Phosphatidylserine (PtdSer) is a phospholipid that is incorporated in the membrane of eukaryotic cells and exhibits a range of important regulatory functions within mammalian cells (2). The addition of PtdSer to mammalian tissues inhibits the production of proinflammatory cytokines (1,11) and induces antiinflammatory responses (13) in in vitro preparations. In addition, intravenously injected PtdSer has been demonstrated to have an inhibitory effect on the immune response to endotoxin-induced inflammation in rodents (22). Although the mechanism(s) by which PtdSer inhibits the immune response remain unclear, recent data suggest that PtdSer inhibits immune responses by acting on cell types (e.g., macrophages, fibroblasts, neutrophils, endothelial cells, epithelial cells) at the site of inflammation (12). Furthermore, in vitro studies have demonstrated that PtdSer has the potential to act as an antioxidant with the ability to protect cells against oxygen-derived free radicals (18) and suppress iron-dependent lipid peroxidation (6).
Soybean-derived PtdSer (S-PtdSer) has been established, using established biochemical and hematological parameters, as a safe oral supplement for human consumption (14); additionally, no side effects following various supplementation regimes have been reported. Fahey and Pearl (9) reported that chronic oral S-PtdSer attenuated the perception of muscle soreness that followed intense weight training. Although the mechanism(s) responsible for this action were unclear, these authors suggested that reductions in circulatory cortisol concentrations following exercise might have been associated with decreased protein degradation.
The aforementioned antiinflammatory and antioxidant properties of PtdSer led us to investigate the effects of S-PtdSer supplementation on the magnitude of lipid peroxidation following exhaustive intermittent running (17). Changes in serum hydroperoxide concentrations suggested that this exercise elevated lipid peroxidation to an equal extent before and following 750 mg·d−1 S-PtdSer for 10 d. However, exercise performance tended to be improved following S-PtdSer supplementation, and because the blood samples were taken after the completion of maximal exercise, free radical production was probably higher after S-PtdSer supplementation. Consequently, it was plausible that the potential antioxidant properties of PtdSer might have been concealed in this study. In addition, supplementation was ceased on the day of exercise, and it is possible that continuing S-PtdSer supplementation during the early stages of an ensuing acute inflammatory response may afford greater protection.
Eccentric muscle activity causes muscle injury and leads to an acute inflammatory response (5). During this response, neutrophils and macrophages migrate to the location of muscle damage, penetrate the damaged tissue, elevate free radical production, and activate cytokines (19). Additionally, exercise produces increases in mitochondrial oxygen flux that have been implicated as a source of free radicals during the reduction of molecular oxygen in the electron transport chain (7). Free radicals can lead to oxidative damage in a wide range of molecular structures including lipids, proteins, and DNA, where the antioxidant defences are overwhelmed (15). Therefore, downhill treadmill running has the potential to initiate inflammation and increase oxidative stress.
The purpose of the current study was to investigate the antioxidant and antiinflammatory effects of chronic S-PtdSer supplementation on markers of muscle damage, inflammation, and oxidative stress following prolonged downhill running.
Eight healthy male volunteers (age: 21.0 ± 0.3 yr; height: 1.79 ± 0.02 m; body mass: 81.2 ± 3.2 kg) completed all of the study requirements. All subjects were informed about the potential risks of the study and gave written informed consent for their participation in the study, which was approved by a university ethics committee and completed in accordance with the policy statement of the American College of Sports Medicine. No subject had prior history of cardiovascular or respiratory disease, and all subjects were nonsmokers. Potential subjects attended an interview prior to undertaking the study and were subsequently excluded if they had taken nutritional supplements in the last 8 wk.
Over a period of approximately 11 wk, each subject completed five downhill runs on a motorized treadmill. Approximately 14 d after an initial familiarization trial, the subjects completed four main trials (trials 1-4). Trials 1 and 3 provided presupplementation control trials because the washout kinetics of PtdSer are currently unknown. Following trials 1 and 3, the subjects were randomly assigned in a double-blind and balanced order crossover fashion to receive either 750 mg·d−1 S-PtdSer (PS) or a weight-matched glucose polymer placebo (P). Supplementation was taken for a 10-d period that began 7 d prior to trials 2 and 4. The duration between trials 1-2 and trials 3-4 was exactly 14 d. Trials 2 and 3 were separated by a 4-wk washout period. Venous blood and ratings of perceived soreness and feelings were assessed prior to exercise (preexercise), after exercise (postexercise), the day following exercise (post 24 h), and the second day after exercise (post 48 h) during each trial. The experimental design is illustrated in Figure 1.
