Creatine kinase (CK) is often used as a biochemical marker for myocardial infarction or skeletal muscle damage after exercise (1,5,7,10,13,17). Following high force eccentric exercise, CK is released into the circulation and remains elevated for several days after the exercise protocol (6,14). Plasma CK levels generally peak on the third or fourth day following high force eccentric exercise (6), and enzyme clearance by the reticulo-endothelial system (17) is responsible for the return of CK to preexercise levels.
Muscle damage after exercise has also been documented directly by myofibrillar disruption and indirectly by the perception of soreness and a prolonged loss in strength and range of motion (6,10). Repeating the same bout of exercise (using the same muscle group) 2 wk to several months later results in a reduced response to the exercise such that the loss in strength and range of motion are not as prolonged, and less soreness is experienced. However, the CK response is completely negated following a second bout (5,6).
Although it is not known what causes the dramatic "repeated bout" effect for CK, the reduced CK response has been attributed to an adaptation effect in the exercised muscle. However, Nosaka and Clarkson (12,13) noticed a blunted response even when the second bout was performed with the opposite arm. They analyzed serum CK activity following a second bout of exercise performed on day five after the first bout, when CK levels were still elevated. Although it was expected that CK levels after the second bout would surpass peak CK activity observed from the first bout, the CK response never exceeded the first peak. It was suggested that accelerated clearance may have influenced CK levels.
CK is a dimeric protein found as three principle isoenzymes: muscle (MM), heart (MB), and brain (BB). Within skeletal muscle, CK occurs mostly in the MM isoform (approximately 90% of TCK) and only minutely in the MB and BB forms (18). CK-MM is further distinguished into three isoforms (MM1, MM2, MM3). Although it is possible to separate the isoforms from the plasma, only the pure gene product (MM1) is found within the muscle. In the blood, cleavage of either a lysine or arginine residue from the carboxyl terminus of one subunit of the CK enzyme will yield CK-MM2. Further release of either amino acid residue from the other subunit yields CK-MM3 (9,16). The presence of MM1 in the blood indicates newly released CK from the muscle.
Following muscle damage, there is a significant leakage of MM1 into the circulation (1,15). Within hours, MM1 is structurally modified into the two other isoforms. Several researchers have employed the MM1:MM3 ratio to analyze CK release and clearance in the circulation following exercise-induced muscle damage (1,2,4). Ratios equal to 1 indicate that CK release equals its clearance, whereas ratios >1 indicate that release is prominent, and ratios <1 indicate faster enzyme clearance from the circulation than release from the damaged muscle.
The present study investigated CK-MM isoform release and clearance dynamics following repeated bouts of eccentric exercise using a modified exercise protocol from Nosaka and Clarkson (13). In one condition, subjects exercised the same arm for both bouts; in the other condition, subjects exercised the opposite arm for the second bout. It was hypothesized that a large, initial increase in MM1, and hence the MM1:MM3 ratio, would occur in both groups after the first bout. Following the second bout, it was believed that there would be some MM1 release, but the amount would be smaller compared with bout 1. However, the subjects using a new muscle group for the second bout were hypothesized to show new circulating MM1, similar to bout 1, despite a lower total CK response. These expected findings would provide support for enhanced enzyme clearance as a partial explanation for the dramatic "repeated-bout" effect for CK.
Subjects and exercise protocol. Ten college-age males voluntarily participated in this investigation. Subjects abstained from any resistance training 6 months before this study. Before engaging in the testing exercise, the subjects were informed of the benefits and potential risks involved and asked to sign an informed consent form consistent with the requirements for human subjects at the University of Massachusetts, Amherst.
Subjects performed two sets of 50 voluntary maximal eccentric muscle actions of the elbow flexor muscles on a modified preacher-curl bench 6 d apart (6). Each contraction occurred for approximately 3-5 s with a rest period of 10-12 s between each repetition to avoid the effects of fatigue on the exercising muscles. Each set of 50 contractions was separated into two sets of 25 contractions with a 5-min rest between each set. On the sixth day following the first bout, the subjects were randomly assigned to one of two groups: subjects assigned to the control group (CON) performed the second bout with the same arm that was used in the first bout, and subjects in the experimental group (EXP) repeated the exercise protocol described above with the opposite arm. Arm preference was balanced between groups. Criterion measures were assessed for a total of 12 d.
