CHANGES IN TISSUE DEGRADATION MARKERS AND SUBJECTIVE REPORTS OF PAIN RESULTING FROM ECCENTRIC MUSCLE CONTRACTIONS

Abstract

The delayed onset of muscular soreness (DOMS) following a heavy eccentric exercise was studied with the aim to verify the “muscular structure” and the “connective tissue” theories explaining the development of DOMS. Die responses of creatine kinase (CK; “structural theory” marker) and hydroxyproline (OHP; “connective tissue theory” marker), as well as level and location of the perceived soreness, were determined following eccentric exercise. Plasma CK activity was elevated 48 and 72 h after the acentric exercise compared with the pre-exercise values, while OHP concentrations remained unchanged. Examination of pain location reports revealed two groups of responders (distal vs. mid-point muscle pain). Distal pain responders were found to have significantly higher post-exercise CK activity than mid-point pain responders, the OHP levels being alike. These findings are supportive of the “muscular structure” theory for DOMS development. However, the non-uniform location for DOMS pain confounds the overall data interpretation.

Introduction

Delayed-onset muscular soreness (DOMS) is the body’s way of indicating the overuse of muscle and is characterized by muscular tenderness and pain. In severe cases, a temporary loss of functional ability (inability to move exercised/damaged muscles in everyday activities without pain) has been reported [2,3,12]. The precise cause of DOMS was long thought to be due to the excessive buildup of lactic acid within the exercised muscles [19,31,34]. Although many exercisers still believe this notion today, recent evidence has now ruled out the existence of this mechanism [2,12,34].

The lengthening process of a contracting muscle, or eccentric muscular contraction, has been implicated in numerous studies concerning the pathophysiology of DOMS [1,15,25,26], Many of these studies used direct comparisons with pure concentric exercise and found eccentric exercise to be the predominant inducer of DOMS As a result of these findings, it is commonly believed that eccentric exercise is the primary factor leading to the onset of muscular soreness 24 – 48 hours post-exercise. Although the relationship between DOMS and eccentric exercise is well documented, the precise mechanism underlying this phenomenon is still unclear. It is generally believed that the initial damage caused by eccentric exercise is mechanical in nature, and that further sequelae occur as a result of this trauma [10,12]. The accumulation of oedema [5,10,15,20] and delays in the rate of glycogen repletion [29] have also been proposed as being secondary reactions to the mechanical damage. The precise events occurring during exercise, immediately after the exercise, and those leading up to the delayed perception of pain, are still unknown and research on this phenomenon continues to be equivocal.

Connective tissue and intramuscular tissue (i.e. muscle fibers) are the two structural units thought to be the recipients of the initial mechanical trauma during muscular contraction, Damage to either or both of these structures can produce delayed pain and soreness and, as a result of these observations, two dominant theories of DOMS development have emerged. Structural damage to the elastic component of the musculo-tendinous junctions constitutes the “connective tissue theory” [2,3,22,28]. The presence of increased levels of collagen degradation products (e.g. hydroxyproline) in the blood and urine of eccentrically exercised subjects provides biochemical support for this mechanism [1,4,22].

Ultrastructural damage to the muscle fibers themselves is the other well supported theory of DOMS pathophysiology [16,19,23,33], Support for this mechanism has been found in studies using electron microscopy to examine the structural changes of eccentrically exercised muscles [12,16,27,33]. All of these studies found evident structural damage within the muscle cells and attributed these changes to eccentric muscular contractions. Additional biochemical support has been found in studies measuring increases in blood levels of structural proteins, enzymes, or degradation markers (e.g. creatine kinase. 3-methylhistidine). following exercise associated with DOMS [12,13,14,33].

Although there are two distinctly different theories explaining how the eccentric muscle contractions cause DOMS, no consensus has been reached on the precise nature of the structural involvement. No study has apparently considered the possibility of these two mechanisms interacting to induce DOMS and this prompted us to undertake the present study. The purpose of this study was:

1. To obtain a subjective rating of the location of DOMS, by determining if either the muscle mid-point (i.e. high muscle fiber area) or the musculo-tendinous junctions presented higher ratings of pain and soreness over 72 hours;

2. To determine which blood markers of tissue damage (creatine kinase, hydroxyproline) were more prominent over the 72 hours following stressful eccentric exercise, and

3. To determine if any relationship existed between subjective (peak levels and location of DOMS) and objective (tissue degradation markers) symptoms of DOMS.

Material and Methods

Subjects:

The subjects were all college aged males (n= 10) with no history of regular weight training. Every subject gave his written consent to participate in the study. The physical characteristics (means ±SD) for the subjects were: age, 22.5±3.0 years; body height, 180.6±7.6 cm; body mass, 74.6±9.3 kg. body mass index, 23.2±0.7 kg·m⁻². Subjects were instructed not to take any analgesic and/or aspirin during the entire experimental period (before, during, or after the exercise).