The S-PtdSer supplements were manufactured using the method of specific transesterification of soybean lecithin and then blended with additional soybean lecithin to provide a concentration of 20% PtdSer (Lucas Meyer; Hamburg, Germany). Both supplements were administered in capsules and placed in generic packaging. Subjects were instructed to maintain their normal diet and activity patterns throughout the study. Subjects weighed and recorded the food that they consumed for 2 d prior to exercise and for 3 d afterwards. Weighed food records were analyzed using commercial software (CompEat v5.8.0; Nutrition Systems, UK). In addition, the subjects were instructed to abstain from strenuous exercise for 3 d prior to and 2 d following exercise. At the completion of the study, all subjects gave their verbal assurance that they had complied with all instructions.
The familiarization exercise trial served to reduce the anticipated trial order effects that are apparent following eccentric exercise protocols and to define individualized exercise intensities for trials 1-4. Exercise started with a 3-min stage of horizontal running at 5 km·h−1. Subsequent 3-min stages were undertaken at −17.5% at a speed that began at 5 km·h−1 and increased at a rate of 1 km·h−1 until the subject reached a heart rate of 70% of age-predicted maximal heart rate. After a 3-min passive rest period, each subject completed a −17.5% treadmill run for 30 min at the terminal speed reached during the progressive section of the protocol. This individualized exercise protocol was recorded and repeated during each subsequent trial (trials 1-4).
Main trial procedures.
On the day of each main exercise trial, the subjects reported to the laboratory at approximately the same time of day (± 1 h) having fasted overnight. Subject mass (model 712; Seca, Germany) and height (Portable Stadiometer; Holtain, UK) were recorded. The intensity of muscle soreness for whole-body general soreness (GenS), hamstrings soreness (HS), quadriceps soreness (QS), and gluteal soreness (GS) were rated on an 11-point scale ranging from 0 (not sore) to 10 (very, very sore). The subjects completed exercise-induced feeling inventories (EFI) as described in detail by Gauvin and Rejeski (10). Briefly, the subjects rated their feelings using the 12-item adjective scale on an analog scale from 0 (do not feel) to 5 (feel very strongly). The appropriate adjectives were averaged to obtain four perceived feeling states (positive engagement, revitalization, tranquillity, and physical exhaustion).
Following a venepuncture (Vacutainer system; Becton-Dickinson Ltd, UK) taken from an antecubital vein in the right arm, the subjects completed the eccentric exercise protocol (mean exercise time, gradient, and speed were 51.0 ± 1.5 min, −16.5 ± 0.0%, and 8.7 ± 0.3 km·h−1, respectively). Heart rates (Polar S610; Polar Electro, Finland) and subjective ratings of perceived exertion (3) were monitored throughout the eccentric exercise protocol. Ambient temperature and humidity were recorded at the beginning and end of exercise (ETHG-912; Oregon Scientific). Postexercise EFI were obtained, and venous blood samples were taken from an antecubital vein in the left arm as previously described.
Subjects returned to the laboratory on the day following (post 24 h) and the second day following (post 48 h) each main exercise trial at approximately the same time (± 1 h) after overnight fasts. During these visits blood samples were drawn and ratings of soreness and EFI were completed as previously described.
Blood sampling and analysis.
Venous blood was collected in a 5-mL container (Becton-Dickinson Ltd, UK) containing the anticoagulant ethylenediamine tetraacetic acid (EDTA). Several small aliquots were removed for the triplicate determination of blood lactate concentration, glucose concentration (YSI 2300, Yellow Springs Instruments), hemoglobin concentration (Hemocue Ltd, UK), and hematocrit (Micro hematocrit MK IV, Hawksley, UK), and changes in plasma volume were estimated as previously described (8). The remaining blood was centrifuged at 3000 g for 15 min to obtain plasma. An aliquot of plasma (100 μL) was added to 900 μL of freshly prepared 10% metaphosphoric acid, mixed, and frozen at −70°C for subsequent vitamin C analysis. The remaining plasma was frozen and stored at −70°C prior to subsequent analysis. Two additional 7-mL blood samples were collected in serum separation tubes (Becton-Dickinson Ltd, UK), left to stand for 15 min, then centrifuged at 3000 g for 15 min to obtain serum. The serum was transferred to appropriate containers and subsequently stored at −70°C prior to analysis.
