Oxidative stress is a biological phenomenon marked by an imbalance between reactive free radicals (often oxygen-based molecules) and antioxidant defenses. The term oxidative stress indicates some combination of accelerated free radical production and/or exhaustion of antioxidant defenses (10). A severe or prolonged oxidative stress can lead to oxidatively modified lipids, proteins, and DNA. Acute exercise has long been associated with a transient oxidative stress response (21). The magnitude of oxidative stress following aerobic type exercise is generally proportional to exercise intensity (13,22). In the realm of physical activity and exercise training, much research has been directed at countering activity-related oxidative stress. As this research sub discipline matures a fuller understanding of the activity-induced oxidative stress is needed prior to efficacious antioxidant countertherapy development or testing. This fact is particularly true with respect to the oxidative stress response to resistance exercise, which has been investigated far less frequently than aerobically based exercise.
To date, very little is known about the acute oxidative stress response to resistance exercise training. Currently available literature on the topic of resistance exercise and oxidative stress are equivocal with some studies demonstrating a readily identifiable oxidative stress (2,3,14,18), whereas other research shows no effect (17). Reassessment of these initial investigations reveals that, as with aerobic exercise, the oxidative stress response to resistance exercise is intensity dependent. Evidence indicates that high-intensity resistance exercise, involving a large muscle mass such as squat exercise, consistently elicit a measurable blood oxidative stress (2-4,14). In contrast, the oxidative stress response to lower-intensity resistance exercise, 40-60% 1RM, have been mixed (17,18).
To date, the relationship between resistance exercise intensity and the subsequent oxidative stress response is not well described. Hoffman et al. (11) have performed a preliminary investigation of resistance exercise intensity and oxidative stress, though their findings are limited to a single blood oxidative damage marker (malondialdehyde). In light of the questionable value of this oxidative damage marker, further investigation is needed to better clarify the impact of exercise intensity on blood oxidative stress following resistance exercise of different intensities. Hence, the purpose of this investigation was to quantify the blood oxidative stress following volume equated resistance exercise sessions representing set and repetition combinations that reflect real world hypertrophy and strength type resistance exercise. We hypothesized that the hypertrophy protocol would induce a higher oxidative stress response as compared with the strength protocol. Our rationale for this hypothesis is based on the combined moderate-intensity and short-duration recovery between sets in hypertrophy protocol relative to the strength protocol.
Ten subjects were recruited for study based on resistance training experience and proficiency in the back squat exercise. The mean participant age was 21.8 ± 1.9 yr, height 176.3 ± 7.0 cm, body mass 92.4 ± 9.5 kg, body fat 13.2 ± 4.2%, 1RM squat 170.8 ± 24.9 kg, and strength-to-body-mass ratio 1.9 ± 0.2. During the 1RM testing, subjects that were unable to complete the back squat with acceptable form were excluded from the investigation. Additional inclusionary criteria required subjects to perform a back squat one-repetition max (1RM) of at least 1.5 times their body mass. All 10 subjects were instructed to refrain from lower- and upper-body training and exercise during the 24 h prior to 1RM testing and throughout the remainder of the study period. Prior to data collection, written and verbal informed consent was obtained from each subject. All experimental procedures were performed in accordance with the policy statement of the American College of Sports Medicine on research with human subjects as published by Medicine & Science in Sports & Exercise® and were approved by the Appalachian State University human subjects committee.
Baseline Strength Testing
Subjects were familiarized with the testing procedures during the study orientation, and completed a familiarization trial consisting of back squat exercise with a knee angle of 90°. Baseline strength testing was performed 1 wk after the familiarization session. All testing was completed in the mornings under 12-h fasting conditions, and it was suggested that subjects receive 8 h of sleep the night before testing. Baseline strength was determined by assessing 1RM for the back squat exercise as previously described by Matuszak et al. (16). Briefly, successful back squat criteria required subjects to obtain a knee angle of 90°. Knee angle verification was monitored by a single study investigator throughout the various trials. Additionally, participants were verbally motivated throughout each exercise protocol. Preliminary strength measurements were used as reference for workload prescription during the subsequent strength and hypertrophy oxidative stress trials.
