Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in progressive muscular dystrophies

Journal of the Neurological Sciences, 1978, 36:349-362 © Elsevier/North-Holland Biomedical Press

349

S E R U M P Y R U V A T E - K I N A S E (PK) A N D C R E A T I N E - P H O S P H O K I N A S E (CPK) I N P R O G R E S S I V E M U S C U L A R D Y S T R O P H I E S

MAYANA ZATZ1,4, LARRY J. SHAPIRO1, DAVID S. CAMPION 2, ETSUKO ODA a,~ and MICHAEL M. KABACK1 1Department o f Pediatrics, Division o/ Medical Genetics, U.C.L.A. School of Medicine, Harbor General Hospital Campus, Torrance, Cal. (U.S.A.) 2Department of Medicine, U.C.L.A. School of Medicine, Center for Health Sciences, Los Angeles, Cal. (U.S.A.) 3Department of Medicine, Division of Hematology, U.C.L.A. School of Medicine, Harbor General Hospital Campus, Torrance, Cal. (U.S.A.) 4Laboratdrio de Gendtica Humana, Instituto de Bioci~ncias, Cidade Universitdria, Caixa Postal 11.461, S~o Paulo (Brazil) (current address) SThird Department of Internal Medicine, Yamagushi University of Medicine, Ebe Yamagushi, 755 (Japan) (current address)

(Received 17 October, 1977) (Accepted 19 January, 1978)

SUMMARY P K and C P K have been determined in the serum from 208 individuals including 70 normal controls (61 adults and 9 children) and 138 patients with a variety of neuromuscular disorders. In adult controls the mean activity ( ± S E ) for P K is 1.2 ± 0.05 #mol/ml/h. In normal children P K activity was about twice as high as in normal adults and decreases with increasing age. In 26 patients with Duchenne dystrophy the range of serum P K was 4.0-150.4 and in 17 individuals with the Becker type, 3.0 to 148.7. All had elevated P K and C P K levels, Eighteen of 20 patients with the facio-scapulo-humeral (FSH) from of muscular dystrophy had increased P K while only 9 had elevated CPK. Regression analyses have shown an inverse correlation between P K levels and age (or degree of disability in DMD). Kinetic and electrophoretic studies indicate that the P K isozyme found in the serum from affected patients and from heterozygotes for the D M D gene is mainly the M1 type PK, which is the only P K isozyme found in skeletal muscle and brain and the major component from myocardium.

This work was supported in part by a grant from Conselho Nacional de Desenvolvimenot Cientifico e Technol6gico, (CNPq), and The National Foundation-March of Dimes Genetics Center Grant, C-114.

350 It is concluded that the concomitant use of serum PK and CPK determinations may be very useful for clinical diagnosis of certain types of muscular dystrophies and may be of great importance in the management of affected patients and their families.

