Abstract
Objective
Athletes undergoing weight reduction are recognised as being more prone to infection. Few studies exist for athletes on the weight reduction‐mediated changes in neutrophil function and related activities such as reactive oxygen species (ROS) production capability, phagocytic activity (PA) and serum opsonic activity (SOA).
Methods
16 Japanese female university judoists were examined in the early morning of the first day (pre‐values) and the last day (post‐values) of a 20‐day pre‐competition training period. Of the 16 subjects, 8 needed to reduce weight (WR group) and the other 8 did not (control group). The parameters assessed were the neutrophil count, serum immunoglobulins and complements, myogenic enzymes, ROS production capability, PA and SOA.
Results
Comparing the post‐values with the pre‐values, ROS production significantly increased in both groups (p<0.01 for both). PA significantly decreased in the WR group (p<0.05); it also decreased in the control group but the decrease was not significant. SOA significantly increased in the control group (p<0.05), but showed no significant change in the WR group.
Conclusions
The changes in the WR group were probably a direct consequence of the weight‐reduction regimen coupled with the exercise regimen, suggesting that neutrophil parameters (ROS production, PA and SOA) had tended to deviate from their typical compensatory changes to maintain immune system homoeostasis.
Neutrophils are cellular factors in the host which have an important role as the first line of defence against invading foreign substances including microorganisms. Neutrophils engulf microorganisms (phagocytic activity, PA) and produce reactive oxygen species (ROS).1,2 Serum opsonic activity (SOA) contributes to this microbicidal activity through opsonisation of microorganisms—that is, acceleration of adhesion of neutrophils to opsonised substances via IgG, C3 and others. Although appropriate levels of ROS from neutrophils can destroy invading microorganisms,3,4 at higher levels, on the other hand, ROS can cause oxidative damage to normal body tissues and organs.5,6
There are many studies that have examined the effect of exercise and sports including judo on neutrophils and neutrophil‐related functions. However, only three studies have reported on the relationship between weight reduction and SOA (one of the neutrophil‐related functions) in university judoists.7,8,9
Mochida et al10 reported that athletic training‐induced changes in immune functional activities of neutrophils and related factors, such as ROS production, PA and SOA, may compensate for each other to maintain the overall integrity of the neutrophil immune function. A study examining the same three neutrophil‐related components after a period of pre‐competition compulsory weight reduction in judoists would therefore be of interest.
In this study, we simultaneously measured three major neutrophil functions—namely, ROS production capability, PA and SOA, in female university judoist during weight reduction.
Subjects and methods
Research object and period
The study subjects comprised 16 Japanese female judoists who were taking part in a competition in June 2000. Eight subjects who required weight reduction to meet their class requirements were defined as the weight reduction (WR) group, and eight subjects without a need for weight reduction were defined as the control group. The assessments were performed on the first (pre‐values) and last (post‐values) days of a 20‐day period just before a competition, the second and final assessment being carried out on the morning of the day before the competition.
The physical characteristics of the16 subjects were as follows: mean (SD) height, 162.3 (8.3) cm; body weight, 65.3 (8.3) kg; percentage of body fat, 20.7% (3.2%); quantity of body fat, 13.8 (3.6) kg; and fat‐free body mass (FFM), 51.4 (3.6) kg.
Approval for the study was obtained from the ethics committee of Hirosaki University School of Medicine, Aomori, Japan. The study protocol and purpose were explained to all subjects, and written informed consent was obtained from all of them before the study.
Research content
Measurement of the physical characterisitcs of all subjects (weight, body density, percentage of body fat, quantity of body fat and fat‐free body mass) was performed on days 1 and 20 under fasting conditions early in the morning before training, and blood samples were collected for the blood biochemistry assay, including ROS and PA (measured by flow cytometry) and SOA (measured by the luminol‐dependent chemiluminescence method). The dietary intake of the subjects during the research period was also investigated.
Weekly training programme during the research period
All subjects performed their usual weekly practice regimen during the research period, after having rested for 2 weeks beforehand. Table 1 shows the weekly regimen in detail. It consisted of 6 days of exercise, alternating running (distance and sprinting) and weight training in the mornings, judo practice in the afternoons, and 1 day of rest. The 2½ h judo practice, which is classed as intermittent anaerobic exercise, consisted of warm‐up (stretching) for 15 min; “uchikomi” (the same technique practiced repeatedly, such as throw‐down, push‐down and hook‐down) for 20 min; “randori” (exercise training in the form of mini‐matches) for 100 min; and cool‐ down for 15 min.