Plasma cortisol (kit EIA-1887; DRG Instruments, Germany) and adrenocorticotropic hormone (ACTH) (kit 7023; Biomerica) concentrations were determined in duplicate using solid-phase enzyme-linked immunosorbent assays (ELISA). IL-6 concentrations in serum were assayed in duplicate using commercially available high-sensitivity ELISA kits (Diaclone Research, France). An automated spectrophotometer (MiraS; ABX Diagnostics, UK) was used to measure plasma myoglobin concentrations and creatine kinase (CK) activity in duplicate with commercially available kits (A11A00165 and A11A00008, respectively; ABX Diagnostics, UK). Serum hydroperoxide concentrations were measured using the method of Wolff (1992) as described in McEneny et al. (21). Low-density lipoprotein was isolated from plasma and oxidized according to the method of McDowell et al. (20). Subsequently, the production of conjugated dienes was followed in duplicate at 234 nm (SpectraMax 190; Molecular Devices Corp) using the computer software SoftMax Pro Version 3.0 (Molecular Devices Corp); the change in absorbance (from baseline to the end of reaction) was used to quantify change in conjugated diene concentration, and the time taken to reach half of the maximum oxidation (t½max LDL oxidation) was taken as a measure of the resistance of the particle to oxidation. Vitamin C concentrations were determined using a fluorimetric assay using a centrifugal analyzer with fluorescence attachment according to the method of Vuilleumier (30). Plasma concentrations of α-tocopherol, γ-tocopherol, retinol, α- carotene, β-carotene, and lycopene were measured by high-performance lipid chromatography with electrochemical detection according to the methods of Catignani and Bieri (4) and Thurnham et al. (28). The intraassay coefficient of variance (%CV) for these assays ranged from 1.4 for α-tocopherol to 12.0 for lycopene.
Statistical analysis was carried out using SPSS software (version 13.0; SPSS Inc., Chicago, IL). Group data were expressed as mean ± SEM, and statistical significance was set at the P < 0.05 level. All data were assessed for normality (Shapiro-Wilk's test), and data that were not normally distributed (myoglobin and CK) were log transformed prior to analyses. Data from trials 1 and 3 (presupplementation control trials) were analyzed using mixed-model repeated-measures ANOVA (within-subject factors: trial × time of sample; between-subject factor: group). If a significant P value was identified for the three-way interaction (group × trial × time of sample), the subject groups reacted differently, and supplementation order was deemed to have had a significant effect. The data from all trials were analyzed using repeated-measures ANOVA (within-subject factors: trials × time of sample). Mauchly's test was consulted and Greenhouse-Geisser correction was applied if the assumption of sphericity was violated. Where a significant P value was identified for the main effect of trial or time (time of sample), multiple pairwise comparisons were made using Bonferonni confidence interval adjustment. Pearson product-moment analysis was used to determine the strength of correlation between plasma ACTH and cortisol concentrations.
Ambient temperature and relative humidity were similar during all main exercise trials, with mean values during all trials being 20.1 ± 0.3°C and 71 ± 3%, respectively. All three-way interactions from trials 1 and 3 were nonsignificant (P ≥ 0.172). The mean exercising heart rates ranged from 136 ± 4 to 137 ± 3 bpm and were similar during all trials (P = 0.951). Blood lactate concentrations were not different between trials (P = 0.548), with postexercise values ranging from 0.9 ± 0.1 to 1.1 ± 2 mmol·L−1. Blood glucose concentrations did not differ between trials (P = 0.057) or timing of sample (P = 0.113), with postexercise values ranging from 4.2 ± 0.1 to 4.4 ± 0.2 mmol·L−1. Estimated changes in plasma volume were significantly lower at postexercise when compared with all other time points in the study (P ≤ 0.039); however, no differences were observed between trials (P = 0.972). No differences were observed in the calculated amount or composition of the food consumed during any trial. The average daily diet comprised of 10.3 ± 0.3 MJ·d−1, of which 44 ± 1, 30 ± 1, 24 ± 1, and 2 ± 1% of energy intake was obtained from carbohydrates, fats, proteins, and alcohol, respectively.
Postexercise plasma ACTH and cortisol concentrations were 20 ± 6 and 27 ± 3% below preexercise values, respectively (time of sample effect, P = 0.015 and P = 0.001, respectively) (Fig. 2). These values did not differ between trials (trial effect, P = 0.150 and 0.166, respectively). Plasma ACTH concentrations and plasma cortisol concentrations were significantly correlated (r = 0.412; P < 0.001).