INTRODUCTION In 1959, Ebashi, Toyokura, Momoi and Sugita first described elevated serum CPK activity in patients with progressive muscular dystrophies. Many reports since have supported these findings, particularly in the X-linked forms of muscular dystrophy [Duchenne (DMD) and Becker (BMD)]. In 1973, Harano, Adair, Vignos, Miller and Kowal reported increased serum pyruvate-kinase (PK) activity in 20 patients with DMD and in 5 patients with limbgirdle muscular dystrophy (LGMD). Extending these observations, Alberts and Samaha (1974) found elevated serum PK levels in 43 of 51 patients with 3 types of muscular dystrophy [29 DMD, 7 facio-scapulo-humeral (FSH) and 7 myotonic dystrophy I.MyD) and in 15 of 26 female relatives of D M D patients]. Hence, the determination of serum PK activity provides a potential additonal diagnostic parameter in patients with suspected muscular dystrophy. Therefore, further characterization of the serum PK activity in these patients is desirable. Several different PK isozymes are present in human and other mammalian tissues. Electrophoretic and immunological studies (Marie, Kahn and Boivin, 1976) have distinguished 2 groups of pyruvate-kinases. The first group consists of an L type (major isozyme in liver) and of an R type (erythrocyte PK). The second group has been designated as "M" type. In adult tissues at least 4 types of M-PK are found: M1 which is the only one found in skeletal muscle and brain and the predominant one in heart; M2 which exists in lung, adrenal, thyroid, fat cells, leukocytes, platelets, spleen and skin, and 2 intermediate forms designated as M8 and M 4 found in smooth muscle, testicle, kidney and other organs. Previous kinetic studies have identified serum PK in progressive muscular dystrophy as being of the M type (Harano et al. 1973). However, no efforts have been reported to further characterize which type of M isozyme is predominant in the serum of affected patients. We have conducted a comparative study of PK and CPK activities in sera from a large sample of patients affected with a variety of neuromucular diseases. It was our intention in these investigations to assess the usefulness of serum PK determination in the clinical diagnosis of various types of muscular dystrophy, to compare PK with CPK for this purpose, and to determine if the PK found in sera from affected patients is of muscular origin. MATERIAL AND METHODS One hundred thirty-eight patients with different types of myopathies attending the University of California, Los Angeles, Clinic for Neuromuscular Dystrophy, were included in this study. The patients' diagnoses were determined by clinical examimttion,

351 mode of inheritance and by electromyography and/or muscle biopsy. The control group consisted of 61 normal adults (42 females and 19 males) and 9 healthy children with no history of neuromuscular disease in their families. Blood samples were drawn by venipuncture without any anticoagulant and centrifuged shortly after collection. None of the individuals had undergone vigorous exercise during the 24 hr prior to sampling. Sera that showed any sign of hemolysis were discarded. Most of the PK and CPK determinations were performed in fresh sera within 3-6 hr of venipuncture. If not assayed the same day, the samples were aliquoted and frozen at --20 °C until the next day. Other aliquots were kept frozen for extended periods of time to evaluate the stability of serum PK activity. All PK and CPK determinations were done in duplicate. Assay of total PK activity was performed at 21 °C by measuring the decrease in absorbance of reduced nicotinamide adenine dinucleotide at 340 nm with a Gilford recording spectrophotometer. The assay was similar to that used by Valentine and Tanaka (1966) as modified by Alberts and Samaha (1974). The only difference was that normal saline was used to adjust the reaction volume instead of distilled water. All enzyme activities are reported as #mol/ml serum/hr. Homogenates of normal muscle and liver were prepared in 0.1 M KC1 containing 20 mM Tris-HC1 (pH 7.4), 5 mM MgSO4 and 1 mM EDTA, with a Brinkmann polytron homogenizer according to Inamura and Tanaka (1972). The tissues used were obtained incidentally at surgery (muscle) or at postmortem (liver) and stored at --20 °C. The liver was extracted in 5 volumes of cold buffer as above and the muscle in 10 volumes. Extracts were centrifuged at 17,000 x g at 40 °C for 30 min prior to assay or electrophoresis. Serum P K electrophoretic studies were done in polyacrylamide gels, according to the method of Inamura and Tanaka (1972). Gels were run for 5-6 hr instead of 2 or 3 hr. Sera from 20 patients with different types of muscle diseases and 3 heterozygotes of the Duchenne gene were studied by electrophoresis and compared with samples of normal muscle and liver homogenates. CPK activities were determined with Sigma kits in the same sera as those used for P K determinations. The colorimetric method used is described in Sigma Bulletin, 520 C (1976). The results are expressed in Sigma units. RESULTS In Table 1, the agregate serum PK and CPK activities in controls and patients with a variety of muscle disorders are summarized. Data from each individual are illustrated in Figs. 1 and 2. The mean P K activity in the adult control group was not significantly different between males and females. The CPK levels, however, were significantly higher in normal men than in normal woman (P < 0.005) as described previously (Rosalki 1967; Meltzer 1971). In 9 normal children (1 girl and 8 boys), ages 3-14 years, PK;levels were significantly higher than in normal adults (P < 0.001) and decreased with increasing age (r = 0.87). In the 2 children (1 boy and 1 girl) over 10 years old the P K levels were