The first visit (day 1) consisted of familiarizing the subject with the testing procedure and completion of an informed consent document. A physical activity readiness questionnaire was also completed, and the subjects were verbally reminded of their rights and responsibilities as a subject participating in this investigation. Day 1 concluded with a preexercise blood draw to be used for baseline measurements. Day 2 involved the exercise protocol, and postexercise criterion measures subsequently followed. Blood collection on this day occurred at hours 2 and 6 following the exercise. Days 3 through 6 were for collection of the criterion measures and blood samples. On day 7, subjects were randomly assigned to one of the two groups and performed the second bout of exercise. Criterion measures and blood sampling on day 7 were identical to day 2, and days 8 through 12 were identical to days 3 through 6.
Criterion measures. Range of motion (ROM) was assessed through relaxed and flexed arm angles (RANG and FANG, respectfully). A goniometer was placed at the elbow joint and used to assess arm angles (11). Mid- and distal-biceps circumferences (MID-CIR and DIS-CIR, respectfully) were measured approximately 4 and 8 cm above the elbow joint. Muscular strength (STR) was assessed by a maximal isometric voluntary contraction (MVC). Using a modified preacher curl bench, the subject was seated, and the arm angle was set at 90°. The bench was attached to a strain gauge and connected to a Jackson evaluation system (Lafayette Instruments, Lafayette, IN). Three MVCs were performed with a 45-s rest between each contraction. The average of three trials for ROM, CIR, and STR was used as the score for statistical analysis. Subjects were asked to rate soreness using a visual analog scale (VAS) consisting of a 100-mm scale ranging from 0 (not sore) to 100 (very, very sore). Subjects assessed muscular soreness by self-palpation (SOR-PALP) and movement (SOR-MVT) through flexion and extension of the exercised arm. All criterion measures were assessed preexercise, immediately postexercise, and every 24 h for 5 d following each bout of exercise.
Approximately 5-7 mL of blood was collected using standard venipuncture from a forearm vein. Blood vacutainers contained EDTA to act as an anticoagulant and to prevent conversion of CK-MM isoforms during centrifugation and storage (3). Plasma storage was at −20°C. Blood samples were collected at 2, 6, 24, 48, 72, 96, and 120 h postexercise. Blood was placed on ice and centrifuged for approximately 10 min. Plasma was drawn off and stored until further analysis. Sampling of blood occurred at the same time points for bouts 1 and 2.
Biochemical techniques. Total plasma CK (TCK) was assessed spectrophotometrically with a commercially available enzymatic kit (Sigma). All samples were run in duplicate at 37°C using a Spectronic 1001 spectrophotometer (Bausch and Lomb, Rochester, NY). The average of the assays was used for statistical analysis.
Isoelectric focusing techniques used were modified from Clarkson et al. (4). TCK samples were diluted to protein concentrations <1000 IU·L−1 with an 0.85% saline solution and applied to ampholine gels (pH gradient, 5.5-8.5) (Pharmacia Biotech, Piscataway, NJ) and focused isoelectrically on a flatbed electrophoresis Multiphor System II (LKB Instruments, Gaithersburg, MD). Plasma samples were applied on sample application pads 1 cm from the cathode and run at 25 W for 2 h at a constant power supply. If the TCK determinations were equal to 100 IU·L−1, 15 μL of plasma was applied to the gel. If samples were >100 IU·L−1, 5 μL of plasma was applied to the gel. The gels were maintained between 4 and 5°C during focusing by water cooled by an isotemp refrigerated circulator (model 9000) (Fischer Scientific, Medford, MA) passing through a cooling plate on which the gels were placed. Contact between the wire electrodes and the gel was made via paper wicks, which were presoaked. Anode wicks were soaked in 0.4 mol·L−1 N-(2-hydroxyethl)piperazine-N′-2-ethanesulfonic acid (HEPES) solution, and cathode wicks were soaked in 0.1 mol·L−1 sodium hydroxide solution. CK-MM variants were transferred to Universal 8-track gels (Ciba Corning, Norwood, MA) coated in CK reagent (Ciba Corning, Norwood, MA). Any CK reacting with the reagent appeared as fluorescent bands when quantified using scanning densitometry.
The intensity of the CK-MM variants was assessed using a densitometer (Ciba Corning, Norwood, MA). Each lane of separated CK protein was analyzed and quantified as a percentage of total protein specific for that lane. These percentages were then converted into IU·L−1 for absolute concentrations of each MM variant and expressed as the MM1:MM3 ratio.
Statistics. A three-way repeated measures analysis of variance (ANOVA) was used to assess significance for the criterion measures and blood parameters. A post hoc Tukey's honest significance difference test (HSD) was used to locate the differences detected by the ANOVA. Significance was set at P < 0.05.
