The Effects of Acute Exercise on Neutrophils and Plasma... : Medicine & Science in Sports & Exercise (original) (raw)
Acute exercise-generated oxidative stress has been well documented over the last decade (1,2,12,15,22). Previous studies have identified elevations in blood oxidative stress markers after acute exercise, indicating that oxidative stress is not limited to the cellular compartment (1,2,15,22). Further, very high intensity exercise appears to exaggerate the blood oxidative stress response (1). Some investigators (22) have suggested that elevations in blood oxidative stress markers are the direct result of superoxide (O2−) generation in skeletal muscle thought to be produced at a rate of 1–3% of total metabolic activity (21,27). Superoxide production at a rate of 1–3% may become detrimental to cellular function during exercise because total oxygen consumption can increase 10- to 20-fold, depending on individual ability and exercise intensity. In light of a recent study that indicated blood oxidative stress was not related to energy expenditure (1), however, other exercise-induced sources for the blood oxidative stress need to be investigated.
As reviewed by Ji (13), high-intensity exercise results in neutrophil translocation to the active skeletal muscle, subsequently resulting in an oxidative stress. Because endurance exercise performed at intensities above lactate threshold dramatically elevates circulating neutrophil numbers, it is plausible that neutrophilia contributes to the blood oxidative stress observed with high-intensity exercise. In support, gross blood levels of neutrophil oxidative enzyme levels (MPO and elastase) (6,11,12,22) and free radical production (O2−) (12,24) can be elevated immediately postexercise. Importantly, exercise-induced elevations in these oxidant markers can occur despite decrements in neutrophil oxidative capacity, indicating that an increased number of circulating neutrophils might impose an oxidative stress in blood despite depressed or unchanged degranulation and oxidative burst. Thus, we tested the hypothesis that an exercise-induced increase in circulating neutrophils imposes an oxidative stress on the blood when exercise intensity exceeds lactate threshold (LT).
To date, the relationship between exercise intensity, circulating neutrophils, and the blood oxidative stress response to acute exercise has not been comprehensively examined. Accordingly, the purpose of this investigation was to determine whether exercise-induced neutrophilia imposes an oxidative stress in blood as measured by decreased water-soluble antioxidant defenses and increased lipid peroxidation markers. Further, the influence of exercise intensity versus total energy expenditure was investigated to better clarify the origin of exercise-induced blood oxidative stress. We specifically examined exercise performed at intensities above and below lactate threshold to better elucidate this critical threshold in regard to neutrophilia and oxidant production as they may have particular relevance to the blood oxidative stress response to exercise.
METHODS
Subjects.
Nine male subjects between the ages of 18 and 30 completed this study. Study clearance was approved by the East Tennessee State University/Veterans Administration Medical Center Institutional Review Board. Each subject granted written informed consent before testing. Several questionnaires were completed before exercise testing to determine subject preparedness for exercise (physical activity readiness questionnaire, PAR-Q), dietary intakes (Nutritionist 4 dietary survey, and a survey of dietary supplements), and for the quantification of physical activity histories (The Aerobics Center Longitudinal Study). To further characterize subjects, hydrostatic weighing was performed to assess body composition.
Experimental protocol.
The exercise testing protocol is presented in Figure 1. Data were collected from exercise sessions performed over the course of 5 wk. For each subject, one exercise session was performed each week with all sessions beginning between 5:30 and 6:30 a.m. For session 1 (Max), the Broeder treadmill protocol was used to elicit a V̇O2max response. The Broeder treadmill protocol was designed to provide a 3-min warm-up period (4). The initial speed was 2.0 miles·h−1 and was increased each minute by 0.5 miles·h−1. Initial treadmill grade was 0% and with increases to 3%, 6%, and 9% at min 4, 6, and 8, respectively. Blood was drawn before (PRE) and after (immediately post = POST, 1 h post = 1 h, and 2 h post = 2 h) exercise in order to quantify the neutrophil and oxidative stress responses to maximal-intensity exercise. All postexercise antioxidant, lipid peroxidation, and MPO variables were corrected for changes in plasma volume (9). For Max exercise, plasma volume changes were −15%, 4%, and −2% for POST, 1 h, and 2 h, respectively. Exercise-induced alterations in plasma volume were similar across all submaximal-intensity sessions (−6%, 2%, and −1% for POST, 1 h, and 2 h, respectively).
