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Estradiol and Exercise-Induced Creatine Kinase Activity
JEPonline
Journal of Exercise Physiologyonline
Official Journal of The American
Society of Exercise Physiologists (ASEP)
ISSN 1097-9751
Volume 4 Number 2 May 2001
EFFECTS OF CIRCULATING ESTRADIOL ON EXERCISE-INDUCED CREATINEKINASE ACTIVITY STEPHEN M. ROTH1, RICHARD GAJDOSIK2 AND BRENT C. RUBY1 1 Human Performance Laboratory, Department of Health and Human Performance & 2 Motor Control ResearchLaboratory, Department of Physical Therapy, The University of Montana, Missoula, MT 59812, USA ABSTRACT
STEPHEN M. ROTH, RICHARD GAJDOSIK AND BRENT C. RUBY. Effects Of Circulating Estradiol
On Exercise-Induced Creatine Kinase Activity. JEPonline. 2001;4(2):10-17. The present investigation
sought to determine the effects of circulating estradiol (E2) and oral contraceptive use on serum creatine kinase
(CK) activity following acute eccentric exercise. Ten college-aged female subjects were grouped according to
their use (OBC, N=5) or non-use (NOBC, N=5) of an oral contraceptive. Both groups performed strenuous
eccentric muscle actions (5 sets with 10 repetitions/set; ≥100% of concentric 1RM) during the menstrual cycle
phase corresponding to low E2 concentrations. The OBC group had significantly lower E2 concentrations than
the NOBC group (11.6 versus 27.2 pg/mL, respectively; p<0.01) prior to exercise. The results indicated that
the OBC group (lower E2) had significantly higher CK activity at 48 and 72 hr post-exercise when compared to
baseline CK activity (p<0.05). Although the NOBC (higher E2) group showed a slight increase in CK activity
at 24, 48 and 72 hr post-exercise, values were not significantly different from pre-exercise. Perceived soreness
values were generally dissociated from CK activity, but demonstrated a similar post-exercise response between
groups. The results indicate that pre-exercise concentrations of circulating estradiol can affect serum CK
activity following damaging eccentric exercise.
Key Words: eccentric exercise, estrogen, muscle damage, oral contraceptive, soreness INTRODUCTION
Strenuous exercise, especially exercise involving eccentric muscle actions, has been shown to elicit histologicaland ultrastructural muscle damage (1,2). One consequence of damaging exercise is the release of variousskeletal muscle proteins into the blood. Increased serum creatine kinase (CK) activity has been used as a markerof muscle cell disruption, and has been indirectly associated with increased membrane permeability (2,3).
However, other factors are known to affect membrane structure, including vitamin E and estradiol (E2), andthus these agents could affect serum CK activity following strenuous activity (4,5).
Estradiol and Exercise-Induced Creatine Kinase Activity
Lower levels of serum creatine kinase activity following strenuous exercise have been shown in femalescompared to males (6), indicating a possible role for estrogens in the CK response (7). Moreover, research inanimals has indicated a role for estradiol in reducing membrane permeability following damaging exercise. Forexample, Amelink et al. (8) and others (9-11) have concluded that higher E2 concentrations decreased serumCK activity following stimuli that induced muscle damage. Recently, female rats demonstrated lower levels ofhistological muscle damage than male rats following eccentrically biased downhill running (1).
Research in humans on the direct role of estradiol in creatine kinase activity is limited. Buckley-Bleiler et al.
(12) and Miles and Schneider (13) found no effect of E2 on CK activity following exercise, but both studies areassociated with limitations including exercise intensity and E2 variability, respectively. Hayward et al. (14)reported that women taking an oral contraceptive had lower CK activity following eccentric muscle actionscompared to controls, but the results were limited due to high variability in both CK activity and E2concentration. Thompson et al. (15) reported that estradiol concentration was not related to CK releasefollowing stepping exercise, despite the fact that the group with higher E2 concentrations had a peak CKactivity value six times lower than the low E2 group. In an attempt to clarify these discrepancies, the purpose ofthe present investigation was to assess the role of oral contraceptive (OBC) use and circulating estradiolconcentration in creatine kinase activity and muscle soreness following strenuous eccentric exercise.