Oxidative Stress Trials
One week after these strength assessments the first experimental trial took place. The subjects completed two experimental protocols designed to produce different metabolic challenges within the activated skeletal muscle groups. The experimental resistance exercise protocols (hypertrophy and strength) were completed in a randomized crossover design separated by 1 wk. The hypertrophy protocol included four sets of 10 repetitions of parallel back squat at 75% of the 1RM, with 90-s rest periods between sets. The strength protocol consisted of 11 sets of three repetitions of parallel back squat at 90% of the 1RM, with 5-min rest periods between sets. Set number, repetition count, and intensity levels between the hypertrophy and strength trials were designed to expose the subjects to equated session work volume but with varied intensity and rest periods between sets. Specifically, our aim was to achieve variations in the metabolic difficulty experienced over the duration of the oxidative stress trial. As with the baseline testing, repetitions not meeting predetermined form standards were considered incomplete requiring the subject to perform an additional repetition. If volitional failure was achieved prior to completion of a prescribed set, the load was reduced by 5% for the subsequent set.
Determination of Work
The mechanical work performed during the eccentric and concentric portion of the back squats was calculated using techniques previously described (15). Briefly, eccentric and concentric work was determined by integrating the area under the curve of the force-displacement graphs attained during the eccentric and concentric phases of each squat (15). Both vertical and horizontal displacement was measured during all back squats through the utilization of two linear position transducers (9). The linear position transducers were mounted anterior and posterior above the subject on a custom built power rack, then attached to the bar that was held across the subject's back during the back squats. Vertical ground reaction force (Fz) was measured throughout the series of back squats via force plate (AMTI, BP6001200, Watertown, MA). Analog data from the linear position transducers and force plate were recorded by a shielded BNC adapter chassis (National Instruments, BNC-2090, Austin, TX) and an A/D card (National Instruments, NI PCI-6014, Austin, TX) at 1000 Hz. LabVIEW (National Instruments, Version 7.1, Austin, TX) was used for recording and analyzing the digital data in which specifically designed programs were used to extract and determine the mechanical work (9).
Blood was collected prior to and following both trials for biochemical determination of oxidative stress and oxidative damage. The term oxidative stress indicates measurable redox perturbations that may be limited to a loss in plasma antioxidant capacity, but it may also include frank oxidative damage to protein and lipids (10). Specifically, a 7-mL EDTA and a 7-mL heparinized blood sample were obtained via Vacutainer apparatus from the antecubital vein by a phlebotomy trained study representative. Blood was collected prior to (PRE), immediately after (IP), and 60 min after (60POST) oxidative stress trials. To obtain the blood plasma fraction, tubes containing whole blood were centrifuged at 3000 rpm (5000g) for 10 min. Plasma was aliquoted and immediately frozen at −80°C until analysis. Finger prick blood samples were also collected at the time points stated above for analysis of blood lactate.
Lactate and plasma redox assays.
Whole-blood lactate concentrations were analyzed using an automated lactate analyzer (Sport Lactate Analyzer 1500, Yellow Springs Instruments, Yellow Springs, OH). Plasma antioxidant capacity was measured by the plasma trolox-equivalent antioxidant capacity (TEAC) technique whereby a radical cation of 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) reaction is quenched by existing antioxidant fortification (8). Total plasma antioxidant potential was determined by the ferric reducing antioxidant potential (FRAP) assay according to the methodology of Benzie (1). Both TEAC and FRAP produce colorimetric solutions that were quantified spectrophotometrically. Plasma urate was determined by the spectrophotometric assay of Kovar et al. (12). Here, the addition of uricase to plasma catalyzes the oxidation of plasma urate to allontoine and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide catalyzes a color generating reaction with 3-methyl-bonzothiazoline-2-one hydrazone (MBTH) and 3-ditmethylaminobenzoic acid (DMAB). Intraassay and interassay coefficients of variation for the TEAC and FRAP assays were 9 and 5%, respectively.
Plasma oxidative damage markers.