Exercise testing protocols; * denotes trials 3 and 4 were randomized; B, blood draw.
Session 2 was a lactate threshold (LT) test that included six to seven 5-min stages of progressively increasing exercise workloads (increments of 10% V̇O2 max/stage). Blood was drawn during the final minute of each stage for determination of plasma lactate (Sigma, St. Louis, MO). Lactate values were expressed in mM according to a lactate standard curve. Lactate threshold was determined by plotting blood lactate against each exercise workload. The V̇O2 elicited in the stage before a rise in blood lactate >1 mM above baseline concentration represented LT (29). The determination of LT was necessary for the prescription of three individualized submaximal exercise workloads at intensities both above and below LT.
Exercise sessions 3, 4, and 5 were of submaximal-intensity and maintained for 45–60 min to mimic exercise recommended for improved health and fitness. During the three steady state trials, each subject exercised at approximately 10% above LT for 45 min (LT+), approximately 10% below LT for 45 min (LT−), and approximately 10% below LT until caloric expenditure equaled that expended during the trial performed above LT (LT-kcal). The order of LT+ and LT− trials was randomized to prevent a training effect on study outcomes. As with the Max trial, blood was drawn before (PRE) and after (POST, 1 h, and 2 h) the three submaximal-intensity exercise sessions for quantification of neutrophil and oxidative stress responses to exercise. Blood concentrations of all postexercise variables were corrected for changes in plasma volume as previously described (9). For all exercise sessions, physical activity and caffeine ingestion were discontinued in the 48 h before exercise. In addition, subjects reported for exercise testing at least 12 h fasted.
Phlebotomy and blood handling.
For the Max and submaximal exercise sessions, blood was drawn from the antecubital vein into an 8-mL Vacutainer tube (EDTA) and a 4-mL Vacutainer tube (heparin) for all time periods. An aliquot of heparinized whole blood was removed and refrigerated (4°C) until assay (< 4 h). After centrifugation of the remaining blood, plasma was aliquoted and stored at either 4°C for day of analysis or at −80°C for later assay. During the LT test, blood was drawn from a 1.25-inch, 20-gauge vinyl venous catheter, inserted into the antecubital vein.
White blood cell counts and biochemical analysis.
Neutrophilia/white blood cell (WBC) counts were generated by multiplication of leukocyte counts and WBC differentials (leukocyte percentages). Manual leukocyte counts were performed using a hemocytometer (American Optical Corp., Buffalo, NY) as viewed under an Olympus (Japan) microscope equipped with 100× magnification. Final values were expressed as cells × 109 L−1 whole blood.
Heparinized whole-blood smears on glass slides (Fisher Scientific, Pittsburgh, PA) were used for WBC differential analysis. Smears were stained using Hema-quick (Biochemical Sciences, Swedesboro, NJ) and air dried. Cell differentials were performed using an Olympus microscope equipped with 1000× oil immersion lens. Specifically, the leukocyte counts including segmented and band neutrophils, eosinophils, basophils, monocytes, and lymphocytes were recorded.
Superoxide (O2−) production by phagocytes was measured in heparinized whole blood using the luminescence technique of Tosi and Hamedani (26). Immune cells were stimulated with 1 μg·mL−1 phorbol myristate acetate (PMA). Importantly, previous investigations have determined that 95% of the O2− measured using this technique is neutrophil generated (25). Luminescence values, produced in the presence of 10-4M lucigenin (Sigma), were read in a Galaxy Floustar multipurpose plate reader. Pilot data collected before starting this study demonstrated that the addition of superoxide dismutase (100 μg·mL−1) to the assay solution inhibited over 97% of the luminescence value. Other pilot experiments demonstrated that this procedure was reproducible and sensitive enough to distinguish differences in the O2− response to exercise sessions of variable intensity.
Plasma MPO levels were measured using Orgentec enzyme-linked immunosorbent assay (ELISA) kits. Absorbance values were read at 660 nm with final concentrations expressed in U·mL−1 according to a standard curve.