Plasma myoglobin concentrations were elevated by 78 ± 12% above preexercise at postexercise in all trials (time of sample effect, P < 0.001; Fig. 3). Plasma CK activities peaked at post 24 h in all trials (time of sample effect, P < 0.001; Fig. 3), being 200 ± 39% above preexercise. There were no significant differences between trials for either myoglobin or CK (trial effect, P = 0.865 and 0.971, respectively).
Perceived ratings in GenS were increased from preexercise values at post 24 h (time of sample effect P = 0.002; Fig. 4). There were no differences between trials (trial effect, P = 0.766). Additionally, exercise caused increases in perceived soreness at post 24 h in all of the reported lower-body locations (QS, HS, and GS); however, no differences were identified between trials (Table 1).
Serum IL-6 concentrations were elevated at postexercise by approximately 123 ± 39% above preexercise values (time of sample effect, P < 0.001) in all trials (Fig. 5). No differences were identified between the trials (trial effect, P = 0.871).
Serum hydroperoxide concentrations were significantly elevated at postexercise by approximately 27 ± 8% (time of sample effect, P = 0.003) in all trials (Fig. 6). There were no differences between trials (trial effect, P = 0.789).
Average changes in conjugated diene concentrations at preexercise, postexercise, post 24 h, and post 48 h were 14.2 ± 0.6, 15.6 ± 0.8, 14.8 ± 1.0, and 14.2 ± 0.8 μmol·L−1, respectively, for all trials (time of sample effect, P = 0.077; trial effect, P = 0.338). Postexercise t½max LDL oxidation times were elevated from preexercise (time of sample effect, P = 0.001) with average preexercise, postexercise, post-24 h, and post-48 h values for all trials being 79.2 ± 2.2, 87.2 ± 2.5, 86.1 ± 2.6, and 82.5 ± 2.7 min, respectively. No differences existed between trials (trial effect, P = 0.192).
Plasma concentrations of vitamin C, α-tocopherol, α-carotene, β-carotene, and lycopene were similar throughout the duration of each trial and were not significantly different between trials (Table 2). Plasma γ-tocopherol concentrations were significantly higher in PS when compared with all other trials (trial effect, P = 0.008) (Table 2).
No significant temporal changes were observed in revitalization (time of sample effect, P = 0.295), positive engagement (time of sample effect, P = 0.357), tranquillity (time of sample effect, P = 0.671), or physical exhaustion (time of sample effect, P = 0.081). Additionally, no differences were identified between trials in any of the EFI subscales (trial effect, P ≥ 0.121).
The main findings of this study were that the supplementation regime (750 mg·d−1 S-PtdSer administered for 7 d prior to and continued for 2 d following exercise) was not effective in attenuating markers of muscle damage, inflammation, and oxidative stress that followed prolonged downhill treadmill running.
In light of our previous finding that supplementation with 750 mg·d−1 S-PtdSer for 10 d tended to improve performance during exhaustive intermittent running (17), the current exercise protocol was chosen to provide each subject with a repeatable dose of exercise intended to cause muscle damage, oxidative stress, acute inflammation, and DOMS. Indeed, this exercise protocol led to increases in markers of muscle damage (plasma myoglobin concentrations and CK activities), lipid peroxidation (serum hydroperoxide concentrations), inflammation (serum IL-6 concentrations), and DOMS (perceived soreness).
The three-way interactions for the analysis of trials 1 and 3 (presupplementation control trials) were all nonsignificant. These data confirmed that the subject groups reacted similarly during these trials. Although the washout kinetics of PtdSer are currently unknown, there is no evidence to suggest that an effect of supplementation lasted longer than the washout period provided.
As anticipated, plasma cortisol concentrations were positively correlated with plasma ACTH concentrations. Plasma concentrations of cortisol and ACTH decreased after eccentric exercise. This finding, which probably reflects the low metabolic demand associated with downhill running at the current relative intensity, is concurrent with previous data (25). Alterations in the concentrations of these hormones within the circulation have been interpreted to reflect changes in the hypothalamo-pituitary-adrenal (HPA) axis (23). Consequently, it is likely that this bout of exercise did not cause a stress-induced increase in hypothalamic corticotrophin-releasing factor that has concurrent stimulatory effects on a range of regulatory hormones from the anterior pituitary (including growth hormone) and sympathoadrenal system (e.g., epinephrine and norepinephrine). Therefore, it is unlikely that the intensity of exercise was sufficient to augment the mobilization of neutrophils into the peripheral circulation as mediated by exercise-induced increases in catecholamines, cortisol, and growth hormone (26). Interestingly, S-PtdSer supplementation did not alter the pattern of ACTH and cortisol release. Fahey and Pearl (9) reported that similar doses of S-PtdSer reduced serum cortisol concentrations following resistance training, leading these investigators to support the efficacy of PtdSer to modulate the neuroendocrine response to overtraining. The disparity in these findings may be partly explained by the different modes of exercise used in these respective studies; downhill running, unlike resistance training, did not stimulate the stress-induced activation of the HPA axis.