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3.0-24.0 3.0-8.5 3.0-24.0 4.0-13.5

Range

M e a n + SE

Mean 4- SE

Range

C P K (Sigma units)

P K (/,mol/ml/h)

19 42 61 9

Number

By double sided Student t-test versus control ap < 0.05; bp < 0.02; ep < 0.001.

A. Controls 1. Adult males 2. Adult females 3. Total adults 4. Children (4-14 yr old) B. Patients 1. Duehenne 2. Beeker 3. Facio-scapulo-humeral 4. Limb-girdle syndrome 5. Myotonic dystrophy 6. Kugelberg-Welander and adult onset spinal muscular atrophy 7. Myotonia eonsenita 8. Polymyosifis 9. D~maatomyositis 10. Possible myositis 11. Unclassifiexl neuromuscular disorders

Individuals

S E R U M P K A N D C P K ACTIVITIES

TABLE 1

5 2 5 2 1 13

26 17 18 17 3

PK

5 1 6 1 2 7

26 17 9 17 3

CPK

7 2 6 2 2 14

26 17 19 19 5

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N u m b e r abnormal

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354 in the upper limit of the adult group (2.1/zmol/ml/hr). The CPK activities, although higher in children, did not differ significantly from the adult group. There was a progressive loss of serum PK activity in samples that had been stored frozen at --20 °C. Sera stored and reassayed after 2 months showed approximately 50 ~o decrease in PK activity. We have found, therefore, that it is essential for sera to be tested within 24 hr of venipuncture for maximum activity to be observed.

Duchenne and Becker dystrophy All patients with D M D and BMD (Fig. 1) had serum PK and CPK activities markedly higher than the upper limit of the control group (P < 0.001). Patients older than 14 years were compared with the adult male controls. There were no significant differences in mean PK values between the DMD and BMD groups. The CPK mean, however, was significantly higher among the D M D patients (P < 0.02). A strong correlation was observed between PK and CPK in both Duchenne (r == 0.77) and Becker dystrophies (r = 0.88).

FSH muscular dystrophy In the group of 20 patients with FSH muscular dystrophy (Fig. 1) 18 had elevated PK levels while only 9 had increased CPK activity. Only 1 patient had normal activity for both enzymes. In this group we included a 21-year-old female whose diagnosis of FSH was probable but not certain since the only clinical sign was some facial weakness, Her mother and her only brother are severely affected. The three of them had increased P K and normal CPK. In addition, a 25-year-old male, whose mother was severely affected, had an increased serum PK (3.6 #mol/ml/hr) with normal CPK (11.0 Sigma units). This individual was not included in this sample because he refused to be clinically examined.

Limb-girdle syndrome Among 21 patients with the limb-girdle syndrome (Fig. 1), 14 had the classical autosomal recessive form of limb-girdle muscular dystrophy and 7 a rarer neurogenic form with dysphagia which is inherited as an autosomal dominant trait as described by Furukawa, Tsukagoshi, Sugita, and Toyokura (1969). The mean PK, although higher in the autosomal recessive form (5.8 4- 1.2) was not significantly different from that in the dominant variety (4.0 ~ 1.5). For CPK, however, the mean was significantly greater in the classical autosomal recessive form (means: 43.5 ± 10.6 and 14.6 ± 6.6; respectively P < 0.025).

