SOA immediately beforeand after an actual 2.5 h daily training session. Measurementsimmediately before training were taken under fasting conditions.The ambient temperature and relative humidity in the trainingroom were 25.9 ± 2.3°C and 58.6 ± 9.3%, respectively.

Training regimen. The contents of the training menu areshown in Table 3. The training regimen consisted of stretchingand limbering for 10 min before and after the training togetherwith specific excercises to strengthen physical fitness throughtechnical training for sumo matches. The total time of the train-ing session was about 2.5 h. Technical training for sumo matchescontains traditional training methods such as ‘shiko’, a stompingexercise that is excellent for developing hip and leg strength,both of which are crucial in sumo, ‘suriashi’, a leg strengthener

Table 1. Previous studies regarding the relationship between exercise and SOA

SOA

Author Subject Exercise LuminolPH

LucigeninPH

Saito et al. (28) Marathon runner (male 24) 30 km running Increase No changeMochizuki et al. (29) Crosscountry skier (male 9, female 2) Maximum loading by treadmill Increase —

Speed skater (male 4)Kumae et al. (30) Not habitually exercising subject

(male 18)1500 m running with maximum exertion

Decrease−increase —

Sato et al. (13) 100 km-marathon runner (male 8, female 10)

Marathon race No change —

Table 2. Changes in anthropometric parameters beforeand after training in 17 male university sumo wrestlers

Pre-exercise Post-exercise

Age (years) 20.2 ± 1.5 —Height (cm) 176.3 ± 6.2 —Body mass (kg) 112.3 ± 27.7 111.1 ± 27.7**Relative body fat (%) 21.3 ± 7.1 —Fat-free mass (kg) 86.7 ± 13.8 —

Values are the means ± standard deviation.**p < 0.01, significantly different from the pre-value.

Table 3. Contents of 2.5 h sumo training in this study

Contents of exercise The number of times or length of enforcement

1. Warm-up 10 min2. ‘Shiko’ 100 times3. ‘Suriashi’ 10 times4. Game 25 times5. ‘Butsukari-geiko’ 10 times6. ‘Shiko’ 100 times7. Jumping squat 50 times * 5 sets8. Push-up 30 times * 5 sets9. Cooling down 10 min

Warm-up and cooling-down mainly consisted of calisthenicsand stretching exercises.‘Shiko’: a stomping exercise that is good for developing hipand leg strength, and which is crucial in sumo. ‘Suriashi’: a leg strengthener performed in a crouching posi-tion with the hands bent at the elbows. The wrestler slideshis feet forward, alternating legs.‘Butsukari-geiko’: when one wrestler runs into another andpushes him to the edge of the dohyo. Usually, the one beingpushed will throw his attacker down so the attacker can prac-tice falling properly.

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which is performed in a crouching position with the hands bentat the elbows—the rikishi (sumo wrestler) slides his feet forward,alternating legs—and ‘butsukari-geiko’, when one rikishi runsinto another and pushes him to the edge of the dohyo. Usually,the rikishi being pushed will throw his attacker down so he canpractice falling properly.

Body composition. Body mass air was measured using anelectronic scale (AD6205, A & D Co. Ltd, Tokyo) to the nearest0.01 kg, with subjects wearing the same attire. Body density wasdetermined using BOD POD (MAB-1000, Life Measurement, Inc.,USA). The percentage of fat mass and fat-free mass were calcu-lated from body density using the Brozek equation (31).

Hematological parameters. Blood samples were collected beforemeals in the early morning to measure the following parame-ters: (i) white blood cell (WBC) and neutrophil counts in wholeblood (using a blood cell autoanalyzer, MicroBiff-II, Coulter Co.,Ltd, Califolnia, USA); (ii) lactate dehydrogenase (LDH), asparateaminotransferase (AST), alanine aminotransferase (ALT) andcreatine kinase (CK) (ultraviolet method in each); (iii) immuno-globulins (IgG, IgA and IgM, nepherometry method); and (iv)complements (C3 and C4, nepherometry method) in serum.

Measurement of SOA. Serum samples were separated fromblood using Vacutainer blood-collection tubes (Becton Dickin-son, Franklin Lake, USA) by centrifugation at 1000g for 10 min,after allowing the blood to clot for 30 min at room temperature.These samples were stored frozen at –80°C until analysis, whensamples were rapidly thawed at 37°C.

Zymosan from Saccharomyces cerevisiae, a well-known activatorof the alternative pathway of the complement system (31, 33),was employed for opsonized particles. Zymosan A (Sigma, USA)was suspended in Hank’s balanced salt solution (HBSS) at a con-centration of 5 mg/mL and then opsonization was performedby adding to the serum samples to a final concentration of 20%and incubating at 37°C for 30 min. The particles were thenwashed twice with HBSS and resuspended in HBSS at a concen-tration of 5 mg/mL .

