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Received 15 October 2002; revised 22 November 2002; accepted 2 December 2002

ABSTRACT: Exhaustive exercise such as long-distance running has been shown to increase susceptibility to infection. In order toinvestigate whether serum opsonic activity plays a role in such conditions, we utilized luminol-dependent and lucigenin-dependentchemiluminescence (LmCL and LgCL). We took serum samples from 24 male marathon runners before and after running 30 km.Neutrophils were isolated from the peripheral blood of healthy volunteers. Serum opsonic activity was examined by measuringneutrophil ROS stimulated with zymosan particles opsonized by the serum samples. Immunoglobulin and complement levels in theserum were also measured. After a 30 km run, the maximum light emission was increased and the time to reach the maximum lightemission was shortened significantly (p � 0.05) in LmCL. However, there were no significant changes in the immunoglobulin andcomplement levels. The increase of ROS production may suggest that serum opsonic activity is accelerated after running 30 km.Thus, serum opsonic activity might not play a significant role in the susceptibility to infection after long-distance running. Copyright� 2003 John Wiley & Sons, Ltd.

KEYWORDS: long-distance running; serum opsonic activity; chemiluminescence; neutrophil

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There has been a considerable increase in the number ofpeople who exercise to develop physical strength and tomaintain their health. Although exercise is generallyconsidered to be beneficial to health, exhaustive exercisehas been shown to increase susceptibility to infection(1–4). Humans have specific and non-specific immunesystems. Phagocytosis by phagocytes, particularly byneutrophils, plays an important role in non-specificimmunity. Both immunoglobulins and complement areinvolved in phagocytosis of neutrophils. The adhesion ofimmunoglobulins and complement to foreign bodies iscalled ‘opsonization’. Neutrophils express receptors toimmunoglobulins and complement on their surfaces, sothat opsonized foreign bodies are engulfed efficiently.Previous studies have shown that phagocytosis of neutro-phils is suppressed by exhaustive exercise (1, 2, 5). Thedecrease of serum opsonic activity might cause insuffi-cient phagocytosis and consequent susceptibility toinfection.

Neutrophils engulf foreign bodies and attack foreignbodies with reactive oxygen species (ROS) and lyso-

somes. However, excessive production of ROS possiblyinjures human tissues as well as foreign bodies (6). Inparticular, when serum opsonic activity is enhanced,oxidative damage might occur easily after phagocytosis.Many studies have examined the relationship betweenexercise and production of ROS by human neutrophils.Several studies have demonstrated that the production ofROS is increased by rapid and intensive exercise (5, 7, 8).Therefore, it is possible that increase of serum opsonicactivity affects ROS production after intensive exercise.

However, the influence of exhaustive exercise, such asa marathon, on serum opsonic activity has not been fullyinvestigated. In this study, we chose long-distancerunning as a model of exhaustive exercise and examinedthe influences of exhaustive exercise on serum opsonicactivity by measuring neutrophil ROS production. Weevaluated the production of ROS using luminol-depen-dent (LmCL) chemiluminescence and lucigenin-depen-dent CL (LgCL). We also examined the changes of thelevel of serum immunoglobulins and complement whichcan affect serum opsonic activity.

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The subjects were 24 male marathon runners, whousually train an average of 17.4 h (SD 4.1 h) and run

Luminescence 2003;17:122–124Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bio.438

Copyright 2003 John Wiley & Sons, Ltd.

ORIGINAL RESEARCH

*Correspondence to: T. Umeda, Department of Hygiene, HirosakiUniversity School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.Email: sdai@cc.hirosaki-u.ac.jp

Contract/grant sponsor: Ministry of Education, Culture, Sports,Science and Technology, Japan; contract/grant number: 10307009.

175 km (SD 33.8 km) per week. The average age, heightand weight of the runners were 22.9 years, 171.1 cm and56.5 kg, respectively.

Blood samples were taken from the forearm veinbefore and after running 30 km. The haematocrit (Ht) wascalculated using K-2000 (Sysmex, Kobe, Japan). Serumsamples were obtained by centrifugation at 2000 � g for10 min and stored at �60°C until use. We measured theserum level of immunoglobulins (IgG, IgA, IgM) andcomplement (C3) using commercially available turbido-metric immunoassay kits (Nittobo Medical Co. Ltd,Tokyo, Japan). The results were corrected using the Htvalue, in order to take the possibility of dehydration intoconsideration. Approval was obtained from the EthicsCommittee of Hirosaki University School of Medicine.The study protocol and purpose were explained, andwritten consent was obtained from all subjects before thestudy.

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CL has been used as a method to detect ROS sensitively(8, 9). In this study, serum opsonic activity was examinedby measuring neutrophil ROS production using LmCLand LgCL. In brief, 5.0 mg/mL zymosan (zymosan A;Sigma, St Louis, CA, USA) in Hanks balanced saltsolution (HBSS) was incubated with each serum sampleat a final concentration of 13% at 37°C for 30 min foropsonization. Neutrophils were isolated from the periph-eral blood of two healthy volunteers, using Histopaque1077 and 1119 (Sigma, St. Louis, CA, USA), followingthe manufacturer’s instructions. Neutrophil suspensionswere prepared by adjusting neutrophil number to

2.0 � 103 cells/�L through dilution with HBSS. Thechemiluminogenic probes used were bis-N-methyl-acridinium nitrate (lucigenin; Sigma) and 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol; Sigma). Luci-genin was adjusted with HBSS to the concentration of0.5 mmol/L, pH 7.4. Luminol was dissolved with 1 mol/LNaOH and the solution was adjusted to an isotonic stateof 12.5 mmol/L at pH 7.4 by adding HCl, ultrapure waterand NaCl. Luminol solution was diluted with HBSS to0.5 mmol/L before use.