Lipid peroxidation was determined by the ferrous oxidation-xylenol orange (FOX) assay of Nourooz-Zadeh et al. (19). In the FOX assay, ferrous ions are oxidized by lipid hydroperoxides to ferric ions, which subsequently react with the ferrous sensitive dye containing xylenol orange. In the presence of lipid hydroperoxides, this reaction forms a complex that is readily identified spectrophotometrically. Protein carbonyls, a measure of protein oxidation, were analyzed using a commercially available ELISA kit (Zentech Technology, Dunedin, New Zealand). Prior to analysis, all plasma samples were assayed for protein concentration based on the methods of Bradford (5) and adjusted to 4 mg·mL−1 protein using a phosphate buffer. Intraassay and interassay coefficients of variation for lipid hydroperoxide and protein carbonyl assays were 9 and 5%, respectively.
Participation was contingent on refraining from drug use (pharmaceutical or illicit) and vitamin/mineral or antioxidant supplements. To rule out potentially confounding effects of diet, subjects completed a diet recall for 3 d before baseline testing and both oxidative stress trials. The diet logs were analyzed using a computer diet analysis program (Food Processor SQL ESHA; Salem, OR).
A two-way repeated-measures analysis of variance (ANOVA) was used to analyze between-condition differences. Follow-up paired-sample t-tests were performed with a Holm's sequential Bonferroni procedure to adjust for significance levels. All statistical analyses were performed using a statistical software package (SPSS, version 13.0, SPSS Inc., Chicago, IL). Data are presented as means ± SEM.
Oxidative stress trial performance data and blood lactate responses.
Comparison of strength exercise intensity, total repetitions, and rest periods is displayed in Table 1. Blood lactate was not different at rest (1.76 ± 0.24 vs 1.49 ± 0.46 mM) (mean ± SEM) between the hypertrophy and strength exercise protocols, respectively. As expected, both hypertrophy and strength protocols produced a significant rise in IP blood lactate concentrations as compared with baseline values where lactate increased IP in both exercise protocols (13.04 ± 0.46 L vs 7.13 ± 1.19 mM, P < 0.05) but was substantially higher in the hypertrophy exercise protocol compared with the strength exercise protocol. By 60Post, blood lactate concentrations had returned to near resting values (2.78 ± 0.44 vs 2.82 ± 0.56 mM) in both the hypertrophy and strength exercise protocols.
Plasma antioxidant/redox status.
In response to the hypertrophy protocol antioxidant capacity, as measured by plasma TEAC, increased IP. This rise in plasma TEAC was also significantly higher when compared with the IP TEAC response to the strength protocol. At the 60POST time period following the hypertrophy challenge, TEAC values returned to baseline (Fig. 1A). Also following the hypertrophy protocol, plasma FRAP rose significantly at the 60POST time period as compared both with baseline values and the corresponding 60POST FRAP levels from the strength protocol. Additionally, the hypertrophy protocol resulted in a dramatic lowering in plasma urate IP as compared with baseline levels and the IP urate following the strength protocol. Furthermore, following the hypertrophy protocol, plasma urate returned to baseline values at the 60POST time point (Fig. 1C). In contrast to the hypertrophy protocol, plasma TEAC was not altered by the strength protocol IP, but dropped below baseline values at the 60POST time point. Plasma FRAP levels were also unaffected by the strength protocol (Fig. 1B). As with FRAP, plasma urate values were not altered during the time points sampled following strength exercise.
Plasma oxidative damage markers.
Statistical analysis of our marker for oxidative damage to lipids, lipid hydroperoxides, did not reveal significant time effects for either hypertrophy or strength trials. Notably, the apparent rise in plasma lipid hydroperoxides at the 60POST time point of the hypertrophy trials was not significant (P = 0.156). Accordingly, no significant differences were observed between the hypertrophy and strength protocols at any time point, PRE, IP, or 60POST (Fig. 2A). In response to the hypertrophy protocol, plasma protein carbonyls were not significantly different from baseline in the IP plasma sample but increased by more than threefold above baseline at 60POST (Fig. 2B). In contrast to lipid hydroperoxides, plasma protein carbonyl levels were affected by both hypertrophy and strength treatments. Immediately following strength exercise, a threefold increase in plasma proteins carbonyls was observed. When compared with baseline, this rise in protein carbonyls continued to a fivefold increase at 60POST strength protocol.
