Soccer is a multiple-sprint sport that requires high-intensity, intermittent activity to be undertaken over an extended period of time. The rapid decelerations and changes in direction undertaken during soccer match play generate large eccentric forces that have frequently been associated with muscle damage (10), leakage of unbound iron from the cytosolic region (12), and delayed onset of muscle soreness (DOMS) (3). Free radical production has been associated with muscle damaging exercise (1) and the ensuing inflammatory response (18). In addition, the elevation in oxygen consumption and the rapid changes in mitochondrial oxygen flux that are associated with prolonged intermittent exercise have the potential to greatly increase the production of free radicals (6). These radicals have the potential to cause oxidative damage to a wide range of molecular structures including lipids, proteins, and DNA, where the antioxidant defenses are overwhelmed (16). Furthermore, prolonged intermittent shuttle running designed to simulate multiple-sprint sports has been previously demonstrated to cause muscle soreness and elevate blood markers of muscle damage and lipid peroxidation in accustomed individuals (27).
Phosphatidylserine (PtdSer) is an essential phospholipid, mainly located within the cytosolic monolayer (7), that has been shown to have important regulatory functions within mammalian cells (2). Exogenous PtdSer supplementation has shown benefits in diverse measures of cognitive functions. Furthermore, intravenously and orally administered bovine cortex-derived PtdSer have been shown to blunt the cortisol response to acute exercise stress (21,22) and, therefore, may attenuate the stress-induced activation of the hypothalamopituitary-adrenal axis in humans. Moreover, Fahey and Pearl (9) reported that chronic oral supplementation with 800 mg·d−1 of soybean-derived PtdSer (S-PtdSer) was effective in reducing serum cortisol concentrations and muscle soreness following intensive resistance training. In addition to a myriad of membrane related functions, in vitro studies have demonstrated that PtdSer has the potential to act as an antioxidant (5,8). Therefore, it is plausible that exogenous supplementation with PtdSer may provide additional defense against the oxidative stress caused by soccer match play. In light of the above, the aim of the present study was to determine whether antioxidant status, tissue oxidation, muscle damage, and muscle soreness following exhaustive prolonged intermittent exercise would be affected by chronic oral S-PtdSer supplementation in familiarized active university soccer players.
Sixteen young males who participated regularly in soccer match play and practice volunteered to participate in this study, which was approved by a university ethics committee. All subjects were informed in writing about the potential risks of the study and gave written informed consent for their participation in the study. No subject had a history of cardiovascular or respiratory disease and all subjects were nonsmokers. Potential subjects attended an interview before undertaking the study and were subsequently excluded if they were smokers or had taken nutritional supplements in the past 8 wk. The subjects’ physical characteristics are presented in Table 1.
All subjects completed two preliminary visits to the laboratory before undertaking the main exercise trials. Maximum oxygen uptake (V̇O2max) was estimated using a multistage fitness test (MSFT) (26) during the initial visit. These V̇O2max values were used in conjunction with the linear regression developed by Ramsbottom et al. (26) to calculate appropriate running speeds for the exercise protocol as described previously by Nicholas et al. (23). During their second preliminary visit, the subjects underwent a familiarization exercise trial in order to minimize anticipated trial order effects. Subjects then performed two main exercise trials (T1 and T2), separated by 14 ± 1 d. The subjects were assigned, in a randomized, double-blind fashion, to receive either 750 mg·d−1 S-PtdSer (PS) or a weight-matched glucose polymer (P) for exactly 10 d before the T2. 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. Food was weighed and recorded by the subjects for the 2 d before and after each main exercise trial. These food records were subsequently analyzed using commercial software (CompEat version 5.8.0; Nutrition Systems, U.K.). In addition, the subjects were instructed to abstain from strenuous exercise for 3 d before and 2 d following each trial. At the completion of the study, all subjects gave their verbal assurance that they had complied with all instructions.
Main trial procedures.