Table 1 Training programme per week during the research period.
| Day | Training period | ||
|---|---|---|---|
| 06:30–07:30 | 09:00–11:30 | 17:30–20:00 | |
| Monday | Training A | Rest | Training D |
| Tuesday | Training B | Rest | Training D |
| Wednesday | Training C | Rest | Training D |
| Thursday | Training A | Rest | Training D |
| Friday | Training B | Training D | Training D |
| Saturday | Training C | Rest | Training D |
| Sunday | Rest | Rest | Rest |
Training A, interval training consisting of sprint running (800 m ×1, 400 m ×3, 200 m ×3, 100 m ×4) and jogging.
Training B, weight training.
Training C, distance running for 30 min and short sprint running (repeated 30 m/50 m sprint running).
Training D, judo training practice.
Rest, take a rest or attend lectures.
Physical characteristics and energy intake
Body weight was measured using an A&D Company digital scale system (AD6205, Tokyo, Japan). All subjects had their body weights measured early in the morning before training.
Table 5 shows the changes in blood leucocyte counts, Igs and complements. No significant changes in these parameters were seen in either of the groups.
Table 5 Changes in blood leucocytes, neutrophils, Igs and complements.
| Pre | End | |
|---|---|---|
| Total leucocytes (per μl) | ||
| WR | 6537.5 (911.7) | 6245.3 (917.9) |
| Control | 5975.0 (882.8) | 6084.7 (979.7) |
| Neutrophils (per μl) | ||
| WR | 3931.4 (946.2) | 3575.0 (847.4) |
| Control | 3452.5 (467.7) | 3588.2 9646.1) |
| IgG (mg/dl) | ||
| WR | 1246.0 (91.2) | 1297.5 (42.5) |
| Control | 1252.4 (123.6) | 1244.8 (118.7) |
| IgA (mg/dl) | ||
| WR | 175.3 (19.0) | 181.0 (44.8) |
| Control | 261.8 (22.9) | 261.9 (24.6) |
| IgM (mg/dl) | ||
| WR | 159.1 (18.5) | 171.3 (5.9) |
| Control | 207.9 (43.0) | 197.4 (36.7) |
| C3 (mg/dl) | ||
| WR | 108.4 (2.7) | 97.1 (4.6) |
| Control | 111.4 (5.6) | 95.0 (4.1) |
| C4 (mg/dl) | ||
| WR | 22.8 (1.7) | 23.3 (22.0) |
| Control | 22.0 (2.4) | 20.0 (1.9) |
End, 1 day before the competition; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
Results are expressed as mean (SD).
The body composition was measured with B‐mode ultrasound equipment, as it is impossible to use underwater weighing for females over a research period of approximately 1 month because of problems associated with menstruation. Subcutaneous fat thickness was measured with B‐mode ultrasound equipment (Echo Camera SSD‐500, ALOKA, Tokyo, Japan) using 3.5 or 7.5 MHz at six sites on the right side: the biceps and triceps (on the anterior and posterior surface 60% distal between the lateral epicondyle of the humerus and the acromial process of the scapula); the abdomen (at an area 2–3 cm to the right of the umbilicus); the subscapular region (at an area 5 cm directly below the angulus inferior of the scapula); and the quadriceps and hamstrings (on the anterior and posterior surface at the midpoint between the lateral condyle of the femur and the greater trochanter). Body density was calculated using the total subcutaneous fat thickness of these six points from the equation of Abe et al,11 and percentage fat, FFM and fat mass were calculated from measured body density using Brozek's equation.12 All these measurements were performed by the same trained person. All sites were measured three times. Furthermore, Abe et al11 previously reported that the results of the ultrasound technique correlated well with the results of the underwater weighing method.
Dietary survey
The subjects recorded their meals and the weight of their food intake every day during the study. Daily nutrient intake and total energy intake were calculated using the fourth revision of the standard Tables of Food Composition.13 The mean of total energy intake during the 3 days before each measurement was used to determine the total energy intake.