METHODS
Subject Characteristics
Ten college-aged (19-23 yr) females read and signed a written informed consent approved by the Institutional
Review Board at The University of Montana. The subjects were moderately active physical education students
who had not previously engaged in an eccentric exercise program or activities associated predominantly with
eccentric muscle actions. The subjects were grouped according to their use (OBC, n=5) or non-use (NOBC,
n=5, free from use for at least 6 months) of an oral contraceptive. All OBC subjects used a 28-day prescription
containing ~30 mg Ethinyl E2. Exercise testing occurred according to the menstrual cycle phase associated
with low E2 concentration. Thus, the OBC group was tested approximately 8-2 days prior to menses in
accordance with the OBC-induced progestin phase. The NOBC group was tested approximately 0-2 days
following menses in accordance with early follicular levels of E2.
Eccentric Exercise Protocol
All subjects completed a unilateral eccentric hamstring exercise protocol using a KIN-COM isokinetic
dynamometer (Chattanooga, TN). Prior to the experimental protocol, all subjects performed a concentric one-
repetition maximum (1RM) of the dominant hamstrings, which was used as a ‘goal’ force output for the
eccentric exercise protocol. Before each test, the subjects performed a 3-min warm-up on a stationary cycle
with light resistance and 5-10 min of static stretching of the hamstrings and quadriceps to help prevent injury.
The 1RM consisted of repeated bouts (6-8) of one concentric repetition at steadily increasing resistance until
failure, with 1-2 min rest periods between each bout. After one failed attempt at the final resistance, the highest
resistance attained was defined as the 1RM.
Following the 1RM test, subjects rested for 5 min before performing five sets of 10 eccentric muscle actionswith both the dominant and non-dominant hamstrings. Each repetition required that the subject maximally resistthe KIN-COM's movement arm starting from full knee extension, through a range of motion approximating 90oat an arm velocity of 60o/s. The repetitions were performed in the seated position with the subject's non-dominant leg and upper body secured to the KIN-COM device. The graphical output corresponding to theelicited force output was in view of each subject and subjects were verbally encouraged to meet or surpasseither the concentric 1RM force level or the level of the previous eccentric action throughout each set. Setswere separated by 2-3 min to allow recovery. The exercise protocol was repeated for the non-dominant legafter a 5-min rest period using the same conditions. Verbal encouragement was provided for all subjects by the Estradiol and Exercise-Induced Creatine Kinase Activity
test administrator in an attempt to elicit maximal efforts at each muscle action, such that the resistance attainedwas equal to or higher than the dominant leg's concentric 1RM. The same individual administered all strengthtests and was blind to the OBC condition of each subject.
Subjects were asked to refrain from physical activity for 48 hr prior to, and 72 hr following the exercise
protocol to decrease the influence of physical activity on the serum CK activity elicited by the exercise
protocol. Baseline serum CK activity of approximately 50 U/L indicated that no prior activity occurred in either
group (16).
Blood Sampling and Analysis
Blood sampling occurred immediately before exercise (pre-exercise), immediately after exercise (post-exercise)
and at 24, 48 and 72 hr post-exercise. Whole blood was allowed to clot at room temperature and the serum
was stored at -20oC until analysis. Serum CK activity was measured using standard spectrophotometric
techniques (U/L at 30oC, assay kit #47-20, Sigma Corp., St. Louis, MO). Serum E2 was measured using
radioimmunoassay (pg/ml at 30oC, assay kit # TKE21, Coat-A-Count, Diagnostic Products, Los Angeles, CA)
with a detection limit of 8 pg/mL. All subjects had E2 concentrations >8 pg/mL. All analyses were performed in
duplicate.
Muscle Soreness
Perceived muscle soreness was assessed pre-, immediately post- and 24, 48 and 72 hr post-exercise. The
questionnaire used for this assessment was adapted from a questionnaire developed by Morgan et al. (17). The
questionnaire required the subject to recall how her muscle 'felt' throughout the morning prior to the workout
(pre-exercise value) or blood sampling (post-exercise) time points, and then asked the subject to assign a
number from one to seven based on her perception of muscle soreness. Each subject assigned a numeric value
corresponding to her perceived soreness, with '1' referring to 'very, very good' and '7' referring to 'very, very
sore.' Subjects were allowed to use half points to more accurately express their level of soreness.
Statistics
Statistical analysis included a priori planned comparisons between cell means across time and OBC status for
both CK activity and soreness. An a priori planned comparison for the main effect of time utilized a 2 x 5 split
plot (1 within = time; 1 between = OBC status) repeated-measures ANOVA. Student's t-tests were performed
on all other between group comparisons. Statistical significance was accepted at p<0.05. Data are presented as
means±SE.