On the day of both main exercise trials, the subjects reported to the laboratory in pairs (matched by estimated V̇O2max and paired in different supplementation groups) at approximately the same time of day (± 1 h) and following an overnight fast. Subjects were asked to void, then mass (model 712; Seca, Germany) and height (Portable Stadiometer; Holtain, U.K.) were recorded. Subsequently, the intensity of muscle soreness for the whole body general soreness (GS), hamstrings (HS), and quadriceps (QS) were rated on an 11-point scale ranging from 0 (not sore) to 10 (very, very sore).
Following a preexercise venipuncture (Vacutainer system; Becton-Dickinson Ltd., U.K.), taken from an antecubital vein in the right arm (after maintaining a seated position for approximately 10 min), the subjects completed an exercise protocol that consisted of two sections (part A and part B). Part A was based on the Loughborough Intermittent Shuttle Test (23), but included additional components designed to further replicate the physiological demands of soccer match play. Briefly, the protocol consisted of variable intensity shuttle running over a 20-m distance that included walking, jogging (55% V̇O2max), cruising (85% V̇O2max), backward cruising (85% V̇O2max), zigzag sprinting, rest periods, and timed sprinting (Brower; Utah) undertaken in a repeated sequence dictated by prerecorded audio compact disks. This sequence was undertaken for 45 min, followed by a 10-min recovery period (half time), and then continued for a subsequent 30-min exercise period. Immediately after the intermittent exercise, the MSFT was completed until volitional fatigue (part B), thus the total exercise time was approximately 90 min.
HR were continuously recorded at each 5-s interval using short-range telemetry (Polar S610; Polar Electro, Finland). Subjective RPE and capillary blood samples were obtained at the beginning of the half-time recovery period (half time) and immediately following volitional fatigue (post-MSFT). Ambient temperature and humidity were recorded at the beginning and end of exercise (ETHG-912; Oregon Scientific, U.S.). Ad libitum water consumption was encouraged throughout T1; this volume was measured and each individual was required to match his fluid intake during T2. Fifteen minutes following the termination of exercise, venous blood samples were taken, in a seated position, by venipuncture from an antecubital vein in the left arm as previously described.
On the day following (post–24 h) and the second day following (post–48 h) each main exercise trial, the subjects returned to the laboratory at approximately the same time (± 1 h) after overnight fasts. Blood samples were drawn by venipuncture, and the subjects rated the intensity of soreness as previously described.
Blood sampling and analysis.
Venous blood was collected in a 5-mL container (Becton-Dickinson Ltd., U.K.) containing the anticoagulant ethylenediamine tetraacetic acid (EDTA). Several small aliquots were removed for the triplicate determination of blood lactate and glucose concentrations (YSI 2300 Stat, Yellow Springs Instruments, U.S.), blood hemoglobin concentration (Hemocue Ltd., U.K.), hematocrit (Micro Hct MK IV, Hawsley, U.K.), and changes in plasma volume 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 before subsequent analysis. Two additional 7-mL blood samples were collected in serum separation tubes (Becton-Dickinson Ltd.), left to stand for 15 min and then centrifuged at 3000 × g for 15 min to obtain serum. The serum was transferred to appropriate containers and subsequently stored at −70°C before subsequent analysis.
Serum Mb concentrations and CK activity were measured with commercial available kits (A11A00165 and A11A00008, respectively; ABX Diagnostics, U.K.) using an automated spectrometer (MiraS; ABX Diagnostics, U.K.). Serum cortisol concentrations were determined using an automated time-resolved fluoroimmunoassay (AutoDELFIA™ Cortisol kit, Perkin Elmer, Life Sciences, U.K.). Serum hydroperoxide concentrations were measured using the method of Wolff (1992) as described in McEneny et al. (20). LDL was isolated from plasma and oxidized according to the method of McDowell et al. (19). Subsequently, the production of conjugated dienes was followed at 234 nm and the lag time taken as a measure of the resistance of the particle to oxidation. Vitamin C concentrations were determined using a fluorometric assay using a centrifugal analyser with fluorescence attachment according to the method of Vuilleumier (30). Plasma concentrations of α-tocopherol, γ-tocopherol, retinol, and β-carotene were measured by high-performance lipid chromatography with electrochemical detection according to the methods of Thurnham et al. (29).