Plasma ascorbic acid was determined spectrophotometrically using the technique of Benzie (3). Samples were added to an assay solution containing ferric-tripyridyltriazine, resulting in a chromophore that absorbs light at 593 nm. Specific ascorbic acid content for a given sample was determined as the difference between absorbance values for aliquots mixed with either ascorbate oxidase or distilled H2O. Final concentrations were expressed in μM according to an ascorbic acid standard curve. Determination of plasma uric acid concentrations was performed spectrophotometrically using the assay of Kovar and El-Yazbi (14). Samples were added to an assay solution containing 3-dimethylaminobenzoic acid and 3-methylbenzothiazoline-2-1 hydrazone HCl, resulting in a chromophore that absorbs light at 590 nm. Specific uric acid content for a given sample was determined as the difference between absorbance values for aliquots mixed with either uricase or distilled H2O. Final uric acid concentrations were expressed in μM according to a uric acid standard curve. Malondialdehyde measurement was performed using the Bioxytech LPO-586 technique developed by Esterbauer and Cheeseman (10). Final MDA values were expressed in μM using a 1.1.3.3-tetremethoxypropane standard curve. Lipid hydroperoxide levels were determined using the Bioxytech LPO-560 technique developed by Nourooz-Zadeh et al. (19). Lipid hydroperoxide values were expressed in μM according to a cumene hydroperoxide standard curve.
Statistical analyses.
Repeated measures ANOVA were used to assess within-treatment and between-treatment differences. When significant main effects were present, interaction effects were also analyzed. Least significant difference post hoc analysis was used only when _F_-test found significance. Multiple stepwise regression was performed to determine complex interactions. All analyses used a P ≤ 0.05 as a criterion for significance.
RESULTS
Subject characteristic data are presented in Table 1. Subjects were of average height and weight and had normal body fat percentages for 18- to 30-yr-old males. Physical activity histories indicated that subjects performed various types of aerobic exercise an average of 4 d·wk−1 and resistance exercises an average of 2 d·wk−1. The average V̇O2max was 51.2 ± 6.7 (mL·kg−1·min−1), indicating that these subjects were fit with an average ranking in the 90% percentile of age-matched counterparts (16). As a further indication of fitness, the LT response was 64.4% of V̇O2max (33.1 ± 5.8 mL·kg−1·min−1) on average.
Subject characteristics.
Based on the data presented in Table 2, the experimental design was successful in producing maximal, above, and below LT V̇O2 responses (Max = 100%, LT+ = 72%, LT− = 55%, and LT-kcal = 54%). Immediate postexercise blood lactate data further support the finding that the prescribed exercise intensities were achieved for each trial (Max: pre = 0.9 ± 0.3, post = 9.9 ± 2.1 mM; LT+: pre = 0.8 ± 0.4, post = 4.2 ± 1.6 mM, LT−: pre = 0.9 ± 0.4, post = 1.2 ± 0.3 mM, and LT-kcal: pre = 0.9 ± 0.5, post = 1.0 ± 0.5 mM). For all trials, the average POST exercise plasma lactate concentration was positively correlated with the average peak V̇O2 (r = 0.997, P = 0.003). With respect to the submaximal-intensity trials, LT− and LT-kcal were successfully matched for exercise intensity (V̇O2 and POST lactate). In addition, LT+ and LT-kcal were successfully matched for total caloric expenditure (total energy expenditure). Because of the different exercise intensities between LT+ and LT-kcal trials, the LT-kcal trial was maintained an average of 15 min longer than LT+ to achieve this paring in total energy expenditure.
Peak maximal performance data.
Immediately postexercise neutrophil counts were elevated in an intensity-dependent fashion as demonstrated by a positive relationship between POST exercise blood lactate levels and POST neutrophil counts (r = 0.97, P = 0.009). In contrast, total energy expenditure (r = 0.59, P = 0.229) was unrelated to circulating neutrophils levels. This POST exercise intensity-dependent neutrophil response is also highlighted in Figure 2 where only the trials performed above LT resulted in neutrophil count elevations over baseline (Max = 73%, LT+ = 43%). At the 2-h time period, a second wave neutrophil rise was noted, though the response was not intensity-dependent (Max = 32%, LT+ = 35%, LT− = 22%, LT-kcal = 24%). Data presented in Figure 3 top show an elevation in O2− values for both Max (POST and 2 h) and LT+ trials (2 h). This finding indicates that more neutrophil-generated O2− was present after high-intensity exercise only. When O2− values were normalized for neutrophil numbers (O2−/neutrophils, Fig. 3 bottom), however, significant increases over baseline were present for Max and LT+ at 2 h only. This result suggests that neutrophil O2− capacity was not increased immediately post Max exercise, and that elevated O2− at that time period are due to the dramatic increase in neutrophil counts. At the 2-h time period, however, the increased O2−/neutrophil ratio may indicate that the neutrophil free radical production capacity was responsible for the rise in O2−. In contrast to O2−, no exercise trial resulted in an elevation in MPO values outside of normal limits as specified by the assay manufacturer (data not shown). Further, when MPO values were normalized for neutrophil number (data also not shown) only the values post Max and LT− were significantly lower (−40% and −24% for Max and LT−, respectively) than baseline. In total, these data clearly illustrate the importance of high-intensity exercise on blood free radical production via neutrophil demargination and activation.
