J Neurol (1990) 237:234-238

Joumal of

Neurology

© Springer-Verlag 1990

Myoglobin is a sensitive marker of increased muscle membrane vulnerability M. F. Driessen-Kletter, G.J. Amelink, P. R. Bär, and J. van Gijn University Department of Neurology, Utrecht, The Netherlands Received November 24, 1989 / Received in revised form March 22, 1990 / Accepted April 3, 1990

Summary. Changes in muscle proteins in serum after exercise were studied to evaluate the use of such proteins as indicators of increased nmscle m e m b r a n e vulnerability. Seventy-one w o m e n were asked to perform bicycle exercise for 45 min at a moderate load; four proteins (creatine kinase - CK, myoglobin - Mb, aldolase - Ald and pyruvate kinase - PK) were measured in serum up to 24 h after exercise. Twenty-one women were carriers of D u c h e n n e ' s muscular dystrophy ( D M D ) ; these are known to show an elevated serum CK activity at rest, as well as an increased C K response after exercise. Fifty w o m e n without a family history of neuromuscular disease were tested to obtain normal values: they showed a small p e a k (18%) of C K activity 8 h after exercise, and an even smaller peak of Mb (9%) I h after exercise. The m e a n post-exercise increase for both CK and Mb in the 21 D M D carriers was significantly higher than in controls; the m a x i m u m of Mb, on average 70% of baseline levels, was reached 1 h after exercise and was higher than that for C K (48%), which was reached 8 h after exercise. It is concluded that myoglobin levels after exercise a r e a good index of increased vulnerability of the muscle m e m b r a n e . Key words: Muscle damage - Creatine kinase - Myoglobin, exercise - Duchenne muscular dystrophy - Exercise test

Introduction Several muscle proteins can act as indicators of increased permeability of the muscle m e m b r a n e : examples are creatine kinase (CK), myoglobin (Mb), aspartate aminotransferase (AST) and lactate dehydrogenase ( L D H ) [2, 28]. The appearance of one or more of these proteins is Offprint requests to: P.R. Bär, Research Laboratory Neurology, AZU, Room G03.228, Postbox 85500, NL-3508 GA Utrecht, The Netherlands

generally considered to reflect muscle damage that occurs in certain diseases, for example, after myocardial infarction [19, 24] or in patients with neuromuscular disease [5, 10, 18]. A raised serum CK activity at rest, observed in some but not all carriers of Duchenne's muscular dystrophy ( D M D ) , presumably reflects a partial and subclinical expression of the same defect that is manifest in patients with D M D , leading in carriers to a spontaneous leakage of C K into the circulation. Elevation of CK after exercise is a well-known phen o m e n o n in healthy subjects - in humans [7] as well as in animals [2, 3]. The increased permeability of the muscle m e m b r a n e in D M D carriers is manifested by the observation that even normal daily activity can result in a raised CK. H e r r m a n n et al. found that D M D carriers reached a significantly higher peak after exercise than controls [14]. This difference was used as an aid to detect carriers, first in D M D and tater in Becker's muscular dystrophy [13]. The authors claimed that their "muscle provocation test" was more sensitive than detection based on the use of serum CK levels at rest, which is known to miss 3 0 % - 5 0 % of D M D carriers [29]. A recent development is the use of D N A analysis: these tests, based on D N A polymorphisms, are very sensitive but are not always available for routine diagnosis. Bakker et al. reported that 75% of carriers could be detected with a reliability better than 98% [6]. Still, the birth of a Duchenne boy has been reported, despite prenatal D N A analysis [9]. Several other indices of muscle damage have been studied, orten in combination with CK, with the aim of detecting D M D carriers; for example, myoglobin [19, 20] and pyruvate kinase [12, 35]. Mb is an especially attractive candidate since it is unique to skeletal and heart muscle [25]. It has been proved to be a useful index of muscle damage in a variety of neuromuscular diseases [20]. Hypermyoglobinaemia has been demonstrated in 63% of D M D carriers, including those with normal CK values at rest [1], and also in female relatives of D M D patients [19, 23], but neither of these studies succeeded