Procedure:

On the assigned day, subjects reported to the laboratory dressed in nonrestrictive clothing. After the pre-test blood sample has been obtained, a DOMS questionnaire administered, and background information collected, the subjects were instructed on how to perform the exercise. After proper stretching and resistance-free warm-up repetitions, each subject was allowed to perform 3 to 5 biceps arm curl repetitions in an attempt to reach maximum concentric strength. The subjects started with using one-quarter of their respective body weights for their first attempt, and then increased the weight accordingly until the lifter failed one attempt. The last fully completed repetition was marked as the maximum concentric strength. This maximum strength determination was used as the individual work intensity for the eccentric trial. The mean (±SD) maximum one-repetition concentric strength for the two-armed biceps curl was 23.7±4.3 kg.

After 15 min of rest, the subjects performed 4 sets of 15 eccentric repetitions. The first set was done with 100% of their previously obtained maximum concentric strength, while the latter 3 sets decreased in resistance by 2.27 kg each. The sets were separated by rest periods of 5 min in order to allow an adequate recovery.

All subjects were requested to return to the laboratory 24, 36, 48, and 72 h afterwards for completing the DOMS rating questionnaire and to have blood samples taken.

Ratings of the perceived muscular soreness and the location of peak soreness were assessed by using the subjective rating scale of Abraham [1]. Clarkson et al. [8] have established this scale as a valid and reliable tool to use in assessing the degree of DOMS. This form was adapted to include the subjective location of DOMS in the exercised muscles. Subjects were instructed to indicate one of the following locations of the most pronounced soreness: the proximal origin, mid-point, or distal insertion of the biceps musculature.

Blood samples (about 20 ml) were withdrawn under sterile conditions from the antecubital vein. Care was taken to use entry points in the veins that were distal to the elbow joint, so that the joint in question would not be otherwise disturbed. Serum was obtained by centrifugation and kept frozen at −54°C until assayed. Serum levels of creatine kinase (CK) were measured using the quantitative, kinetic method (37°C) and commercial kits (Sigma, U.S.A). Hydroxyproline (OHP) determination was carried out using a modified version of the colorimetric method of Murguia et al. [24] and of Woessner [35], as modified by Hackney [17]. All biochemical assays were carried out in duplicates.

Design and analysis:

The results of biochemical determinations and subjective ratings of the perceived muscular soreness have been presented as means ±SD. The numbers of DOMS locations indicated were also presented. The three parametric dependent variables (DOMS peak pain. CK and OHP) were subjected to one-way MANOVA vs. time as the independent variable. One-way ANOVA was used to detect the significant within-group differences between the post- and pre-exercise values. The Tukey’s post-hoc test was used to detect the between-group differences at the consecutive time points. The level of significance was set at P≤0.05.

Results

Muscle Soreness:

Peak results of the perceived delayed muscular soreness (DOMS) are summarized in Table 1. The values recorded 24, 36, 48 and 72 h post-exercise were significantly higher than the pre-exercise one rated as zero. In addition, both the 36 and 48 h responses were significantly greater than the 72 h rating.

Table 1.

Subjective ratings of peak DOMS scores and numbers of subjects indicating location (n=10)

Sampling period (h, post-exercise)	Peak DOMS (Mean ± SD)	DOMS location
		Proximal	Mid-point	Distal
Pre-exercise	0	0	0	0
24	1.6 ± 0.7*	1	5	4
36	2.2 ± 0.8*	1	5	4
48	1.9 ± 0.9*	1	5	3
72	0.9 ± 0.7*	1	4	2

^*Significantly different from the pre-exercise value (P<0.05)

Regarding the DOMS location (Table 1), 5 subjects reported their muscular soreness to occur in the mid-point of the biceps muscle, 4 subjects - the distal musculotendinous location and one subject - the proximal region, as the primary source of pain (24 and 36 h post-exercise). At 48 and 72 h, not all subjects reported pain, therefore no DOMS location scores were reported for these individuals. In general, the responders could be classified as those who reported pain in the mid-point musculature (n=5) or in the distal connective tissue (n=4).

Blood markers:

Mean values of CK activity and of OHP concentration in plasma are predented in Table 2. The elevations in CK activity were significantly highest 48 and 72 h post-exercise compared with those before or 24 h after the exercise The post-exercise OHP levels in plasma show no significant changes with respect to the pre-exercise ones following this acute bout of eccentric exercise.

Table 2.

Mean (±SD) creatine kinase (CK) activity and hydroxproline (OHP) concentrations in plasma over the time course of the study

Variable	Sampling time (h)
	Pre-exercise	24	36	48	72
CK (U·1−1)	101±62	255±259	569±581	1020±1180*	1154±1238*
OHP (mg·1−1)	3.38±0.94	3.66±1.64	3.06±0.66	3.65±0.39	3.50±0.82

^*Significantly different from the respective pre-exercise value (P<0.05)

The two groups of muscle soreness responders (distal or mid-point locations) did not differ from one another regarding plasma OHP levels but the CK responses 36, 48, and 72 h post-exercise were in the distal point responders significantly higher than in the mid-point responders (see Fig. 1).