Haematological and biochemical assays
Blood samples were taken from the forearm vein to measure the following parameters: (i) white blood cell and leucocyte counts in whole blood (using a blood cell autoanalyser, MicroBiff‐II; Coulter, Fullerton, California, USA); (ii) Igs (IgG, IgA and IgM, nephelometry method); and (iii) complements (C3 and C4, nephelometry method) in serum. Furthermore, serum samples were used to determine the concentrations of asparate aminotransferase, alanine aminotransferase, lactic acid, lactate dehydrogenase, and creatine kinase. These serum parameters were measured using the ultraviolet method.
Neutrophil oxidative burst and PA
Neutrophil oxidative burst (ROS production) and PA were measured with FACScan (Becton Dickinson, San Jose, California, USA) using two‐colour flow cytometry. Hydroethidine (HE; 44.4 μmol/l; Polyscience, Warrington, Pennsylvania, USA) was used as an indicator for oxidative burst activity, and opsonised zymosan particles (Sigma Chemical Co, St Louis, Missouri, USA) labelled with fluorescein isothiocyanate (FITC; Sigma Chemical) were used as indicators for PA. In brief, 100 μl of heparinised whole blood was mixed with 22 μl of HE to a final concentration of 8 μM, and incubated at 37°C for 35 min. After the addition of 25 μl of FITC‐labelled opsonised zymosan (FITC‐OZ; final concentration of 5 mg/ml), the sample was incubated at 37°C for 35 min. The same amount of whole blood labelled with only HE was prepared to measure the basal oxidative burst activity. After incubation, Lyse and Fix (Immunotech, Marseille, France) was added for haemolysis of red blood cells and fixation of white blood cells. The sample was washed twice in phosphate‐buffered saline with sodium azide. The fluorescence intensity in the activated neutrophils was measured with FACScan (Becton Dickinson). Extracellular fluorescence was quenched by adding 30 μl of trypan blue (0.25 mg/ml, pH 4.5) just before the assay to differentiate between attached and ingested FITC‐OZ in the neutrophils.14,15
FACScan
Neutrophils were analysed on a standard flow cytometer (FACScan). In each sample, 10 000 neutrophils were analysed. Neutrophil oxidative burst activity (ROS production) and PA data were collected using a logarithmic amplifier and estimated as the mean fluorescence intensity channel number of activated neutrophils. The percentages of positive cells producing ROS and incorporating OZ were calculated.
Measurement of SOA
Chemiluminescence has been used as a method to detect ROS sensitively.16,17 In this study, SOA was examined by measuring neutrophil ROS production using luminol‐dependent chemiluminescence.
Zymosan from Saccharomyces cerevisiae, a well‐known activator of the alternative pathway of the complement system, was employed for opsonised particles. Zymosan A (Sigma) was suspended in Hank's balanced salt solution (HBSS) at a concentration of 5 mg/ml and then opsonisation was performed by adding to the serum samples to a final concentration of 20% and incubating at 37°C for 30 min. The particles were then washed twice with HBSS and resuspended in HBSS at a concentration of 5 mg/ml. Luminol was prepared by dissolving 5‐amino‐2,3‐dihydro‐1,4‐phatalazinedione (Sigma) initially in 1 M NaOH to give a clear solution and then adjusted using HCl and HBSS to give a final concentration of 2 mM (pH 7.4). Normal pooled human neutrophils were obtained from the peripheral blood of a healthy adult male volunteer, whereby whole blood centrifuged through Mono‐Poly resolving medium (Dainippon Pharmaceutical, Tokyo Japan) has been modified. The neutrophils were suspended to 3×106 cells/ml using an automatic blood cell counter (Coulter MD II, Coulter Co, Tokyo, Japan).
OZ suspension and chemiluminigenic probes prepared as above were added to each well of black flat‐bottom microplates (Greiner Japan, Tokyo, Japan), and 50 μl of standard neutrophils was added. The plates were automatically measured on the Auto Luminescence Analyzer, Alfa system (Tokken, Funabashi, Japan).18 All measurements were performed at 37°C. As peak height and area under the curve have been widely used and are reliable parameters,19,20,21 the results were evaluated using the maximum light emission (peak height) and the area under the curve of chemiluminescence response. Each sample was run in duplicate and values were expressed as means.