235 in reliably d e t e c t i n g carriers b a s e d o n the d e t e r m i n a t i o n of b a s e l i n e M b levels. T h e m e a s u r e m e n t of s e r u m pyruvate k i n a s e (PK) a n d C K activity seems less useful t h a n m e a s u r i n g C K a l o n e , as the c o m b i n a t i o n of P K a n d C K d e t e c t e d o n l y 45% of 39 carriers [12]. C a r b o n i c a n h y d rase ( C A I I I ) has r e c e n t l y b e e n a d v o c a t e d as a s e r u m m a r k e r for muscle d a m a g e ; C A I I I is e v e n m o r e specific t h a n M b , as it is p r e s e n t o n l y in skeletal muscle [32]. H o w e v e r , w i d e - s p r e a d use of this m a r k e r is h a m p e r e d b e c a u s e at p r e s e n t , t h e r e is n o c o m m e r c i a l l y available m e t h o d for q u a n t i f y i n g C A I I I in s e r u m . B a s e d o n earlier o b s e r v a t i o n s that h a d s h o w n that M b levels increase b e f o r e C K levels do a n d fall m o r e r a p i d l y after exercise [4, 10] or after m y o c a r d i a l infarction [18], we h y p o t h e s i z e d that M b m i g h t n o t o n l y be a m o r e sensitive m a r k e r for subclinical muscle d a m a g e t h a n the e n z y m e s m e n t i o n e d earlier, b u t also w o u l d req u i r e s h o r t e r o b s e r v a t i o n periods. T o test this h y p o t h e sis, we s u b j e c t e d h e a l t h y w o m e n to a muscle p r o v o c a t i o n test: D M D carriers, b e c a u s e it is k n o w n that their muscle m e m b r a n e is m o r e v u l n e r a b l e , so that they w o u l d act as positive controls, a n d n o n - c a r r i e r s to o b t a i n the " n o r m a l " p a t t e r n of p r o t e i n efflux after exercise.

Materials and methods The test persons were asked to refrain from sports and other strenuous physical activity on the day before the test and on the morning of the test; a light breakfast was allowed before the exercise test. Exercise was performed on a bicycle ergometer for 45 min at 1.6 W/kg body weight without previous training; the pedalling frequency was kept between 60 and 80/min. During the test the pulse rate was kept below an age-dependent maximum [80% of (220-age in years) beats/min], if necessary by adjusting the workload (this was only the case for two individuals). A cardiologist was consulted when subjects were older than 45 years. Venous blood samples were taken before the test, and 1, 2, 4 and 8 h after the test. No anticoagulant was used, and the blood was centrifuged immediately. The activity of CK, Ald and PK in serum was assessed as described before [2]. The concentration of Mb in serum was mea-

sured by means of a commercially available radioimmunoassay method (Ria-mat, Byk-Sangtec Diagnostica, Dietzenbach, FRG). Two groups participated: the control group consisted of 50 women (mean age 31 years, range 22-48) without a family history of neuromuscular disease. The carrier group consisted of 21 women (13 definite and 8 probable carriers according to the criteria of Walton [33], mean 43 years, 28-64). We used 95% confidence intervals (CI) to detect significant differences in and between groups according to recommendations of Gardner and Altman [11].

Results

Control subjects T h e results of the controls are s h o w n in T a b l e l A . C K i n c r e a s e d steadily after exercise, r e a c h i n g a p e a k in all b u t four controls 8 h after exercise; the a v e r a g e i n c r e a s e was 18% ( + 8 . 1 U / 1 , 95% CI 5 - 1 1 U / 1 , P < 0 . 0 0 1 ) . C K was also m e a s u r e d 24 h after the test in 27 of the controls: the activity h a d r e t u r n e d to starting values. M b was i n c r e a s e d at 1 h after cycling in 26 w o m e n , w h e r e a s in the o t h e r 24 w o m e n it r e m a i n e d c o n s t a n t or s h o w e d a decrease. T h e average increase, 9 % , was small b u t significant ( + 3.1gg/l, 95% CI 0 . 5 - 5 . 8 , P < 0.05). This p e a k h a d d i s a p p e a r e d 2 h after the test, a n d M b levels e v e n significantly d e c r e a s e d 4 a n d 8 h after exercise ( T a b l e 1, Fig. 1). T h e next day, M b levels were b a c k to n o r m a l (n = 26, data n o t shown). A l d o l a s e s h o w e d a n increase 8 h after the test of 20% ( + 0 . 2 8 U / 1 , 95% C I - 0 . 0 4 - 0 . 6 0 , NS); P K did n o t show significant c h a n g e s in the 24 h it was m o n i t o r e d .