Statistical analysis was carried out using SPSS software (version 12.0; SPSS Inc., Chicago, IL). Group data were expressed as mean ± SEM and statistical significance was set at the P < 0.05 level. Subject characteristics were compared under supplementation groups using independent samples t-tests (Table 1). Environmental conditions were compared using paired samples t-tests. The percentage of change in exercise times to exhaustion during part B (individual delta values) was compared under PS and P conditions using an independent samples t-test. The remaining data, which contained multiple time points during each trial, were analyzed using mixed-model repeated-measures ANOVA (within-subject factors: trial × time of sample; between-subject factor: supplementation groups). Mauchly’s test was consulted and Greenhouse–Geisser correction was applied if the assumption of sphericity was violated. If a significant P value was identified for the interaction effect (supplementation group × trial × time of sample), supplementation was deemed to have a significant effect. If a significant P value was identified for the main effect of time (time of sample), multiple pairwise comparisons were made using Bonferroni confidence interval adjustment, with statistical significance set at P < 0.01.
The environmental conditions were not significantly different during all trials, ambient temperature being 19.6 ± 0.7 and 21.3 ± 0.5°C, and humidity 51.5 ± 2.6 and 54.0 ± 2.8%, respectively, for T1 and T2. Mean and peak exercising HR were not significantly different for PS and P during T1 (PS, P: 151 ± 6, 153 ± 6; 196 ± 4, 196 ± 4 bpm, respectively) and during T2 (PS, P: 149 ± 5, 149 ± 7; 196 ± 4, 195 ± 3 bpm, respectively). Blood lactate concentrations were similarly elevated from preexercise (1.3 ± 0.1 mmol·L−1) at half time (2.4 ± 0.2 mmol·L−1), post-MSFT (6.1 ± 0.2 mmol·L−1) during all trials (P < 0.01). Blood glucose concentrations were not significantly different for preexercise, half -time, post-MSFT, and postexercise, being 4.2 ± 0.1, 4.5 ± 0.1, 5.3 ± 0.1, and 4.0 ± 0.1 mmol·L−1, respectively, for all trials. Ratings of perceived exertion were not different between trials at half time or at exhaustion, being 14 ± 1 and 18 ± 1 units, respectively, for all trials. Estimated changes in plasma volume were not different between trials or at any point during the course of the data collection (Table 2). No differences were observed in the calculated amount or composition of the food consumed during any trial (Table 3).
Exercise times to exhaustion during part B of the exercise protocol were similar between the groups before supplementation (P: 11.3 ± 0.6; PS: 11.2 ± 0.7 min; P = 0.894). There was a nonstatistical tendency for the change (i.e., T2-T1, expressed as a percentage of T2) in the exercise times to exhaustion to be longer in PS when compared with P, being 4.2 ± 0.7 and −3.7 ± 4.2%, respectively (P = 0.084) (Fig. 1). Supplementation had no significant effect on sprint speeds during part A of the exercise protocol (supplementation group × trial × time of sample, P = 0.143). The changes (i.e., T2-T1, expressed as a percentage of T2) in sprint speeds during sprints 1–6, 7–11, and 12–17 were 3.7 ± 1.3, 4.1 ± 1.4, and 7.3 ± 2.6%, and 3.7 ± 2.8, 4.4 ± 2.8, and 0.4 ± 2.8% for PS and P, respectively (Fig. 2).
Preexercise, postexercise, post–24 h, and post–48 h serum cortisol concentrations for all trials were 392 ± 15, 526 ± 24, 295 ± 17, and 306 ± 17 nmol·L−1, respectively (time of sample effect, P < 0.001) (Fig. 3). Supplementation had no affect on cortisol values (supplementation group × trial × time of sample, P = 0.437).