Two chemiluminigenic probes, lucigenin and luminol, wereemployed for the detection of ROS. Lucigenin was prepared bydissolving bis-N-methylacridinium nitrate (Sigma, USA) in HBSSto give a final concentration of 0.5 mM (pH 7.4). Luminol was pre-pared by dissolving 5-amino-2,3-dihydro-1,4-phatalazinedione(Sigma, USA) initially in 1 M NaOH to give a clear solution andthen adjusted using HCl and HBSS to give a final concentrationof 2 mM (pH 7.4).

Normal pooled human neutrophils were obtained from theperipheral blood of a healthy adult male volunteer. A methodwhereby whole blood is centrifuged through Mono-Poly resolvingmedium was modified (34, 35). The neutrophils were suspendedto 3 × 106 cell/mL using an automatic blood cell counter (CoulterMD II, Coulter Co. Ltd., Tokyo, Japan).

Opsonized zymosan (OZ) suspension and chemiluminigenicprobes prepared as noted above were added to each well ofblack flat-bottom microplates (Greiner Japan, Tokyo, Japan),and 50 μl of standard neutrophils was added. The plates wereautomatically measured on the Auto Luminescence Analyzer,Alfa system (Tokken, Funabashi, Japan) (36). All measurementswere performed at 37°C. Since the maximum light emission,peak height (PH) and the area under the curve (AUC) are widelyused and reliable parameters (37), the results were evaluated

using the PH and AUC of the chemiluminescence response. Eachsample was run in duplicate and values were expressed asmeans.

Statistical analysis

Data were presented throughout as means ± standard errors (SE).The differences in each parameter between the pre- and postvalues were analyzed using the paired t-test. The correlationsbetween parameters were studied with Pearson’s correlationcoefficient. Probability values (p) of less than 0.05 were consid-ered significant.

ResultsPost-training body weight significantly decreased by 1.2 ± 0.6 kg(p < 0.01) in comparison with the pre-value (Table 1). The neu-trophil and leukocyte counts and the neutrophil:leukocyte ratiosignificantly increased (p < 0.01 all; data not shown). Myogenicenzymes, immunoglobulins and complements significantlydecreased (p < 0.01 each), although they significantly increasedin the non-adjusted values (data not shown).

The PH and AUC of SOA using LgCL significantly increased(p < 0.01 each), although those using LmCL showed no signifi-cant changes (Table 4). The change in neutrophil counts signifi-cantly correlated negatively with the change in the CK values(r = 0.711, p < 0.05). A positive correlation was seen betweenthe changes in SOA and IgG/C3 values (r = 0.76, p < 0.05 andr = 0.70, p < 0.05, respectively).

DiscussionIn this study, the values of complements and immunoglobulinssignificantly increased, and those of myogenic enzymes andleukocyte/neutrophil counts also significantly increased after2.5 h of exercise loading in the sumo wrestler subjects. Exercisehas been reported to elevate the levels of such serum enzymesas CK, ASAT, ASLT and LDH through the possible mechanismsof muscular inflammation, increased permeability of musclecell membranes (33, 38), anoxic or hypoxic damage to muscles(39, 40) or production of toxic free radicals (41). Furthermore,Evans et al. reported that CK levels rose more remarkably in non-exercising subjects compared with habitually exercising subjectsunder the same exercise loading conditions, and suggested that

Table 4. Changes in PH, PT and AUC of luminol-dependent(LmCL) and lucigenin-dependent (LgCL) chemiluminescenceresponse before and after training

Pre-exercise Post-exercise

PH (cpm)LmCL 1148.8 ± 37.5 1166.1 ± 47.1LgCL 21.3 ± 8.9 41.1 ± 13.6**AUC (cpm*sec)LmCL 27960 ± 1601 28986 ± 1954LgCL 433.8 ± 284.2 1099.2 ± 493.9**

Values are the mean ± standard deviation.PH, peak height. AUC, area under the curve for 45 min.**p < 0.01, significantly different from the pre-value.

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habitual exercise may strengthen muscular function and tissuestructure so that subjects who exercise regularly are capable ofperforming more intense exercise without exercise-mediatedinjury (42).

The increase in the number of circulating neutrophils withexercise has been well established, and the increase is intensity-dependent (2). This increase may be as a result of stress-relatedrelease of growth hormone, adrenalin or noradrenalin (43), or maybe a part of the inflammatory response via cytokines to exercise-induced tissue damage (44). In the present study, the increasesin neutrophil counts were statistically well correlated with theelevation of the enzyme levels. However, it was beyond the scopeof this study to ascertain whether the increased number of neu-trophils actually caused muscle damage through enhanced ROSproduction or occurred because of the increased number ofcompromised muscle cells which were required to be absorbed.