The LmCL and LgCL responses of each sample werestudied in a 96-well microplate at the same time. Eachwell of a microplate was added 50 �L neutrophilsuspension, 50 �L opsonized zymosan (OZ), 50 �Lluminol or lucigenin solution and 100 �L HBSS. Thefinal concentration of luminol and lucigenin was0.1 mmol/L. CL were measured continuously for60 min at 37°C using a Lumi-Box H-1000 (MicrotecCo. Ltd, Funabashi, Japan) (10). The results wereevaluated using the maximum light emission (peakheight: PH) and the time to reach PH (peak time: PT)in the curve of the CL response (Figure 1).

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All data were presented as mean � SD. The differencesbetween before and after running 30 km were tested bythe paired t-test. The differences were considered to bestatistically significant at p � 0.05.

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In this study, the average time for a 30 km run was 1 h48 min 37 s (SD 3 min 23 s). The changes of PH and PTafter running 30 km are presented in Table 1. In LmCL,the PH was significantly higher (p � 0.01) and PT wasshortened significantly when zymosan was opsonized byserum obtained after running 30 km (p � 0.01). On theother hand, no significant changes were observed inLgCL.

The changes of serum level of immunoglobulins andcomplement are shown in Table 2. There were no

Figure 1. Typical pattern of different luminol-dependentchemiluminescence (LmCL) response using serum samplesobtained before and after running 30 km. Peak height (PH) ofresponse curve is significantly higher (p � 0.01) and peak time(PT) is significantly shorter after running 30 km than beforerunning (p � 0.01).

Table 1. Changes of peak height (PH) and peak time (PT) oflumind-dependent (LmCL) and lucigenin-dependent(LgCL) chemiluminescence response after running 30 km

Before After

PH (cpm) LmCL 133,603 � 16,292 145,360 � 19,120*LgCL 12,139 � 1608 11,511 � 2417

PT (s) LmCL 805 � 75 760 � 67*LgCL 799 � 87 778 � 74

* Significantly different from before values (p � 0.01).

Copyright 2003 John Wiley & Sons, Ltd. Luminescence 2003;17:122–124

Long-distance running increases serum opsonic activity ORIGINAL RESEARCH 123

significant changes in the level of immunoglobulins andcomplement after running 30 km.

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In the present study, we utilized LmCL and LgCL tostudy changes of serum opsonic activity after running30 km, as a model of exhaustive exercise.

LmCL showed an increase of serum opsonic activityafter running 30 km, while no significant changes wereseen in LgCL. Since LmCL and LgCL were measured inthe same conditions, our results might suggest thatrunning 30 km could have different effects on LmCL andLgCL. LgCL mainly reacts with superoxide (O�

2 ) whichis the first substance in a metabolism of ROS (12, 13, 14).Superoxide is metabolized to various ROS by, forexample, myeloperoxidase (MPO) activity. LmCL rela-tively reflects the total production of ROS includinghypochlorous acid (HOCl/OCl�) produced by MPOactivity (11, 12, 13). In this study, LmCL was increasedwithout decrease of LgCL. These results implicate thepossibility of enhanced ROS production after running30 km. Furthermore, ROS measured by LmCL are moretoxic than those measured by LgCL. Therefore, ourresults might suggest that oxidative tissue injury is likelyto occur after running 30 km. Shortened PT was also seenafter running 30 km in LmCL. Increased and rapid ROSproduction may implicate increased opsonic activity afterrunning 30 km.

Serum opsonic activity is influenced by immuno-globulins and complement (5). Previous studies demon-strated that exhaustive exercise decreases serum levels ofimmunoglobulins and complement (5, 14). In contrast,the level of immunoglobulins and complement was notchanged after running 30 km. Therefore, the concentra-tion of immunoglobulins and complement might notaffect serum opsonic activity. However, we can notpreclude the possible influence of complement activitybecause we did not measure this parameter.

In this study, zymosan was opsonized by the serum ofsubjects while neutrophils were obtained from healthyvolunteers. Our results suggested that zymosan opsonizedby the serum after running 30 km was efficiently engulfedby neutrophils. However, since we did not use neutro-

phils from the runners, the results did not mean thatphagocytosis was actually accelerated in the subjectsafter running. Although exhaustive exercise causes anincrease in susceptibility to infection, our results suggestthat alteration of serum opsonic activity is not relevant.

In conclusion, running 30 km possibly affects serumopsonic activity and increases the production of ROSfrom neutrophils, which possibly causes enhancedoxidative tissue damage. The results may indicate apossible application of exercise to ageing. On the otherhand, serum opsonic activity may not play a significantrole in the susceptibility to infection after exhaustiveexercise.

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This work was supported in part by a Grant-in-Aid fromthe Ministry of Education, Culture, Sports, Science andTechnology (No. 10307009), Japan.

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Table 2. Changes of level of immunoglobulins and comple-ments after running 30 km

Before (mg/dL) After (mg/dL)

IgG 1154 � 306 1123 � 265IgA 196.7 � 68.5 188.6 � 66.2IgM 117.5 � 62.4 115.1 � 64.2C3 88.0 � 10.9 87.2 � 11.2C4 18.8 � 3.6 18.6 � 3.8

Copyright 2003 John Wiley & Sons, Ltd. Luminescence 2003;17:122–124

124 ORIGINAL RESEARCH D. Saito et al.