Other myopathies In the group of Kugetberg-Welander and adult onset spinal muscular dystrophies the PK and CPK means were significantly greater than in the control group (P < 0.001 for PK and P < 0.025 for CPK). In polymyositis, PK was greatly increased in patients who clinically exhibited weakness and there was a high degree of correlation with CPK activity (r = 0.99). One patient in this group, with a PK activity ranging from 28-43 #mol/ml/hr on 7 independent determinations had an unusual type of myositis. This

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Fig. 3. Regression analysis of serum PK versus age in patients affected by different types of muscular dystrophies: Becker muscular dystrophy (BMD) (A); limb-girdle muscular dystrophy (LGMD) and facio-scapulo-hurneral muscular dystrophy (FSH) (B); and Duchenne muscular dystrophy (DMD) (D); Regression analysis of serum PK versus degree of disability (Vignos scale) in DMD (D). (Observe the different scales in different patieot groups). was characterized by muscular weakness localized to the more distal muscles of the lower extremities, a muscle biopsy and E M G typical of myositis, and very high CPK levels (40-60 times normal).

Regression analysis Regression analyses of serum PK versus age are shown in Figs. 3a, b and d in four different groups of patients: D M D , BMD, FSH and L G M D . In the latter group, all 14 patients had the classical autosomal recessive form of the disease. In the group of D M D patients, serum P K was also correlated with the degree of disability, according to the Vignos scale (Vignos, Spencer and Archibald 1963). In all patients there was a decrease of serum PK with increasing age, which was most marked in the Duchenne and Beeker patients. In the D M D group, serum PK showed a higher correlation with the degree of disability (Figs. 3c and d), according to the Vignos scale, than with age (r : 0.97 and 0.83, respectively).

Kinetic studies Apparent Michaelis constants for serum P K enzyme activity (as a function of

357 TABLE 2 APPARENT MICHAELIS CONSTANTS (Km) OF PYRUVATE KINASE ACTIVITIES IN SERA FROM MUSCULAR DYSTROPHY PATIENTS, HETEROZYGOTES FOR THE XLINKED GENES AND CONTROL SERA AS COMPARED WITH NORMAL MUSCLE AND LIVER HOMOGENATES Source of material

Km a

Normal muscle Normal liver

6.5~x 10-5 M 5.0"/ 10 4 M

Serum from muscular dystrophy patients Duchenne b Duchenne b Duchenne Becker Facio-scapulo humeral Limb-girdle syndrome

3.7 5.3 5.4 7.6 4.2 6.2

x × x x × x

10-s M 10-5 M 10-5 M 10-5 M 10 5 M 10-s M

Serum from heterozygotes for X-linked genes Duchenne possible carrier 5.8 x 10 5 M Duchenne obligate carrier 7.0 x 10-3 M Becker obligate carrier 6.5 x 10-3 M Serum from normal controls (females) 1

2 3 4

3.6 2.9 4.3 4.9

x 10-s M x105M / 10-5 M ×105M

With respect to phosphoenolpyruvate (PEP) concentration. b Frozen at --20 °C for 2 months.

the c o n c e n t r a t i o n o f p h o s p h e o n o l p y r u v i c acid) were d e t e r m i n e d in the sera o f 6 patients, 2 h e t e r o z y g o t e s for the D M D gene, 1 carrier o f the B M D gene a n d 4 n o r m a l c o n t r o l s a n d c o m p a r e d with similar studies o f P K activities in n o r m a l muscle a n d liver h o m o genates. A l l kinetic studies were p e r f o r m e d on fresh sera with the exception o f two samples f r o m p a t i e n t s with D u c h e n n e d y s t r o p h y which h a d been frozen for 2 m o n t h s at - - 2 0 °C. The a p p a r e n t Michaelis c o n s t a n t s o f serum P K activity are s u m m a r i z e d in T a b l e 2 a n d the L i n e w e a v e r - B u r k plots are illustrated in Fig. 4. The a p p a r e n t K m s f o u n d in the sera o f the patients a n d heterozygotes (as well as in n o r m a l controls) are very similar to t h a t f o u n d in muscle a n d distinctly different f r o m t h a t in liver h o m o genates. I t is o f interest t h a t a l t h o u g h there was a decrease o f a b o u t 50 ~ in t o t a l serum P K activity in the s t o r e d frozen samples their a p p a r e n t K m s r e m a i n e d c o m p a r a b l e to those in fresh s a m p l e s a n d in muscle h o m o g e n a t e s . Also, the a p p a r e n t K m s o b s e r v e d in the muscle a n d liver p r e p a r a t i o n s were in the same range as the ones r e p o r t e d p r e v i o u s l y for purified enzyme p r e p a r a t i o n s (Bucher a n d Pfleiderer 1955; T a n a k a , H a r a n o , Sue a n d M o r i m u r a 1967). Electrophoresis P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (3.34 ~ ) o f serum P K was p e r f o r m e d in 20