Statistical analysis
Data were presented as mean (SD). The changes within each group before and after weight reduction were tested with the Wilcoxon's test, and the Mann–Whitney U test was used to analyse the differences in all parameters between the two groups. The differences were considered to be statistically significant at p<0.05.
Results
Table 2 shows changes in body composition in the WR and control groups. In the WR group, body weight, body fat and FFM significantly decreased after weight reduction (p<0.001, p<0.05 and p<0.05, respectively). There was no significant change in the control group.
Table 2 Physical characteristics and changes in anthropometric parameters.
| Pre | End | |
|---|---|---|
| Body weight (kg) | ||
| WR | 65.5 (12.0) | 62.3 (12.0)** |
| Control | 64.8 (8.4) | 64.5 (8.1) |
| Weight loss rate (%) | ||
| WR | 0.0–0.0 | 4.9 (2.7)**† |
| Control | 0.0–0.0 | 0.5 (1.1) |
| Relative body weight (%) | ||
| WR | 20.1 (1.2) | 19.9 (1.0) |
| Control | 21.3 (1.5) | 20.6 (1.4) |
| Body fat (kg) | ||
| WR | 13.5 (1.7) | 12.6 (1.5)* |
| Control | 14.1 (1.6) | 13.5 (1.4) |
| FFM (kg) | ||
| WR | 52.0 (2.7) | 49.7 (2.9)* |
| Control | 50.7 (1.5) | 50.9 (1.5) |
End, 1 day before the competition; FFM, fat‐free mass; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*p<0.05, **p<0.001, compared with pre‐value, †p<0.001, compared with control.
Results are expressed as mean (SD).
Table 3 shows the changes in nutritional intake per kg body weight during the trial period. In the WR group, intakes of energy, protein, lipid and carbohydrate decreased, with a reduction rate for all of >50%, with the exception of carbohydrates. On the other hand, no changes in these intakes were seen in the control group.
Table 3 Changes in energy intake per kg body weight.
| Pre | End | Reduction rate (%) | |
|---|---|---|---|
| Energy (kcal/kg) | |||
| WR | 37.97 (6.20)† | 18.11 (5.41)* | 52.30 |
| Control | 29.50 (7.20) | 24.95 (8.94) | 15.43 |
| Protein (g/kg) | |||
| WR | 1.23 (0.21) | 0.49 (0.15)*† | 60.08 |
| Control | 1.02 (0.27) | 0.78 (0.23) | 23.40 |
| Lipids (g/kg) | |||
| WR | 1.24 (0.21) | 0.55 (0.17)* | 55.48 |
| Control | 0.94 (0.30) | 0.82 (0.39) | 13.48 |
| Carbohydrates (g/kg) | |||
| WR | 5.35 (1.13) | 3.50 (1.27)* | 33.77 |
| Control | 4.25 (1.05) | 2.84 (1.00) | 33.13 |
End, 1 day before the competition; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*p<0.05, compared with pre‐value.
†p<0.05, compared with control.
Results are expressed as mean (SD).
Table 4 shows the changes in serum myogenic enzymes: creatine kinase, lactate dehydrogenase, asparate aminotransferase and alanine aminotransferase levels had significantly increased on the last (post‐values) day of the 20‐day study period.
Table 4 Changes in serum myogenic enzymes.
| Pre | End* | |
|---|---|---|
| CK (IU/l) | ||
| WR | 174.5 (44.5) | 604.6 (993.6)† |
| Control | 276.8 (108.8) | 211.3 (44.2) |
| LDH (IU/l) | ||
| WR | 348.1 (28.2) | 385.9 (88.6)† |
| Control | 372.4 (25.5) | 367.5 (25.4) |
| AST (IU/l) | ||
| WR | 18.5 (2.1) | 40.4 (49.3)† |
| Control | 20.1 (2.5) | 18.0 (1.4) |
| ALT (IU/l) | ||
| WR | 13.9 (1.2) | 25.0(22.1)† |
| Control | 14.9 (2.0) | 14.4 (1.0) |
ALT, alanine aminotransferase; AST, asparate aminotransferase; CK, creatine kinase; End, 1 day before the competition; LDH, lactate dehydrogenase; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*The post‐values were adjusted for dehydration by plasma volume.
†p<0.05, compared with pre‐value.
Results are expressed as mean (SD).