Macronutrient dietary intakes and selected antioxidants are presented in Table 2. Dietary analysis demonstrated that prior to testing, all subjects met RDA intake standards for dietary energy, macronutrients, and vitamins (including vitamins C and vitamin E). Analysis of vitamins known for their antioxidant capacity did not reveal that subject antioxidant intakes were above normal, with the notable exception of vitamin C. The mean intake of vitamin C is approximately three times the normal recommended dietary allowance of 90 mg·d−1. Nonetheless, this mean value for vitamin C well below the daily dose (500-1500 mg·d−1) often prescribed to attenuate exercise-induced oxidative stress (6,20). Additionally, no significant differences in the reported intake of vitamins A, C, and E existed between the hypertrophy and strength protocols. Therefore, we conclude that it is unlikely that these antioxidant vitamins impacted the results obtained for the dependent variables in this investigation.
The aim of this study was to investigate the impact of work intensity on the oxidative stress response to acute resistance exercise. Our main finding is that both strength and hypertrophy back squat protocols resulted in blood oxidative stress. This finding refutes our hypothesis that oxidative stress would be most significant following the hypertrophy resistance exercise. Importantly, our study is among a select few exercise and oxidative stress investigations to focus exclusively on resistance exercise and to control for exercise intensity (% 1RM) as a means of eliciting oxidative stress. Important facets of the current study design included the following considerations: 1) strength and hypertrophy resistance exercise protocols were volume equated to clearly isolate for the impact of intensity on oxidative stress, 2) for our experimental model, we employed both strength and hypertrophy back squat exercise protocols to elicit an oxidative stress, and 3) we investigated both perturbations in blood plasma antioxidant capacity in addition to oxidative stress-mediated damage to better represent the entire redox milieu of blood plasma. In aggregate, these methodological considerations enabled us to perform a well-controlled examination of resistance exercise intensity on blood oxidative stress.
Exercise intensity and oxidative stress.
Regarding a source for free radical production during acute exercise, conventional wisdom implicates superoxide release from respiring mitochondria as a stiochiometric function of total oxygen flux (22). Recent work in aerobic and combined aerobic/resistance exercise, however, suggests that exercise-induced oxidative stress is more responsive to exercise intensity than total aerobic metabolism (22). The current findings, using two anaerobic exercise protocols, are in agreement with previous investigations that indicate moderate- and high-intensity exercise results in a measurable oxidative stress (2,4,18,23). In support, Bloomer et al. (2) identified a blood oxidative stress following full-body resistance exercise performed at 70% 1RM. This rationale was the impetus for our strength and hypertrophy protocols with a respective 90% 1RM and 5 min of recovery between sets and 75% 1RM and 90 s of recovery between sets. Stated differently, because our strength and hypertrophy protocols were volume equated, the total work performed during the hypertrophy protocol did not differ from the total work performed in the strength protocol, although the hypertrophy protocol was performed in approximately one-fifth the time required to complete the strength exercise. Further, we demonstrated in this investigation that load reduction due to volitional fatigue did not confound total volume performed between trials. The metabolic impact of these work/time ratios is evident the plasma lactate responses to both protocols. Both strength and hypertrophy exercise produced immediate postexercise elevations in plasma lactate as compared with baseline, but the response magnitude was most dramatic following hypertrophy exercise. Thus, despite intensity differences between the two protocols, the energy demand per minute was significantly greater during the hypertrophy protocol as compared with the strength protocol. In support, a recent investigation by Hoffman et al. (11) examined blood oxidative stress following similar resistance exercise regimens of 60% 1RM and 90% 1RM. Unfortunately, only one oxidative damage marker, malondialdehyde, was used, and no increases were elicited by either intensity. Nonetheless, when plasma malondialdehyde values were correlated with an indirect marker of tissue oxygenation, a modest but significant positive relationship resulted (11). This finding implicates a relationship between resistance exercise intensity and oxidative stress and further validates the study rationale of the current investigation.