Serum Mb concentrations and CK activities are shown in Figure 4. Postexercise Mb and CK were above preexercise values in all trials, being elevated by 296 ± 48 and 76 ± 7%, respectively (time of sample effects being P < 0.001 and P < 0.001, respectively). The activity of CK peaked at post–24 h in all trials, whereas Mb values had returned to preexercise values at post–24 h. No significant interaction effects were determined for Mb and CK (supplementation group × trial × time of sample being P = 0.190 and P = 0.596, respectively). General soreness was elevated the day following exercise (post–24 h) above preexercise values and then returned to baseline values at post–48 h (Table 4). The pattern of soreness was not significantly influenced by supplementation.
Serum hydroperoxide concentrations (HPO) were significantly elevated at postexercise by approximately 24 ± 5% (time of sample effect, P = 0.001) in all trials (Fig. 5). Supplementation had no significant effect on HPO (supplementation group × trial × time of sample, P = 0.500). Exercise caused a significant elevation in conjugated diene lag times at postexercise (time of sample effect, P = 0.011), being 67.5 ± 1.4, 69.8 ± 1.1, 68.4 ± 1.0 and 66.5 ± 1.2 min at preexercise, postexercise, post–24 h and post–48 h, respectively. Supplementation did not affect conjugated diene lag times (supplementation group × trial × time of sample, P = 0.489).
Postexercise plasma vitamin C concentrations were significantly elevated when compared with preexercise values in all trials (time of sample effect, P = 0.001), although these values were similar before and after supplementation in PS and P (Table 5). Plasma concentrations of α-tocopherol, retinol, and β-carotene were similar throughout the duration of each trial and were not significantly different between trials (Table 5). Supplementation significantly affected plasma γ-tocopherol concentrations (supplementation group × trial × time of sample; P = 0.008). Following supplementation the change in preexercise and postexercise values were 75 ± 21 and 94 ± 22% higher than T1 values, respectively, in PS (Table 5).
The study primarily aimed to investigate whether chronic S-PtdSer supplementation would influence muscle soreness and markers of muscle damage and oxidative stress following an exhaustive running protocol consisting of intermittent running designed to simulate the physiological demands of soccer that was immediately followed by a progressive run in well-familiarized university soccer players. The supplementation regime significantly increased plasma γ-tocopherol concentrations throughout the day of exercise, but did not cause increases in the concentrations of any other antioxidant vitamin measured. Moreover, supplementation with S-PtdSer had no effect on perceived muscle soreness or markers of muscle damage and lipid peroxidation. An unexpected and potentially useful outcome from this investigation was that oral supplementation with 750 mg·d−1 of S-PtdSer for 10 d tended to improve running performance.
The uptake and biokinetics of PtdSer following oral administration in humans are largely unknown. However, oral administration with PtdSer in rats suggests that although the majority of the exogenous phospholipid content is hydrolyzed and degraded, small fractions of PtdSer remain available (25). Furthermore, the addition of exogenous PtdSer to mammalian cell cultures results in efficient incorporation within a variety of cell membranes (24). Therefore, it is likely that the chronic supplementation regimen used in the current study will have caused transient increases in the PtdSer content within systemic circulation and incorporation of PtdSer within cell membranes in the PS group.
In the present study, exercise caused rises in serum cortisol concentrations that were similar to those previously reported following comparable activities (27), suggesting that the protocol activated the hypothalamopituitary–adrenal (HPA) axis (21). However, supplementation had no effect on serum cortisol concentration at any time during the investigation. This finding does not support those of Monteleone et al. (21,22), who reported that PtdSer supplementation resulted in significant reductions in plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations during submaximal exercise in untrained subjects.