One subject from each group was dismissed from the study, reducing the subject size to four in each group. The subject from CON revealed participating in resistance exercise within the 6 months before volunteering for the study; the EXP subject was dismissed because blood samples following the second exercise could not be obtained. The physical characteristics of the remaining eight subjects were as follows (means ± SD): height, 180.66 ± 7.24 cm; body mass, 74.15 ± 4.38 kg; age, 23.25 ± 2.19 yr.
Noncreatine kinase criterion measures. Figure 1 presents changes in RANG for both groups. RANG and FANG responded similarly, so only RANG is expressed graphically. For bout 1 there was a significant loss (P < 0.01) in each criterion measure for both CON and EXP, with no differences between groups (P > 0.05). However, there was a significant difference (P < 0.05) between CON and EXP following bout 2. In comparing bout 1 and bout 2 for CON, there was a smaller change in RANG and FANG following the second bout (P < 0.01). The exercised muscle groups for EXP did not respond differently between bouts for either RANG or FANG (P > 0.05).
Figure 2 presents changes in CIR-MID for both groups. CIR-MID and CIR-DIS responded similarly, so only CIR-MID is expressed graphically. For bout 1 there was a significant increase (P < 0.01) in each circumference measure for both CON and EXP, with no differences between groups (P > 0.05). However, there was a significant difference (P < 0.05) between CON and EXP following bout 2. In comparing bout 1 and bout 2 for CON, there was a smaller change in CIR-MID and CIR-DIS following the second bout (P < 0.01). The exercised muscle groups for EXP did not respond differently between bouts for either CIR-MID or CIR-DIS (P > 0.05).
Figure 3 presents changes in STR for both groups. For bout 1 there was a significant loss (P < 0.01) in STR for both CON and EXP, with no differences between groups (P > 0.05). However, there was a significant difference (P < 0.05) between CON and EXP following bout 2. In comparing bout 1 and bout 2 for CON, there was a smaller change in STR following the second bout (P < 0.01). The exercised muscle groups for EXP did not respond differently between bouts for STR (P > 0.05).
Figure 4 presents SOR-MVT for both groups. SOR-MVT and SOR-PALP responded similarly following the first bout, so only SOR-MVT is expressed graphically. Both criterion measures were significantly elevated following bout 1 for CON and EXP (P < 0.01), with no differences between the groups (P > 0.05). Both criterion measures were shown to be significantly higher for EXP when compared with CON following the second exercise (P < 0.01). The differences between bouts 1 and 2 were also determined to be significant for CON and EXP (P < 0.01).
The data from non-CK criterion measurements suggest that the second exercise damaged the EXP contralateral muscle groups similarly to those muscles exercised in the first bout. Conversely, in CON the second exercise did not affect the previously exercised muscles as dramatically as the first exercise, thus demonstrating the characteristic repeated-bout effect.
Total creatine kinase and CK-MM isoform parameters. TCK in each group was shown to be significantly elevated following the first exercise (P < 0.01) with no difference between CON and EXP (P > 0.05). There was a significant difference (P < 0.01) between groups following the second bout. In comparing bout 1 and bout 2, EXP and CON exhibited significantly lower TCK activity after the second exercise (P < 0.01). Although no clearly defined TCK peak was observed after the second bout for EXP, the slope of the increase toward the peak following bout 1 is much greater than the slope of ascension following bout 2. It was believed that 120 h postbout 2 would be the peak for EXP (Fig. 5, top). In fact, the TCK for one EXP subject peaked at 96 h postbout 2, suggesting that TCK indeed peaked around 96 or 120 h after the second exercise.
The percentage of TCK for each isoform was analyzed, and there was a gradual rise in %MM1 (Fig. 5, bottom) from preexercise values that was determined to be significant for CON and EXP (P < 0.01). There were no differences detected between the groups after the first exercise (P > 0.01). Following the second bout, there was an additional increase in %MM1 in each group, but these second peaks were significantly lower than those observed in bout 1 (CON, P < 0.01; EXP, P < 0.05). Because the majority of TCK across days was in the form of either %MM2 or %MM3, the changes of these variants closely followed the patterns of TCK in both groups.
Figure 6 depicts the calculated MM1:MM3 ratio for EXP and CON. There was a significant increase following the first exercise for EXP and CON (P < 0.01) with no differences between the groups (P > 0.05). Bout 1 and bout 2 were shown to differ for both groups (P < 0.01). Two smaller increases were observed for EXP (P < 0.01) and CON (P = 0.053) after the second exercise. The ratio was not significant between groups after the second bout (P > 0.05).