358

4-

PATIENTS • • • NORMAL NORMAL

SERA PK LGMD Km = 6xlO-SM FSH K m = 4x 10"SM DMD K m = 5x 10"5M MUSCLE K m = 6.SxlO'SM LIVER K m = 5xlO'4M

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patients with different types of myopathies, 3 heterozygotes for the DMD gene and 2 normal controls. Patients included: 8 with DMD, 5 with BMD, 3 with LGMD, 2 with FSH and 2 polymyositis. In Fig. 5, serum PK is compared electrophoretically with muscle and liver PK. This system separates the PK isozymes into 3 bands, the M2, M1, and L types. PK is serum from a Duchenne carrier and a patient with DMD showed migration identical with the M1 band from muscle and distinctly different from the L band found in liver. In Fig. 6, PK activities in sera of 4 patients with different types of muscular dystrophies are compared with muscle homogenate. In each of these patients, the PK band is similar to the one found in muscle, i.e. Mz type. In 2 other patients with polymyositis, serum PK migrated in a single band similar in position to that found in normal muscle and in the serum of patients affected with muscular dystrophy. In sera with PK activity less than 7 #mol/ml/hr no visible band could be developed following electrophoresis. For this reason, it was not possible to study the electrophoretic mobility of normal serum PK with this system. With a 5-fold cencentration of normal serum from 2 controls, a very faint band similar to the Mx type could be seen. However, serum concentration might affect the electrophoretic mobility and therefore this observation must be interpreted with caution.

359

Fig. 5. Thin-layer polyacrylamide gel electrophoresis of serum PK from a D M D patient, and a D M D carrier compared with PK from normal muscle and liver homogenates.

Fig. 6. Thin-layer polyacrylamide gel electrophoresis of serum PK from patients with different types of muscular dystrophies as compared with PK from normal muscle homogenate.