In light of the aforementioned relationship between exercise intensity and oxidative stress, we were surprised to discover that both strength and hypertrophy protocols elicited significant elevations in plasma protein carbonyl levels. This finding suggests that both protocols exceeded an undefined threshold for eliciting an oxidative damage response based on currently available detection methods. Further, the magnitude of protein carbonyl elevation following strength protocol exceeded the hypertrophy protocol at both the IP and 60POST time points. At first glance, this finding could be interpreted as further support for the positive relationship between intensity and oxidative stress. This approach, however, overlooks differences in the time course to complete and recover from the two protocols. For instance, starting at the PRE time point for both protocols, the strength protocol IP time point virtually coincides with the hypertrophy protocol 60POST time point. Accordingly, when the protein carbonyl data are presented chronologically relative to PRE, the protein carbonyl damage response between the two protocols is identical (Fig. 3).
The current finding that the time course for an elevation in plasma protein carbonyls following both protocols is extremely interesting and supports previous work by Bloomer et al. (4), where the highest postexercise protein carbonyl concentrations occur not immediately following, but instead over an hour after, the start of a similar resistance exercise. Previous investigations of the protein carbonyl response for extended durations (up to 48 h postevent) have produced variable results. For instance, one study observed a peak in protein carbonyl values 24 h postevent (4), whereas another found no effect (2). Considering that these three previous studies originate from the same lab, much of the difference is attributable to variations in exercise protocol, including intensity. Undoubtedly, the current investigation is limited by having no data past the 60POST time point. In an effort to fully document the time course of protein carbonyl elevation following variable-intensity resistance exercise, future investigations would benefit by blood sampling up to 48 h after resistance exercises to better identify the time course for protein carbonyls to return to baseline levels.
The free radical pecking order and blood oxidative stress.
During this investigation we adhered to a definitional approach for the examination of oxidative stress; where oxidative stress begins when antioxidant defenses are compromised. If allowed to persist, an oxidative stress will eventually produce an identifiable oxidative damage; as measured in this study by the biomarkers lipid hydroperoxides and protein carbonyls (10). This scientific approach, originally called the "free radical pecking order" in blood plasma, dictates that oxidative stress will result in oxidative damage, in the case of lipid peroxidation, only after the depletion of local water soluble and then fat soluble antioxidants (7). Based on this rationale, the oxidative stress response to resistance exercise was examined in human plasma.
The redox balance of plasma is sensitive to a multitude of redox sensitive components of both the cellular and extra cellular compartments. Accordingly, two markers of redox status, plasma antioxidant capacity (TEAC) and potential (FRAP), were used. Further, plasma urate levels, known to be the single most significant contributor to water soluble antioxidant capacity, were analyzed (24). The hypertrophy protocol resulted in the most dramatic changes in all three measures of plasma antioxidant capacity. While a postexercise rise in plasma urate would have made intuitive sense, a significant drop in urate was observed. Unfortunately, a biological explanation for this finding in plasma TEAC and urate following the hypertrophy exercise protocol does not exist, and this warrants further study. Finally, the rise in plasma antioxidant potential as evaluated by FRAP was significantly elevated only at the 60POST time point following hypertrophy exercise only. Despite differences in study protocol, our finding is in agreement with McAnulty et al. (17) where plasma FRAP levels increased by nearly 40% 1 h following full-body resistance training session (17). As with the TEAC and urate data, an explanation for the plasma FRAP findings is illusive and required further study.
In summary, the results of this study indicate that acute moderate- and high-intensity back squat exercise simulating traditional hypertrophy and strength training scenarios result in similar oxidative damage response as assessed by the biomarker protein carbonyls. This result seems to be independent of resistance exercise intensity. The exact implications of these findings likely represent complex redox interactions between cellular and extracellular compartments, which are yet to be uncovered. Finally, findings of the current exercise-induced oxidative stress investigation illustrate the importance of a rigorous methodological application to subject testing and biochemical oxidative stress assay.
This work was supported by grants from BeActive North Carolina, The Vaughn Christian Grant, the Office of Student Research of Appalachian State University, and the Graduate Student Research Grant courtesy of the Cratis D. Williams Graduate School of Appalachian State University. We further wish to acknowledge the valuable consultation given by Dr. William Stone and Christian of East Tennessee State University.
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