As for neutrophil and related functions such as the ROS (neutrophil oxidative burst activities) production capability, PA and SOA (tables 6–8), comparing the post‐values with the pre‐values, ROS production significantly increased in both groups (p<0.01 for both), PA significantly decreased in the WR group (p<0.05), and, although it decreased in the control group, the difference was not significant, and SOA significantly increased in the control group (p<0.05), although it showed no significant change in the WR group.
Table 6 Changes in luminol‐dependent serum opsonic activity.
| Pre | End | |
|---|---|---|
| LmCL‐AUC (×105) | ||
| WR | 315.9 (7.4) | 325.4 (13.1) |
| Control | 304.6 (9.6) | 345.2 (7.1)* |
| LmCL‐PH (×104) | ||
| WR | 107.8 (1.5) | 111.4 (3.0)† |
| Control | 107.8 (3.1) | 119.6 (1.5)* |
AUC, area under the curve for 45 min; End, 1 day before the competition; LmCL, luminol‐dependent chemiluminescence; PH, peak height; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*p<0.05, compared with pre‐value.
†p<0.05, compared with control.
Results are expressed as mean (SD).
Table 7 Changes in neutrophil phagocytic rate and activity9.
| Pre | End | |
|---|---|---|
| Phagocytic rate (%) | ||
| WR | 88.2 (16.1) | 94.4 (2.1) |
| Control | 87.2 (17.3) | 94.0 (1.3) |
| Phagocytic activity per cell (FI) | ||
| WR | 737.7 (96.5) | 567.7 (74.6)*† |
| Control | 824.4 (227) | 658.7 (103.6) |
End, 1 day before the competition; FI, fluorescence intensity; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*p<0.001, compared with pre‐value.
†p Value compared with control.
Results are expressed as mean (SD).
Table 8 Changes in neutrophil oxidative burst rate and activity9.
| Pre | End | |
|---|---|---|
| Oxidative burst rate (%) | ||
| WR | 72.5 (18.9) | 96.8 (2.7)* |
| Control | 72.1 (18.5) | 97.4 (2.5)* |
| Oxidative burst activity per cell (FI) | ||
| WR | 169.4 (15.3) | 337.7 (21.5)**† |
| Control | 171.0 (14.3) | 307.5 (22.6)** |
End, 1 day before the competition; FI, fluorescence intensity; Pre, 20 days before the competition; WR, weight reduction.
WR group, n = 8; control group, n = 8.
*p<0.05, **p<0.001, compared with pre‐value.
†Compared with control.
Results are expressed as mean (SD).
Spearman's correlation coefficients among the changes from pre‐value to post‐value for ROS production, PA and SOA of Igs and complements are shown in table 9. No significant correlations were seen among these parameters.
Table 9 Spearman's correlation coefficients among measured parameters.
| IgG | IgA | IgM | C3 | C4 | |
|---|---|---|---|---|---|
| PA per cell | 0.267 | 0.217 | 0.211 | 0.127 | 0.112 |
| ROS production per cell | –0.222 | 0.167 | 0.200 | 0.100 | 0.023 |
| AUC of SOA | 0.198 | –0.045 | 0.212 | 0.267 | 0.121 |
AUC, area under the curve; PA, phagocytic activity; ROS, reactive oxygen species; SOA serum opsonic activity.
Discussion
Some studies have reported that weight reduction for athletes by a combination of dieting and exercise decreases muscle volume and body fat. In the present study, against their total weight reduction, subjects reduced their fat mass and FFM by 34% and 64%, respectively, suggesting severe weight reduction and, in fact, an inappropriate degree of weight reduction, considering the maintenance of performance levels in the competition.
The typical change in the three neutrophil functions examined after a single bout of normal exercise with no dietary restrictions is an increase in both ROS and SOA and decrease in PA,22,23,24 and it has been reported that these three neutrophil functions compensate for each other. For example, when PA decreases, ROS and SOA increase, or when PA increases, ROS and SOA decrease.10,25 Mochida et al10 suggested that such compensatory changes allow a “balancing act” to take place among neutrophil activities and related factors, so that the overall integrity of the neutrophil immune function is maintained; however, it is not known which ones compensate for the others. However, under conditions of severe and prolonged exercise such as a full marathon and a training camp plus weight reduction, neutrophil parameters have tended to deviate from such typical compensatory changes. For example, although PA decreased, ROS also decreased.26,27
In the 20‐day study period, the control group showed the following changes: ROS and SOA significantly increased and PA decreased, although it was not significant. These changes are probably due to the effects of the daily training for this period. On the other hand, although PA significantly decreased and ROS production significantly increased in both WR and control groups, SOA did not show a significant increase in the WR group, unlike the control group.