Blood neutrophil counts before and after maximal and submaximal intensity exercise; N = 9; values with different letters (a,b,c) are significantly different between trials for measurement period, P ≤ 0.05; significantly different from PRE, P ≤ 0.05.
Superoxide (top) and superoxide/neutrophils (bottom); N = 9; values are means ± SE; a, significantly different for Max measurement period, P ≤ 0.05; significantly different from PRE, P ≤ 0.05.
Figure 4 presents water-soluble antioxidant values (ascorbic acid—top, uric acid—-bottom) before and after the four exercise trials. Immediately after Max, both ascorbic acid and uric acid were significantly decreased (ascorbic acid = −26%, uric acid = −29%). During the 2 h of recovery from maximal-intensity exercise, however, ascorbic acid and uric acid responded differently. At the 1-h and 2-h time periods after Max exercise, ascorbic acid values returned to baseline levels (1 h = +2.6 μM, P = 0.189; 2 h = −7.1 μM, P = 0.079), whereas uric acid values were significantly higher than baseline (1 h = +100.9 μM, P = 0.026; 2 h = +97.9 μM, P = 0.03). In contrast to maximal-intensity exercise, no submaximal-intensity trial elicited significant alterations in plasma ascorbic acid values. Similarly, uric acid values were not altered significantly in response to any trial other than Max, with the exception of 2 h post LT− trial (+24%). In this instance, one outlying data point appears to have altered the mean response. Finally, no exercise session resulted in significant alterations in either LOOH or MDA values, presented in Tables 3a and 3b, respectively.
Ascorbic acid (top) and uric acid (bottom); N = 9; values are means ± SE; Values with different letters (a,b) are significantly different between trials for measurement period, P ≤ 0.05; significantly different from PRE, P ≤ 0.05.
Plasma lipid hydroperoxides (μM) before and after maximal and submaximal intensity exercise.
Plasma malondyaldehyde (μM) before and after maximal and submaximal intensity exercise.
DISCUSSION
We tested the hypothesis that an exercise-induced increase in circulating neutrophils imposes an oxidative stress on the blood when exercise intensity exceeds lactate threshold. In regard to exercise intensity and blood oxidative stress, the findings of decreased antioxidants indicate that an oxidative stress was present in response to maximal-intensity exercise only. Concomitant to this finding, elevated neutrophil counts and neutrophil-generated superoxide levels were highest immediately after maximal treadmill exercise. This suggests that exercise-induced neutrophilia may have contributed to the observed oxidative stress. Under the technical constraints of the current study, however, we cannot conclusively demonstrate a causal link between exercise-induced increases in neutrophilia and blood oxidative stress. Further, we cannot rule out the possibility that other potential oxidant sources, such as total oxygen flux, may have contributed to the observed oxidative stress. Inconclusive identification of an oxidant source for the oxidative stress observed immediately postmaximal-intensity exercise highlights a limitation inherent to testing heterogeneous human populations. Moreover, these findings illustrate the complexity of blood redox status; therefore, caution must be exercised in the interpretation of these results. Nonetheless, the results of this study reveal for the first time several important findings in regard to exercise-induced neutrophilia and blood oxidative stress that warrant further discussion.