Circulating E2 concentrationswere low in both groups Table 1. Descriptive data for OBC (n=5) and NOBC (n=5) groups
Group
Mean E2
E2 Range
Eccentric Force
%Ecc/Con
OBC
NOBC
Data are means±SE. %Ecc/Con, percent of eccentric force versus dominant leg’s concentric one-repetition maximum (1RM). Estradiol (E2) concentrations in pg/mL at 30oC. *Significantly different from NOBC, p<0.01 For each leg, all subjects completed 50 unilateral eccentric muscle actions with mean force outputs at or abovethe dominant leg concentric 1RM (Table 1).
Serum CK activity remained at pre-exercise levels immediately post-exercise and began increasing at 24 hrpost-exercise with peak values occurring at 24 hr post-exercise for the NOBC group and 72 hr post-exercisefor the OBC group (Figure 1). The OBC group (lower E2) had significantly higher (p<0.05) CK activity at 48 Estradiol and Exercise-Induced Creatine Kinase Activity
Figure 1. Changes in serum CK activity before (Pre), immediately Post- (0 Pst), and 24, 48 and 72 hr following
eccentric exercise. *Significantly different from pre-exercise, p<0.05; †Significantly different from pre-exercise,
p<0.01; Data are mean CK (U/L at 30 ºC)±SE.
and 72 hr post-exercise when compared to the pre-exercise CK levels. CK values for the NOBC group (higherE2) declined slightly after 24 hr post-exercise and were not significantly different from pre-exercise values atany timepoint. The group x time interaction was significant (p=0.04).
Both groups reported a range of soreness ratings from 2.0 - 5.7 (scale=1-7), with the highest ratings occurringat 24 and 48 hr post-exercise (Figure 2). Soreness values increased significantly in both groups at all post-exercise timepoints (Figure 2; p<0.05). The NOBC group, which had a lower overall CK response to theexercise protocol, exhibited a higher mean rating of muscle soreness (4.4±0.3) following the exercise protocolcompared to the OBC (higher CK response) group (3.8 ± 0.3) (p<0.01).
DISCUSSION
The results of the current investigation indicate that estradiol (independent of OBC use) has a role in creatinekinase activity in humans following damaging exercise, as the subjects with higher E2 levels exhibited nosignificant change in CK activity, while subjects with lower E2 levels exhibited a significant increase in CKactivity at 48 and 72 hr post-exercise. Further, these results support prior research pointing to the lack of anassociation between perceived muscle soreness and CK activity (15,18).
CK activity has been shown to be lower following strenuous exercise in females compared to males (6,7).
Following a marathon, serum CK activity was five-times higher in males versus females (7). The role of E2 wasnot directly assessed in those investigations. Amelink et al. (8-10) completed a series of investigations in ratsboth in vivo and in vitro indicating that E2 reduced the increase in serum CK activity normally observedfollowing damaging exercise or electrical stimulation. Further, the data of Bar et al. (19) showed the protectiveeffects of E2 treatment in male and ovariectomized female rats. Following a 2-hour treadmill run, CK activity Estradiol and Exercise-Induced Creatine Kinase Activity
increased nearly 4-fold for the male and ovariectomized female rats; however, male and ovariectomized femalerats treated with E2 showed less than a 1-fold increase in CK activity in response to the same protocol.
Figure 2. Mean soreness ratings (± SE) for both OBC and NOBC groups (scale = 1-7) at pre-exercise (Pre),
immediately post-exercise (0 Pst) and 24, 48 and 72 hr post-exercise. *Significantly different from pre-exercise,
p<0.05.
Recently, Komulainen et al. (1) reported the first direct evidence of gender differences in muscle damagefollowing eccentric exercise, although estrogen levels were not assessed. They observed no changes in musclemicroarchitecture immediately following and six hours after downhill treadmill running in female rats, whilemale rats demonstrated muscle damage immediately after and six hours after the exercise bout (1). Possiblegender differences in muscle damage in response to strength training were recently reported in humans (20).
Human research addressing the role of E2 in CK activity following exercise has been limited. Buckley-Bleiler etal. (12) found no effect of E2 on CK activity following an isometric exercise protocol. The results of that studyare limited, as CK activity did not increase following the exercise protocol, staying within normal resting levels(highest post-exercise CK = 75 U/L, normal level <100 U/L) (12,16). Miles and Schneider (13) examinedserum CK activity in 15 females following 40 min of stepping exercise. The exercise protocol was notstandardized to menstrual cycle phase and E2 concentrations varied widely between subjects (10-359 pg/mL).