In the control group, a change in the three major neutrophil functions was demonstrated, which is similar to that induced by normal exercise. The difference in the changes between the control and WR group is that the increase in SOA in the WR group was less than that in the control group, even though the decrease in PA was more notable in the WR than in the control group. These changes in the WR group were probably a direct consequence of the weight reduction regimen coupled with the exercise regimen, suggesting that neutrophil parameters tended to deviate from the typical compensatory changes reported previously.
As for weight reduction in athletes, an inhibitory association in male university judoists between SOA and weight reduction plus exercise has been reported only in the study by Ohta et al,7 which is contrary to the results of the present study. The change in Ohta's study may have two explanations. First, the decrease in SOA was to compensate for an increase in PA. Second, the decrease in SOA was a deviation from the typical changes already mentioned above, even though PA had increased. It is impossible to state which of these are correct, because, firstly, PA was not measured in the study by Ohta et al, and secondly, we cannot evaluate whether the exercise/training and weight reduction in that study was normal or not. Furthermore, the difference in the changes between the current study and Ohta et al's study may have been due to gender difference.
From the results of both the current study and that by Mochida et al,10 the measurement of changes in the three major neutrophil functions should allow accurate evaluation of the relationship between exercise loading and the subjects' physical condition.
On the other hand, the values of Igs and complements, although they are recognised as affecting SOA, had no correlation with the changes in SOA levels. Therefore, factors other than Igs and complements may be involved in changes in the SOA in athletes undergoing a combined weight reduction and exercise regimen.
What is already known on this topic
The typical change in the three neutrophil functions (reactive oxygen species production capability, phagocytic activity and serum opsonic activity) examined following a single bout of normal exercise with no dietary restrictions is an increase in both reactive oxygen species production capability and serum opsonic activity, whereas phagocytic activity decreases, and it has been reported that these three neutrophil functions compensate for each other
What this study adds
The changes in the weight reduction group were probably a direct consequence of the weight reduction regimen coupled with the exercise regimen, suggesting that neutrophil parameters (reactive oxygen species production capability, phagocytic activity and serum opsonic activity) had tended to deviate from their typical compensatory changes to maintain immune system homoeostasis
Acknowledgements
We thank the students at the Nippon Sports Science University for their participation in this survey. This study was supported in part by grant‐in‐aid from the Ministry of Education, Culture, Sports, Science and Technology (No 11470092), Japan.
Abbreviations
FFM - fat‐free body mass
FITC - fluorescein isothiocyanate
HBSS - Hank's balanced salt solution
HE - hydroethidine
OZ - opsonised zymosan
PA - phagocytic activity
ROS - reactive oxygen species
SOA - serum opsonic activity
WR - weight reduction
Footnotes
Competing interests: None declared.