Fig. 1.

Mean creatine kinase (CK) activity in distal (full dots) and mid-point (empty dots) pain responders over the lime course of the study

Pre - Pre-exercise value;

* Significant (P<0.05) between-group difference

Discussion

This study examined the delayed onset of muscular soreness (DOMS) following a heavy eccentric exercise to see if subjective reports of soreness level and location supported either the “muscular structure theory” of DOMS, or the “connective tissue theory” of DOMS development. Specifically, blood CK (“structural theory” marker) and OHP (“connective theory” marker) responses were examined following eccentric exercise.

All post-exercise values of DOMS were significantly higher from the pre-exercise ones, the peak level being reported at the 36 h time point. Using the same DOMS scale, Abraham [1] reported values of 1.6 and 2.1, 24 or 36 h, respectively, following an eccentric biceps exercise. The values for DOMS at the same time points post-exercise, reported by us, were 1.6 and 2.2, respectively. DOMS has been reported to peak, generally, between 24 and 48 hours and to persist for up to 72 hours after the exercise [12,21,26,30,31,32]. In concurrence with these reports, the subjects in this study reported peak DOMS to occur at the time point of 36 h, with the pain persisting until the last observation (72 h).

Reporting the precise location of muscle soreness is an indirect means of indicating which structure might be directly involved in the pathophysiology of DOMS [1]. In contrast to other studies [1,2,4,11,22], in which no detailed data on DOMS location have been reported, we have demonstrated that the subjects pointed out various locations of DOMS. Four of them indicated the distal insertions of the forearm flexor muscles and 5 others - the mid-point musculature, to be the most painful sites. This finding suggest that more than one mechanism may be involved in producing DOMS, and that there may potentially be two, or more, different groups of responders. Furthermore, one subject consistently reported proximal forearm flexor pain in this study which could technically be classified as connective tissue pain (because of the proximal tendon and high degree of connective tissue at the proximal musculotendinous junction).

Hydroxyproline values have not changed throughout the experiment, which suggests they are unaffected by eccentric exercise. Contrary to that, Murguia et al. [24] reported changes in OHP levels and this controversy is probably due to the nature of exercise used in the present study. We used an acute bout of eccentric exercise, whereas Murguia et al. found elevated scrum OHP levels following a long-term, chronic exercise (7 weeks of intensive military training). Interestingly, significant elevations in OHP were found by Murguia only in those subjects who were diagnosed as having connective tissue injuries.

The lack of any increase in OHP in the present study may have been due to an insufficient exercise stress or to a low assay sensitivity. Since the same OHP assay was used as in the study of Murguia et al. [24], who found significant elevations with greater stress, the relatively low level of physical stress we induced was probably responsible for our findings. It should be noted, however, that our OHP results seem to support those of Horswill et al. [18] who reported that an acute session of eccentric exercise did not effect OHP excretion. Although no OHP responses to exercise were found in our study, the presence of an acute muscle soreness suggested that it was unrelated to effects in the connective tissue.

Elevated scrum CK activities are thought to represent muscle cell damage [12]. Since CK molecules arc large, the muscle cell sarcolemma must be damaged to some extent to allow passage of these proteins into the lymphatic system. Like in other studies [6,7,8,9]. pronounced, delayed increases in serum CK following eccentric exercise have been noted by us 48 and 72 h post exercise, peaking at 72 h, with an 18-fold increase over the pre-exercise levels. We have found no correlation between peak muscle soreness and peak CK activity which is in agreement with previous findings [8,25], Peak DOMS and peak CK release (36 and 72 hours post-exercise, respectively) were separated by 36 hours, suggesting that the amount of CK present in the extracellular fluids may not be directly related to subjective feelings of pain. We speculate that CK may represent muscle tissue damage, but it does not provide a sensitive enough indicator of its extent.

When the rate and amount of CK release has been examined in the two responder groups (Fig. 1), a much more dramatic CK release was observed in the distal pain responders compared to those who reported the mid-point pain over the post-exercise period of 36 to 72 hours. Throughout the 72 h period of observation, both responder groups had the same levels of subjective ratings of pain (DOMS) and OHP responses. No plausible explanation of these differences in CK activity between the responder groups can be offered and the issue needs further research.

In conclusion, our blood markers do not directly support the “connective tissue theory” for the origin of DOMS; however, our subjective markers of pain location are contradictory. Conversely, the elevation of CK supports the “muscular-structural theory” but our subjective data are at some variance with this concept. Perhaps the subjective location of pain does not indicate the location of damage but may be related to other events. Thus, the blood CK could be a response indicating muscle damage. The pain, however, may be only the product of an inflammatory response to a local “irritant” and not a true indication of the overall damage.