The researchers reported no relationship between E2 and post-exercise CK activity, but noted that low samplesize and data variability may have contributed to that result (13). In an abstract, Hayward et al. (14) reported17 and 59-fold increases in CK activity in oral contraceptive users and non-users, respectively, followingeccentric muscle actions. The variation in these values suggests a relationship between E2 and post-exercise CKactivity; however, because of the variability in circulating E2 levels (50-220 and 60-400 pg/mL for OBC usersand non-users, respectively) (14), the relationship between E2 and CK activity is difficult to interpret.
Thompson et al. (15) examined the effects of OBC use on muscle damage following 50 min of steppingexercise. The researchers measured E2 concentrations in both OBC and NOBC groups, similar to the present Estradiol and Exercise-Induced Creatine Kinase Activity
investigation. In contrast to the current study, the researchers tested subjects during the phase of the menstrualcycle that maximized the E2 environment. Similar to the results reported here, CK activity increased more inthose women with low E2 (OBC, 17 pg/mL) compared to those with higher E2 levels (NOBC, 116 pg/mL).
The peak CK activity was 715 vs 161 U/L for the two groups, respectively. However, baseline CK levelsdiffered substantially in the two groups (123 vs. 51 U/L, respectively) and statistical significance was notattained leading the researchers to conclude that E2 was not related to CK activity following strenuous exercise(15).
The results of the current investigation indicate that estradiol can affect serum creatine kinase activity followingdamaging eccentric exercise. Subjects with low E2 concentrations (OBC - 11.6 pg/mL) demonstratedsignificantly higher CK activity than baseline at 48 and 72 hr post-exercise. The women with higher E2concentrations (NOBC - 27.2 pg/mL) did not show a statistically significant increase in CK activity followingexercise at any time point. The large increases in CK activity reported in the current study are much higher thanthose reported previously (12,15), likely the result of the strenuous nature of the exercise protocol used in thepresent study, which was designed to elicit muscle damage. One limitation of the current investigation is a lowsample size; however, the sample size reported here is similar to that of Thompson et al. (15).
The results presented here also point to the limitations of CK as a marker of muscle damage. While CK hasbeen associated with both soreness and muscle edema (21,22), direct associations between serum CK activityand histological and ultrastructural muscle damage remain unremarkable (2,23) and inter- and intra-subjectvariability are high (24,25). As such, some researchers have moved from associating CK with 'muscle damage'to 'membrane damage' or 'membrane permeability' (2,18). The results of the current study and those of Milesand Schneider (13) and Thompson et al. (15) suggest that CK variability limits the ability to assess statisticaldifferences in CK activity.
The present results support prior research indicating a lack of association between perceived muscle sorenessand serum CK activity (15,18). Perceived muscle soreness (mean value) was higher in the NOBC groupcompared to the OBC group (4.4 vs. 3.8; p>0.05), although both groups showed a similar significant increaseover time compared to pre-exercise values (Fig. 2). These results contradict the CK activity results of eachgroup, as the NOBC group showed a lower increase in CK response over time compared to the OBC group.
The results, however, do support data presented by Thompson et al. (15) that indicated lower ratings ofsoreness (increased pain threshold) in women taking an OBC. The present results point to the inadequacy ofCK as an indicator of muscle soreness, and show that the relationship between membrane damage (interpretedfrom serum CK activity) and actual soreness is tentative at best.
CONCLUSIONS
The present results indicate that circulating levels of estradiol affect post-exercise creatine kinase activityindependent of oral contraceptive use. Further, serum CK activity does not necessarily correspond to perceivedsoreness levels following damaging exercise, although OBC status may be related to soreness ratings. Futurework should determine the effects of E2 administration on serum CK activity in males following damagingexercise to verify the present results. The results presented here clearly suggest that investigators must carefullycontrol for the possible effects of estradiol when assessing serum creatine kinase activity in females.
Estradiol and Exercise-Induced Creatine Kinase Activity
ACKNOWLEDGMENTS
The authors thank the volunteers for their participation in this study. Funding for this investigation was
awarded to B. C. Ruby from the University Grant Program, Office of Research Administration, The University
of Montana. S.M. Roth’s current address is A300 Crabtree Hall, Department of Human Genetics, University of
Pittsburgh, Pittsburgh, PA 15261.
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Address for correspondence: Brent C. Ruby, Human Performance Laboratory, Department of Health and
Human Performance, The University of Montana, Missoula, MT 59812, USA; email: ruby@selway.umt.edu

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