The dose of S-PtdSer used in the current study was similar to the regime employed by Monteleone et al. (22) that used 800 mg·d−1, but the possibility exists that this dose may not have been high enough to attenuate the cortisol response in these active individuals. Alternatively, increased blood cortisol concentration is a generic response to stress from both psychological and physical origin; consequently, as the current experimental design was completed until volitional exhaustion during both trials, the overall stress may have been similar during all trials. It remains plausible, although speculative, that exogenous S-PtdSer might have influenced membrane-mediated factors that may have attenuated the stimulation of ACTH and subsequent cortisol release (22) at some time point during exercise (although serial blood samples were not available during the exercise protocol to evaluate this). Thus, a blunted activation of the HPA axis may have lead to a reduced perception of stress and partially contributed to the finding that S-PtdSer supplementation tended to enhance running performance in PS when compared with P. Also, increases in membrane-bound PtdSer may have the potential to enhance muscle excitation–contraction coupling, potentially through the activation of different protein kinase C isoforms (15) and/or enhanced calcium uptake (25), which might have contributed to the finding that supplementation tended to improve exercise performance.
The exercise-induced increases in perceived soreness and serum concentration of Mb and the activity of CK were comparable to those previously reported for familiarized individuals who completed similar activity patterns (28). Nevertheless, supplementation had no effect on markers of muscle damage (CK and Mb) or the extent of perceived soreness in PS or P. These findings are in spite of a tendency for the PS group to increase exercise performance during part B of the protocol. Exercise elevated serum hydroperoxide concentrations and conjugated diene lag times to a similar extent before and following supplementation, demonstrating that exercise caused free radical production that overwhelmed antioxidant defense and led to comparable lipid peroxidation during all trials. Therefore, it is possible that S-PtdSer might have had some protective effect in reducing muscle damage and inflammation that was masked by the unexpected tendency for performance to be improved in the PS group. Further protection might have been afforded against the delayed onset of muscle damage that has been recently associated with inflammatory processes caused by free radicals (1), had supplementation been continued on the days following exercise.
PtdSer supplementation led to approximately twofold increases in plasma γ-tocopherol concentrations before and following exercise; although concentrations returned to basal levels post–24 h. Oral supplementation with foods containing high concentrations of γ-tocopherol have been demonstrated to elevate blood γ-tocopherol concentrations; however, the mechanism for the increases in plasma γ-tocopherol concentrations following S-PtdSer supplementation are unclear, because soy oil consumption has previously been shown to have no effect on blood γ-tocopherol concentrations in humans (4). Nonetheless, increases in plasma and tissue γ-tocopherol in rats have been demonstrated to attenuate protein and ascorbate oxidation following inflammation-mediated damage without affecting α-tocopherol concentrations (14). Thus, the transient increases in plasma γ-tocopherol concentrations and probable increases in tissue γ-tocopherol concentrations may have augmented the antioxidant defense in PS during T2, although supplementation had no effect on concentrations of the other plasma antioxidant vitamins (vitamin C, α-tocopherol, retinol, and β-carotene) or lipid peroxidation. In addition, evidence has been presented suggesting antiinflammatory effects of γ-tocopherol that are different from its free radical scavenging actions (13) possibly mediated through specific protein-binding interactions (11).
In conclusion, S-PtdSer supplementation had no effect on perceived soreness and markers of muscle damage and oxidative stress caused by exhaustive exercise designed to simulate the movement patterns of soccer. There are several plausible reasons for the lack of effect of S-PtdSer supplementation in the current study. The supplementation regimen used in the current study may not have been optimal in attenuating the activation of the HPA axis, oxidative stress, and inflammation responses to exhaustive exercise. In addition, as the exercise was continued to exhaustion, any effect of supplementation may have been shrouded by differing amounts of external work. The tendency for PtdSer to improve running performance when compared with P suggested that there might be a true effect of PtdSer supplementation; therefore, the potential ergogenic effects of S-PtdSer warrant future investigation (17)
The authors acknowledge Mrs. R. E. Dietzig for her technical assistance during the study and the laboratory staff from Queen’s University and the Royal Group of Hospitals for their assistance in blood analyses. In addition, we thank Lucas Meyer for supplying the supplements.
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