The present investigation is the first to comprehensively examine the blood neutrophil and oxidative stress responses to acute exercise in humans. In addition, there were several unique aspects inherent to the experimental design by which the importance of exercise intensity versus total energy expenditure on blood neutrophil and oxidative stress outcomes were evaluated. From these data, we conclude that exercise intensity appears to be more important than total energy expenditure in the postexercise neutrophil and oxidative stress responses. In fact, maximal-intensity exercise elicited the most dramatic neutrophil demargination and blood oxidative stress response as compared with all submaximal-intensity exercise sessions. The nonsignificant relationship between total exercise energy expenditure and blood oxidative stress markers is in stark contrast to previously held beliefs that blood oxidative stress reflects mitochondria respiration work (22). This finding supports previous evidence that indirectly suggest that mitochondrial-generated free radicals are not directly responsible for blood born oxidative stress after exercise (1) and emphasizes the need to investigate other potential oxidant sources, including neutrophils brought into circulation during exercise.
Previous studies have investigated postexercise neutrophil activity via measures of phagocytosis and oxidative burst in response to artificial (12,24,30) or bacterial stimuli (24) in isolation from the blood plasma environment. In contrast, to characterize the potential for exercise-induced alterations in neutrophil activity on blood redox status, we chose to examine 1) specific measures of neutrophil free radical production (O2−) and oxidative enzyme release (MPO); and 2) O2− release within the whole blood environment. Further, O2− values were normalized for neutrophil counts in order to estimate neutrophil oxidative burst capacity and degranulation as others have done (12). Results from this study indicate that whereas absolute O2− values were elevated immediately post (Max) and 2 h post (Max and LT+) exercise, only the second wave of neutrophilia (2 h) resulted in elevated neutrophil free radical production capacity as assessed by normalized O2−. In contrast, normalized O2− levels were not elevated immediately post Max, whereas absolute O2− values were. Several potential explanations for this variable response immediately post versus 2 h post Max exercise may give insight into the nature of the neutrophil and oxidative stress responses to exercise. First, PMA, used to stimulate neutrophils, activates oxidative burst through a PKC-dependent pathway that is inhibited by elevations in circulating catecholamines likely to be at the highest study levels immediately post Max exercise (18). Another possible explanation for elevated oxidative burst capacity at the 2-h recovery period may be the functional nature of the second wave neutrophilia. The fact that the second wave neutrophil response was not intensity-dependent supports the hypothesis that these cells are brought into circulation from the bone marrow as stimulated by exercise-induced elevations in cortisol (20). In support, Suzuki et al. (23) found that neutrophils mobilized from bone marrow were more oxidatively active than those brought into circulation immediately postexercise. Clearly, this second wave of neutrophilia may represent a functionally different neutrophil subset as compared with the demargination response seen immediately postexercise. Physiological and functional characteristics of neutrophils in these distinct immune responses have yet to be examined and represent an avenue for future research. As a final explanation, the systemic rise in potent oxidative burst stimuli, such as chemotactic elements from acute muscle-contraction damage, released into circulation during the 2 h post Max exercise also may explain the delayed increase in neutrophil activity (20). However, previous research has indicated that the neutrophil response to exercise known to induce muscle damage includes dramatic postexercise increases in plasma MPO levels (6). In this study, MPO elevations during the 2-h recovery from maximal-intensity exercise did not exceed normal values, suggesting that neutrophil degranulation and oxidative burst were not stimulated immediately postmaximal-intensity exercise. Thus, any oxidative stress that may have been imposed by neutrophils would be due to increased numbers rather than increased oxidative burst or degranulation. This finding likely indicates that the nature of the exercises performed by our subjects did not result in significant muscle damage and a subsequent inflammation response to the exercised skeletal muscle (20).
A major finding of this study was that both ascorbic acid and uric acid were significantly decreased immediately after maximal-intensity exercise. This finding is important in regard to exercise intensity and the blood oxidative stress response. Thus, because submaximal-intensity exercise did not affect levels of either antioxidant, one may conclude that the blood oxidative stress experienced at these exercise intensities did not exceed the capacity of the blood to quench those radicals. Equally important was the plasma antioxidant recovery response to Max exercise. The return in ascorbic acid levels to baseline during the recovery from maximal-intensity exercise in the current study is in agreement with the study of Camus et al. (7) In their study, Camus et al. (7) observed a decrease (−40%) in ascorbic acid immediately postexercise (35 min of downhill running at 60% V̇O2max) followed by a return toward baseline after only 20 min of recovery (−17%). As described in a review by Sen (21), the ascorbic acid regeneration phenomenon observed in the present study most likely occurred through interactions with the plasma the glutathione system.