DMD carriers T w e n t y - o n e carriers were tested; their results are s h o w n in T a b l e 1B. T h e values at rest of CK, M b a n d A l d were significantly different f r o m those of the c o n t r o l g r o u p at rest ( P < 0 . 0 0 1 ) ; o n l y P K did n o t differ. N i n e w o m e n h a d a n o r m a l C K at rest (i.e., < 105 U/l, n o r m a l C K ) , 12

Table 1. Serum levels of four muscle proteins before and after exercise in controls and Duchenne carriers Index

t=0

A. Controls CK Mb Ald PK

(50.0 (32.9 (2.37 (32.9

+ 22.3) _+ 9.2) + 0.85) _+15.0)

B. Carriers CK (118 _+84) Mb (70 _+40) Ald (3.53_+ 1.36) PK (32.9 +20.4)

t= 1

t=2

t=4

t=8

N

+ 3.9"** +3.1" +0.06 +0.1

+ 4.5"** -1.1 -0.09 -0.4

+ 7.3*** -4.8** -0.04 -0.6

+ 8.1"** -6.6*** +0.28* -0.8

50 50 50 39

+10 + 49*** - 0.31 + 0.3

+17 + 38*** - 0.05 + 0.4

+31 + 25* - 0.07 - 2.1

+60* + 19 + 0.21 + 7.2

21 21 21 20

Column 1 (Index) contains the four proteins (CK - creatine kinase, Mb - myoglobin, Ald - aldolase, PK - pyruvate kinase) that were measured at rest (t = 0); activities (CK, PK and Ald, in units per liter) or concentration (Mb, in gg/l) before exercise are presented as mean + SD, under the heading t = 0. The changes in absolute value (U/1 or gg/l, "+" indicates an increase, " - " indicates a decrëase) at i h, 2 h, 4 h and 8 h after 45 min of exercise of control subjects (A, Controls) and Duchenne carriers (B, Carriers) are given under t = 1, 2, 4 and 8. The results of statistical analysis are given: * P < 0.05, ** P < 0.01 and *** P < 0.001, based on the confidence interval of the differences between groups; each individual served as its own control. The column N gives the number of persons in each group

236 200

a

b

Ti

180

160

Fig.la, b. The changes in creatine kinase activity (a) and myoglobin concentration (b) in serum of 50 control women (open circles) and 21 DMD carriers (filled circles) after 45 min of exercise on a bicycle ergometer. The changes are plotted as percentage of the values immediately before exercise (t = 0). The bars indicate the SEM, the dashed line indicates the starting level (100%)

140

120

100

0

2

4

6

8

2

4

6

8

HOURS

had a raised C K at rest (high CK); women in this latter category (12/21 = 57%) would have been classified as D M D carriers, based on their CK values at rest alone. The average CK increase in the carrier group was significant only at 8 h after exercise and clearly larger than that of controls (48% vs 18%), with a rather large variation within the group ( + 60, 29 U/l, mean, SEM); no difference was observed between the responses of the high C K and normal C K carriers. Only four w o m e n showed a decrease in C K activity, two from the high CK subgroup and two from the normal CK subgroup. Figure 1 shows that the profile of the CK increase in carriers and controls was comparable up to 1-2 h after exercise, but that the leakage of C K seemed to persist in carriers, whereas it reached a plateau at 2 - 4 h postexercise in controls. The increase in Mb was clearly higher in the carrier group than in the controls, in absolute terms (49, 9.4 vs 3.1, 1.3 ~tg/1), as weil as in relative terms (70% vs 9%, Table 1)" moreover, the Mb increase showed a smaller variation than that of CK. Again, only small and insignificant differences were found between the high CK and normal CK subgroups. A n o t h e r difference with CK was that the Mb peak occurred almost immediately (1 h) after exercise, instead of 8 h after exercise. Only 2/21 carriers (one from each subgroup) showed a decrease in their serum Mb concentration i h after exercise (vs 24/50 controls). Figure 2 shows that the profiles of Mb efflux from muscle of controls and carriers were completely different. The average Mb concentrations in carriers decreased after one hour but, other than in controls, the baseline levels were not reached again over the 8 h monitored. Apparently, the damaging effect of exercise was more outspoken in carriers and lasted longer than in controls: four carriers even showed a delayed rise of Mb 4 - 8 h after the test, a p h e n o m e n o n that was never observed in controls. The increase in aldolase activity in serum after exercise was not different for carriers and controls: although the starting value was significantly higher in carriers (3.53 vs 2.73 U/l, Table 1), both groups showed a small

increase after 8 h. PK was the same for both groups both at rest and after exercise, with a large variation.

Discussion

Our main finding is that myoglobin in serum is a sensitive index for muscle damage, which rapidly appears in the circulation after exercise and reaches a peak value hours earlier than CK. The standardized exercise protocol that we used differed slightly from that of H e r m a n n and Spiegler [14]. Our subjects exercised at 1.6W/kg body weight instead of 2W/kg body weight, because a pilot study had shown that 2 W/kg could not be sustained by all women for 45 min, especially not by the somewhat older or more obese subjects. This difference in workload might explain that we, unlike H e r m a n n and Spiegler, did not find a significant difference in the post-exercise increäse in CK activity between controls and carriers. A more important difference between the two studies is that we measured not only CK, but also myoglobin. Konagaya et al. [23] have measured Mb after 3 min of exercise (70 W) and found no change of C K levels, and in only 5 of 11 carriers an increase in Mb. The difference from our observations are explained by the fact that they used a very small workload (3.5W) compared to our workload (60-90 W), and did not wait long enough after exercise to find a CK rise, if at all present after so littte work. In our standardized muscle provocation test, the Mb increase 1 h after exercise was significantly larger in carriers (17 out of 21) than in controls. It seems that measuring Mb as well as CK allows a less demanding test, without losing diagnostic value as a test for subclinical muscle vulnerability. CK is widely used as a m a r k e r of muscle damage: its activity in the serum of patients with muscular dystrophy in general is probably continually elevated; very high values are observed in D M D patients, in patients with polymyositis and in athletes involved in long distance