360 DISCUSSION AND CONCLUSIONS Most of the comparative enzyme studies in progressive muscular dystrophies have shown that the determination of serum CPK is the best simple test for clinical diagnosis and carrier detection (Aebi, Richterich, Colombo and Rossi 1961 ; Pearce, Pennington, and Walton 1964; Zellweger, Durnin, and Simpson 1972, and many others). However, recent studies (Harano et al. 1973; Alberts and Samaha 1974) have suggested that serum PK determinations may be more sensitive than CPK both for diagnosis and for carrier identification (X-linked forms). Data presented here indicate that PK determinations are equally as reliable as CPK for the clinical diagnosis of the X-linked forms of muscular dystrophy (DMD and BMD) and may be more reliable in the clinical diagnosis of other types of muscular dystrophies, particularly the FSH type, in which the massive elevations of serum CPK seen in Duchenne and Becker dystrophies rarely occur. As demonstrated by this study and by other investigators (Hughes 1971), serum CPK levels are elevated in only about half of the patients with FSH muscular dystrophy. Some of the patients, however, have a very mild form of the disease which can be limited indefinitely to only one or two muscle groups (Walton and Gardner-Medwin 1974). The detection of these forms or ofpresymptomatic cases has obvious importance for family management and reproductive counseling. In our experience 18 of 20 patients affected with FSH muscular dystrophy had elevated serum PK activities while only 9 had increases in CPK. This finding is similar to the data reported by Alberts and Samaha (1974) who found elevated PK levels in 7 of 10 patients with FSH while none had elevated CPK activities. The existence of an apparently muscle specific PK isozyme (type M1) allows an investigation of the origin of circulating PK activity. In the present study PK found in the serum of patients, as well as in Duchenne carriers, was characterized as being the M1-PK isozyme. This is the main form of PK found in heart and the only one which occurs in adult muscle and brain (Marie et al. 1976). Apparent Kms of the 2 serum samples from Duchenne patients which had been frozen for 2 months were very similar to the fresh ones (and to the muscle preparation) in spite of the significant decrease of total PK activity. This observation suggests that serum PK in Duchenne patients is mainly of the M type and that the apparent stability of M-PK in frozen sera, as suggested in previous studies (Harano et al. 1973), cannot be confirmed. The kinetic studies done in the fresh sera of the 4 normal controls suggest that the M-type PK is also predominant in normal serum and therefore the differences found between the serum of affected patients as compared with normals might be only quantitative. The exact mechanism by which muscle enzymes enter the circulation is unknown but one possibility is abnormal permeability of muscle cell membranes to the relevant enzymes. The muscle enzymes elevated in the serum of affected patients are almost always found in sarcoplasm whereas membrane-bound enzymes do not appear in the serum (Rowland 1976). The question of why some patients with normal CPK have elevated PK remains unanswered. An abnormality of membrane permeability, however, might not affect all substances equally. This hypothesis has been supported by the ob-

361 servations that patients with Duchenne dystrophy have normal levels of the muscle constituent carnitine in both muscle and serum when compared to control subjects (Rowland 1976). An increase in the synthesis of PK in dystrophic muscle has also been suggested by Alberts and Samaha (1974). Alternatively, a simple difference in the serum half-life of CPK and PK might account for the observed differences in serum activity. The present study supports the hypothesis that the P K found in the serum of affected patients is derived from muscle through the same mechanisms as is the serum CPK. This is concluded because: (1) the kinetic and electrophoretic data show that the serum P K activity in patients as measured is mainly the M1 type; (2) the regression analysis shows a decrease of serum PK in affected patients with increasing age and with increasing duration of the disease as reported also to occur with CPK levels (Okinaka, Kumagai, Ebashi, Sugita, Momoi, Toyokura and Fujie 1961 ; Pearson 1963; Zatz 1973; Emery and Skinner 1976); (3) there is a high degree of correlation between PK and CPK activities in affected patients. In conclusion, the present study suggests that serum PK determination may be a more sensitive test than CPK for certain types of neuromuscular diseases (such as FSH muscular dystrophy) and therefore of critical importance in the clinical diagnosis and management of patients affected with such conditions and for family counseling. A possible extension of this study would be to the prenatal diagnosis of these conditions in early fetal life. It has been reported recently that the capability of prenatal diagnosis of D M D through intrauterine fetal blood sampling may be possible (Golbus 1977) and a successful application of such techniques utilizing CPK measurements has been reported (Haseltine, Mahoney, Hobbins and Caskey 1977). Our observations of an inverse correlation between serum PK activity and age in Duchenne patients suggest that this enzyme may be elevated in affected fetuses and might be very useful as an adjunct to CPK for prenatal diagnosis. In addition, an extended study (Zatz, Shapiro, Campion, Oda and Kaback 1978) has shown that serum P K (also in combination with CPK) enhances carrier identification in the X-linked forms of muscular dystrophy and may significantly improve our ability to provide genetic counseling for family members of affected patients. ACKNOWLEDGEMENTS The authors wish to express their gratitude to Drs. S. Oda and K. Tanaka for their valuable and helpful suggestions, to all our colleagues who donated their blood as controls, and to Laetitia Wotta for her secretatial assistance in the preparation of this manuscript.

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