As for immunoglobulins and complements, there are manystudies regarding exercise-mediated changes in these parameters,although the studies have presented conflicting results, showingincreased, decreased or unchanged counts (32, 45–47). In thepresent study, one of the major reasons behind this mechanismmay be the mobilization and recruitment of immunoglobulinsand complements to the exercise-mediated denatured anddamaged areas of muscle tissue, which had then entered thefirst inflammatory stage of the wound healing response. Anothercredible possibility is that immunoglobulins and complementsincreased due to exercise-mediated dehydration.

As has already been mentioned above, SOA contributes tothe neutrophil bactericidal activity through accelerating theadhesion of neutrophils to opsonized substances via IgG, C3etc., after which neutrophils engulf foreign bodies and produceROS. Activated neutrophils initiate a ‘respiratory burst’, leadingto the production of superoxide anions (O2

−), which are quicklyconverted to hydrogen peroxide (H2O2). Neutrophil azurophilicgranules contain large quantities of myeloperoxidase, which worksin the presence of H2O2 and chloride ions to produce hydrochlo-rous acid (HOCl). HOCl has a significantly higher oxidizingpotential than its precursors O2

− and H2O2 and contributes to thecomplexity of the oxygen-dependent antimicrobial systems ofneutrophils (48).

As shown in Table 1, SOA has demonstrated a variety ofresults after a severe bout of exercise in previous studies. Thereason for these contradictory findings is still unknown, althoughit has been suggested that it may be due to variations in theexperimental designs that were used in each study. All experi-mental designs differed in the degree of the physical load andits duration and in the specimen sampling conditions such asatmospheric temperature and timing of sampling, especially thelapse from the completion of the physical load. Furthermore, thesubject’s physical condition at the time of the study (fitness andfatigue) may have also contributed to such differences in theresults.

In this study, SOA was increased by 2.5 h sumo training,suggesting an enhanced ROS production capacity. This findingsupports the results of the most previous reports which haveshowed the enhancement of the ROS production capacity ofneutrophils after exercising (13, 14, 18, 19, 25, 29, 45, 49). One ofthe main reasons for this may be due to the increase in immuno-globulins and complements such as opsonins, and that suggestionwas backed up in the present study by the positive correlationbetween changes in SOA and IgG/C3 (r = 0.76, p < 0.05 andr = 0.70, p < 0.05, respectively).

Mochida et al. (16) suggested that major three neutrophilimmune functions such as SOA, ROS production capability andPA may compensate for each other to maintain the overall integrityof the neutrophil immune function, depending on the exerciseloading and subject’s physical condition, such as the degree offatigue. In previous studies including Mochida’s report, the typicalchange in ROS production capability and PA by a single bout ofnormal exercise has been an increase in SOA and ROS whereasPA decreased (16, 20, 21). However, under the conditions of severeand prolonged exercise such as a full marathon and a trainingcamp plus weight reduction, neutrophil parameters have tendedto deviate from such typical compensatory changes. For example,in some reports both PA and ROS decreased (22, 28).

In this study, SOA showed a typical response, e.g. increasedafter the training, suggesting that this training may be appropriatefor males to maintain the neutrophil function by balancing ROSincrease with decreasing PA and increasing SOA.

We investigated the ROS metabolism using two chemilumi-nescence probes, i.e. lucigenin and luminol. Lucigenin is wellassociated with the detection of O2

−, whereas luminol mainlydetects HOCl including the contribution of MPO degranulation.In this study, exercise increased the LgCL in SOA, and no changewas seen in the LmCL. Therefore, the increase in SOA noted inthis study may have contributed to the production of the loweroxidizing potential of substances such as O2

− rather than its pre-cursor HOCl.

The biological significance of increased ROS production fol-lowing exercise through increased SOA may be to assist in thedestruction and degradation of foreign pathogens (48), which inother words could be regarded as a natural advantageous actionfor health and performance. On the other hand, an excessiveincrease of ROS has been associated with injury to normal tissueand acceleration of the aging process, i.e. a disadvantageousaction (12, 48). There is currently not sufficient evidence to showwhether the increase in ROS production seen after exercise isadvantageous for, makes no difference to or is disadvantageousfor the athlete. Further studies are required to determine which ofthese possibilities either alone or in a combination is in fact the case.

Furthermore, the changes in LgCL and LmCL in SOA are differentfrom the results of Sato’s and Kumae’s reports (13, 30) and simi-lar to the results of Saito’s and Mochizuki’s reports (28, 29). Thereason for this might be due to the difference in the exerciseload or the subjects’ physical condition.

This study has three limitations. Firstly there was no controlgroup. Secondly the sample size was small. Thirdly we did notmeasure either the intensity (e.g. percentage maximal oxygenconsumption; %VO2 max) during the sumo training or the physicalfitness of the subjects.

Acknowledgements

The authors would like to thank the subjects for their participa-tion, and former Professor Kazuo Sugawara of the Departmentof Social Medicine, Hirosaki University School of Medicine for hisassistance and his suggestions during the preparation of thismanuscript.

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