References
- 1.Benestad H B, Laerum O D. The neutrophilic granulocyte. Curr Top Pathol 1989797–36. [DOI] [PubMed] [Google Scholar]
- 2.Sawyer D W, Donowitz G R, Mandell G L. Polymorphonuclear neutrophils: an effective antimicrobial force. Rev Infect Dis 198911(Suppl 7)S1532–S1544. [DOI] [PubMed] [Google Scholar]
- 3.Ehlenberger A G, Nussenzweig V. The role of membrane receptors for C3b and C3d in phagocytosis. J Exp Med 1977145357–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Unkeless J C, Wright S D. Structure and modulation of Fc and complement receptors. Contemp Top Immunobiol 198414171–187. [DOI] [PubMed] [Google Scholar]
- 5.Pyne D B. Regulation of neutrophil function during exercise. Sports Med 199417245–258. [DOI] [PubMed] [Google Scholar]
- 6.Duarte J A, Appell H J, Carvalho F.et al Endothelium‐derived oxidative stress may contribute to exercise‐induced muscle damage. Int J Sports Med 199314440–443. [DOI] [PubMed] [Google Scholar]
- 7.Ohta S, Nakaji S, Suzuki K.et al Depressed humoral immunity after weight reduction in competitive judoists. Luminescence 200217150–157. [DOI] [PubMed] [Google Scholar]
- 8.Kowatari K, Umeda T, Shimoyama T.et al Exercise training and energy restriction decrease neutrophil phagocytic activity in judoists. Med Sci Sports Exerc 200133519–524. [DOI] [PubMed] [Google Scholar]
- 9.Suzuki M, Nakaji S, Umeda T.et al Effects of weight reduction on neutrophil phagocytic activity and oxidative burst activity in female judoists. Luminescence 200318214–217. [DOI] [PubMed] [Google Scholar]
- 10.Mochida N, Umeda T, Yamamoto Y.et al The main neutrophil and neutrophil‐related functions may compensate for each other following exercise: a finding from training in university judoists. Luminescence 20062220–28. [DOI] [PubMed] [Google Scholar]
- 11.Abe T, Kondo M, Kawakami Y.et al Prediction equations for body composition of Japanese adults by B‐mode ultra sound. Am J Hum Biol 19946161–170. [DOI] [PubMed] [Google Scholar]
- 12.Brozek J, Grande F, Anderson J T.et al Densitometric analysis of body composition: revision of some quantitative assumption. Ann N Y Acad Sci 1963100113–140. [DOI] [PubMed] [Google Scholar]
- 13.Standard tables of food composition in Japan [in Japanese]. 4th edn. Tokyo: Ministry of Finance 198243–398.
- 14.Hed J, Hallden G, Johansson S G.et al The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods 1987101119–125. [DOI] [PubMed] [Google Scholar]
- 15.Sahlin S, Hed J, Rundquist I. Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay. J Immunol Methods 198360115–124. [DOI] [PubMed] [Google Scholar]
- 16.Kikuchi T, Suzuki K, Abe T.et al Measurement of chemiluminescence from neutrophils in a 96‐well microplate using Lumi Box U‐800 II. J Biolumin Chemilumin 199712149–153. [DOI] [PubMed] [Google Scholar]
- 17.Suzuki K, Sato H, Kikuchi T.et al Capacity of circulating neutrophils to produce reactive oxygen species after exhaustive exercise. J Appl Physiol 1996811213–1222. [DOI] [PubMed] [Google Scholar]
- 18.Kumae T. A study of the fluorescence measurement using a 96‐well microplate by a remodelled parallel luminescent measuring system. Luminescence 199914375–381. [DOI] [PubMed] [Google Scholar]
- 19.Allen R C. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol 1986133449–493. [DOI] [PubMed] [Google Scholar]
- 20.Hasegawa H, Suzuki K, Nakaji S.et al Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96‐well microplate format using lucigenin‐ and luminol‐dependent chemiluminescence. J Immunol Methods 19972101–10. [DOI] [PubMed] [Google Scholar]
- 21.Kumae T, Yamasaki K, Ishizaki K.et al Effects of summer camp endurance training on non‐specific immunity in long‐distance runners. Int J Sports Med 199920390–395. [DOI] [PubMed] [Google Scholar]
- 22.Chinda D, Umeda T, Shimoyama T.et al The acute response of neutrophil function to a bout of judo training. Luminescence 200318278–282. [DOI] [PubMed] [Google Scholar]
- 23.Suzuki M, Umeda T, Nakaji S.et al Effect of incorporating low intensity exercise into the recovery period after a rugby match. Br J Sports Med 200438436–440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Miura M, Umeda T, Nakaji S.et al Effect of 6 months' training on the reactive oxygen species production capacity of neutrophils and serum opsonic activity in judoists. Luminescence 2005201–7. [DOI] [PubMed] [Google Scholar]
- 25.Sugawara K, Suzuki K, Liu Q.et al Production of reactive oxygen species from human neutrophils in many kinds of environment. Hirosaki Med J 199951(Suppl)S31–S42. [Google Scholar]
- 26.Chinda D, Nakaji S, Umeda T.et al A competitive marathon race decreases neutrophil functions in athletes. Luminescence 200318324–329. [DOI] [PubMed] [Google Scholar]
- 27.Saito D, Nakaji S, Umeda T.et al Effects of long‐distance running on serum opsonic activity measured by chemiluminescence. Luminescence 200318122–124. [DOI] [PubMed] [Google Scholar]