In contrast to ascorbic acid, post Max recovery levels of uric acid actually exceeded preexercise values and reflect the transient nature of blood redox status after maximal-intensity exercise. Whereas other investigations have also identified a postexercise uric acid increase (15), the present study appears to be the first to document a postexercise uric acid drop before increase. Previous investigators have hypothesized that exercise-induced elevations in plasma uric acid represent purine metabolism within skeletal muscle (15). In light of the fact that maximal-intensity treadmill exercise produces dramatic energy deficits within active skeletal muscle, this explanation is plausible. Ultimately, the uric acid rebound during recovery from Max exercise in the current study supports the importance of uric acid as a plasma antioxidant (17,28) and may explain why ascorbic acid levels were preserved despite a rise in O2− at the 2-h post Max time period.
In contrast to the plasma antioxidant data, mean values of lipid peroxidation (LOOH and MDA) were not significantly altered by any exercise session, though the percent increase in LOOH was significantly elevated during the 2-h recovery from Max as compared with LT− and LT-kcal. Biologically, the time course of percent LOOH production after an immediate decrease in water-soluble antioxidants supports the free radical pecking order within biological fluids (5). Further, MDA formation results from lipid hydroperoxides oxidative chain reactions (8). Thus, the current finding that MDA was unaltered by any exercise intervention is understandable. In agreement with this investigation, previous studies by Ashton et al. (2) and Allessio et al. (1) have also found immediate postmaximal exercise rises in lipid hydroperoxides (42% and 20%, respectively) with no alteration in MDA levels. In total, use of these two lipid peroxidation markers in the current study suggest that the limit of in vivo blood oxidative stress detection may have been exceeded.
In conclusion, significant blood oxidative stress was observed immediately after maximal-, but not submaximal-, intensity exercise. Concomitant to this oxidative stress, neutrophilia and subsequent increases in neutrophil-generated O2− were also elevated significantly. Although these findings suggest that an exercise-induced increase in neutrophil counts may impose an oxidative stress on the blood environment, other potential sources cannot be ruled out at this time. Nonetheless, a blood oxidative stress response occurred at a threshold between supra-LT and maximal-intensity exercise. This threshold represents an imbalance between blood antioxidant defenses and oxidant production favoring oxidative stress. The fact that an oxidative stress response was not observed after submaximal-intensity exercise may indicate that a blood oxidative stress did not occur for those trials. The more likely alternative, however, is that the sensitivity of the techniques employed in the current study were unable to detect an oxidative stress present at submaximal intensities. This would indicate that more sophisticated techniques may need to better elucidate the magnitude of oxidative stress responses at various exercise intensities. Finally, our finding that the blood oxidative stress response was an effect of exercise intensity rather than absolute metabolic workload may highlight the importance of regular exercise training in preventing activity-related oxidative stress. Stated differently, the activities of daily living, despite absolute metabolic costs, require a lower relative percent of maximal ability in trained versus untrained. Thus, for a trained individual, a given activity is less likely to approach the oxidative stress threshold observed in this investigation. In light of the fact that aging results in a decreased functional capacity independent of training status, this conclusion emphasizes the necessity of lifetime physical fitness as outlined by ACSM criteria for the prevention of oxidative stress as it may relate to disease prevention.
Alternately, the findings of this study also warrant consideration of the idea that high-intensity exercise imposes an obligatory oxidative stress that is necessary for adaptation and potentially, disease prevention. Future studies are needed to determine whether a blood oxidative stress in response to exercise can effect, for example, genetic regulation of endogenous cellular antioxidant defenses within vascular endothelial cells. Further study is also needed to determine how the findings of this study apply the unfit and/or elderly. Clearly, examination of immune and plasma oxidative stress response to exercise is very important to consider in very deconditioned and elderly populations, especially when activities of daily living may approach or even exceed lactate threshold. Moreover, it seems reasonable to presuppose that nutritional and immune status in elderly and unfit persons are more likely favor a plasma oxidative stress at submaximal intensity as compared with the young, healthy subjects in this study.
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Keywords:
ANTIOXIDANT; WHITE BLOOD CELL; SUPEROXIDE; MYELOPEROXIDASE; LIPID HYDROPEROXIDE
©2003The American College of Sports Medicine