The purpose of this study was to investigate the blunted CK release following repeated bouts of eccentric exercise by assessing the activity of the CK muscle (MM) isoforms. To achieve this, a second damage-inducing exercise was performed either on the same arm (CON) or the contralateral arm (EXP) while total plasma CK was elevated as a result of the first bout. One limitation of this study was the small sample size. However, all subjects in a group responded similarly to the respective treatments.
As expected, results of the present study showed that performing a second damage-inducing exercise with the same muscle group (CON group) 6 d following the first exercise had a substantial effect on changes of ROM, STR, CIR, and SOR. The difference between bout 1 and bout 2 for these parameters indicated that changes were more dramatic following the first bout than the second bout. These results are similar to those observed in previous studies that have documented a repeated-bout effect (6,7).
Conversely, both arms in EXP responded similarly after the damage-inducing exercise for ROM, STR, and CIR. This was also expected because different arms were exercised. The differences in SOR between bouts in EXP suggest an increased pain tolerance after the first exercise. The novelty of muscle soreness in the first arm exercised seemed to serve as a reference by which to gauge the degree of soreness after bout 2. Subjects often compared the soreness after the second exercise to what they had experienced following the first bout. Thus, a psychological adjustment most likely attributed to the soreness differences between bouts for EXP.
Performing a second exercise using the same arm within days of the first exercise does not further elevate TCK (6,7). This observation is consistent with results of CON in the present study. EXP exhibited a second rise in TCK as demonstrated earlier (13) following the second exercise bout on the contralateral arm. Nosaka and Clarkson (13) showed a clearly defined blunted second peak in TCK, whereas our results revealed that TCK was still ascending by 120 h postbout 2. Based on the small ascending slope of TCK, however, we believe that this time point represents the second peak observed at 96 h by Nosaka and Clarkson (13). Moreover, the TCK for one EXP subject peaked at 96 h postbout 2, further suggesting that TCK peaked around 96 to 120 h after the second exercise. The blunted response of TCK was suggested earlier to be, in part, the result of accelerated enzyme clearance by the reticulo-endothelial system (7,13). Our analysis of MM isoforms after a repeated bout of eccentric exercise in CON and EXP supports the theory that accelerated clearance is partly responsible for the blunted response.
MM isoforms have been used previously to assess release and clearance of CK following running (1) or a single bout of exercise-induced muscle damage (2,4,15). Apple et al. (2) and Clarkson et al. (4) reported significant elevations in MM1 and the MM1:MM3 within several hours of the damaging exercise. Our results following the first bout in the present study did not detect significant elevations so quickly after the exercise. This may be because Clarkson et al. (4) induced damage using isometric contractions and subjects from Apple et al. (2) performed 120 eccentric contractions at 110% of the subjects' maximal concentric contraction. The extent of damage following these protocols could have elicited different CK release patterns than what was observed in the present study. Instead, %MM1 and the MM1:MM3 ratio were not significantly different from baseline until 48 and 24 h, respectively. In agreement with Page et al. (15), MM1 peaked 1 d before MM2 and MM3 in both groups postbout 1. The early release of MM1 after the first bout, as shown through the percent MM1 data, coincides with the infiltration of neutrophils, which appear in the circulation and within tissue (8). Neutrophil invasion may therefore contribute to the early %MM1 elevations.
The MM1:MM3 ratio indicated that release was prominent between 24 and 48 h and 6 and 72 h for CON and EXP, respectively. At 96 h, the ratio for each group returned to below 1 for the remainder of the study, signifying that clearance was dominant during this period despite the second bout of exercise. The significant rise in %MM1 postbout 2 in both groups indicated that new release did occur. This was expected in EXP, as a new muscle group was subjected to the damaging protocol. The additional, but also smaller, rise in %MM1 for CON may have been because of residual release from either previously damaged fibers that had not fully recovered from the first bout or because of damage to new fibers unaffected during bout 1. This result is in concert with the soreness and loss of strength and range of motion that were also found in CON bout 2. Thus, after bout 2, there was an increase in %MM1, a loss of strength and range of motion, and soreness development that was lower than bout 1.
In summary, analysis of CK-MM isoforms following two bouts of eccentric-damaging exercise suggest that clearance of CK is enhanced after the first exercise. This acceleration would contribute to the dramatic blunting of TCK if a damaging exercise is performed within days of the first bout. Although there were clear differences in TCK patterns following the second bout between the groups analyzed in this study, newly released CK was detected following the second exercise for both groups using MM isoform data. MM variants should therefore be employed in future repeated-bout studies to detect additional CK release that may not be apparent when analyzing TCK alone.
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