The elevations in plasma myoglobin concentrations and CK activities were comparable in magnitude and followed similar temporal patterns to those that have been previously reported following downhill running protocols (24). Our protocol also led to increased soreness (GenS, QS, HS, and GS). Furthermore, the time course of the changes in the ratings of soreness, plasma myoglobin concentrations, and plasma CK activities following eccentric exercise were similar across all trials. Consequently, it was concluded that supplementation with S-PtdSer did not affect muscle damage and other important corollaries induced by an acute bout of eccentric exercise. In contrast, a similar S-PtdSer supplementation regime has been demonstrated to reduce muscle soreness during intense weight training (9). These authors postulated that exogenous PtdSer might have improved cell membrane stability and/or reduced protein degradation by attenuating the stress-induced activation of the HPA axis. Although markers of protein catabolism were not measured in the present study, there was no evidence to suggest that S-PtdSer affected protein degradation following eccentric exercise.
Downhill running led to significant increases in postexercise serum IL-6 concentrations that were comparable with those previously reported to follow similar exercise protocols (24). The magnitude of the IL-6 increases following downhill running were relatively small compared with the changes that have been reported to occur following prolonged endurance exercise (27). This finding probably reflects both the type and duration of activity undertaken. Although the stimulus for eccentric exercise-induced IL-6 remains largely unknown, it is likely that mechanical damage to muscle tissue led to the rapid invasion of neutrophils (29), which produce free radicals that stimulate cytokine production. However, supplementation did not affect circulatory concentrations of IL-6 in the current study.
Serum hydroperoxide concentrations were elevated to a similar extent following exercise in all trials; consequently, exercise caused an increase in free radical production that overwhelmed the antioxidant defences and, independent of supplementation, contributed to comparable amounts of lipid peroxidation.
Exercise did not significantly affect the susceptibility of LDL to copper-induced oxidation as measured by changes in conjugated diene concentrations. However, t½max LDL oxidation times were elevated postexercise. Because t½max LDL oxidation is a measure of resistance to copper-induced oxidation, the increase following exercise might reflect an increase in the mobilization of antioxidant defences following downhill running. Supplementation did not influence susceptibility or resistance of LDL to in vitro peroxidation.
Supplementation led to significant increases in plasma γ-tocopherol concentrations throughout the PS trial. This increase is in agreement with our previous findings (17); however, the mechanism(s) for increasing plasma γ-tocopherol following supplementation are unclear. Nevertheless, there is insufficient evidence to suggest that the elevations in plasma γ-tocopherol concentrations and probable increases in tissue γ-tocopherol affected oxidative stress or inflammation caused by eccentric exercise. In addition, supplementation had no effect on concentrations of the other plasma antioxidant vitamins (vitamin C, α-tocopherol, γ-tocopherol, retinol, α-carotene, β-carotene, and lycopene).
Downhill running did not affect any of the EFI subscales. The EFI was developed to assess distinct feeling states that occur during exercise (10), and we have recently demonstrated that the subscales are sensitive to intermittent cycling in recreationally active males (16). This difference probably reflects the low metabolic demand associated with this downhill running protocol and suggests that DOMS does not change the feeling states that are measured by these subscales. The lack of change in these feeling states may have contributed to the lack of an identifiable difference between trials; nevertheless, the current data does not suggest that S-PtdSer modifies feeling states before or after downhill running.
In summary, S-PtdSer supplementation before and following exercise did not attenuate perceived soreness and markers of muscle damage, inflammation, and oxidative stress caused by moderate-intensity downhill running. These findings indicate that the current supplementation regime is not effective in reducing the deleterious effects of eccentric muscle activity.
The authors wish to acknowledge the laboratory staff from Queen's University for their assistance in the blood analyses. In addition, we would like to thank Lucas Meyer for supplying the supplements.
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Keywords:©2006The American College of Sports Medicine
PHOSPHATIDYLSERINE; ECCENTRIC EXERCISE; DELAYED ONSET OF MUSCLE SORENESS; MUSCLE DAMAGE; INFLAMMATION