237 events. CK may also be elevated in asymptomatic carriers without previous exercise. CK is released slowly from skeletal muscle after exercise, and it has a long half-life in serum: from 28 h up to a week [17, 21, 23]. It is therefore probably a good indicator of chronically increased muscle membrane permeability. In contrast, Mb is released from muscle much sooner after infarction [18], or exercise [4, 23], and is rapidly cleared by the kidneys (half-life 6.7 la, [26]). This may be explained partly by the small size of the Mb protein (MW 17,500) and partly by its negligible degree of binding to serum proteins [30]. As a result, a peak of Mb occurs shortly after damage and disappears rapidly. The difference between the dynamics of CK and Mb is underlined by the observation of Konagaya et al. [23], that both CK and Mb show a diurnal rhythm in boys affected with D M D : CK varied only between 2000 and 3400U/1, whereas Mb went from about 50 ng/ml (early morning) to over 600 ng/ml (afternoon), a more than tenfold increase. Borleffs et al. compared the characteristics of CK and Mb release into serum of patients with polymyositis or dermatomyositis and concluded that whereas Mb is an excellent marker for acute damage, CK is bettet suited for monitoring long-term changes, such as the effect of corticosteroid therapy in these patients [8]. A t this m o m e n t we have no explanation for our finding that in controls Mb, after a rapid initial rise, falls below resting levels; this was not seen in carriers and may be used to differentiate between carriers and noncarriers or, more generally between people with and without an abnormal membrane vulnerability. It seems that the rate at which Mb decreases after i h is similar for carriers and controls, but that they differ only in the absolute amount that is released from muscle, perhaps because the membrane recovers more slowly in carriers. This suggests that the way in which Mb is cleared from the circulation is the same in both groups and that only the larger amount that leaks from muscles of carriers distinguishes between the two groups. In this study we used Mb for detecting an abnormal vulnerability of muscle membrane in D M D carriers at a level where CK determinations were inconclusive: about half the carriers showed a postexercise CK response that was in the same range as that of controls. In contrast, the Mb response of only four carriers (two from each subgroup) could not be distinguished from that of the controls. These observations are in keeping with our initial hypothesis, namely, that Mb is a more sensitive marker of muscle damage after exercise than CK. Our experiments do not indicate the cause of this difference. Dystrophin, the product of the D M D gene [15], may be involved in the coupling of myofilaments and triads during contraction [16]. Alternatively, dystrophin, which is reported to be similar to spectrin [22, 34], may have spectrin-like activity and thus be involved in maintaining the integrity of the plasma membrane during contraction [34, 36]. In both cases it is plausible that a deficiency of this protein leads to a decreased membrane stability and that Mb, being the smaller molecule of the two, leaks from the damaged tissue first. We are currently studying the Mb effiux in patients with Becker's muscular dys-

trophy, a disease in which there is a defect of the same gene as D M D , but less dramatic than D M D . The exercise test we describe here can be used as a general test for assessing increased muscle membrane fragility, occurring under circumstances such as mitochondrial diseases [10], during drug treatment (e.g. with vastatines [27, 31]) or during different stages of training in sportsmen. The substantial difference in mean age between the controls and the carrier group (31vs 43 years) forces us to be cautious in our conclusions, even though there was no correlation between age and resting CK or Mb, or between age and the postexercise increase in CK or Mb. In view of the sophisticated techniques now available to detect D M D carriers, this may no longer be the primary use of the exercise test. We suggest that myoglobin is excellently suited for monitoring any form of acute muscle damage or increased muscle membrane permeability, be it after training or as a consequence of a disease or medication.

Acknowledgements. The authors thank all of the women, the DMD carriers and healthy volunteers, who participated in this study for their cooperation; the Vereniging Spierziekten Nederland, Mrs. M. J. van Tol-de Jager, Mrs. P. F. Ippel and Prof. H. F. M. Busch for information on carriers, the Prinses Beatrix Fonds and Byk Holland for supplying funds for travelling expenses.

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