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

2.4 :~ 0.4 b 2.9 4- 0.7 7.3 4- 3.9 1.9 4- 0.5 1.6 4- 0.2 2.9 4- 0.3 e

8.3 e 8.9 b 1.0 e 0.9 e 0.3 a

10 2 7 3 3 21

44444-

43.1 24.1 4.7 5.2 1.9

0.1 0.1 0.05 0.3

26 17 20 21 8

± ± 44-

1.1 1.3 1.2 3.4

0.8-4.5 2.2-3.7 1.5-30.2 1.0-2.7 1.3-2.1 1.1-6.3

4.0-150.4 3.0-148.7 1.7-18.1 1.1-14.9 0.9-3.7

0.5-1.7 0.5-2.1 0.5-2.1 2.1--4.4

16.1 16.6 46.6 12.5 10.9 21.1

373.9 173.5 22.8 35.6 14.4

8.5 5.2 6.2 7.5 65.8 c 52.6 b 5.1 b 7.7 e 3.2 b

1.1 0.3 0.4 0.9

4- 4.7 a + 0.9 4- 25.9 4- 3.9 ± 0.8 i 6.5 a

44+ ± 4-

± ± ± d-

4.7-53.5 15.7 17.5 10.2-201.0 7.0-20.0 9.7-12.5 3.5-127.0

53.0-1510.0 19.0-700.0 5.5-95.0 7.2-165.0 5.5-28.7

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

P K or CPK

N u m b e r abnormal

t,a

353 SIGMA

o~

UNITS

~ o o ~ ~

~-ooooo

~

I l l l l l l l ' ~ l +t

o

~8o

~

~

o

I

I

I

oo = ~

~ o

3 -t

Ii

¢J

~

~~o®o

ooo

0

÷1

~E o +1

tomo*

q188N~0

0~ r~

m3 E ¢tjO eO00

E

i

O +!

oe

•

•

•

t • ----:..

41

H

°ell°

•

•

..|

i

°

,¢

~

E

o -I-; ¢q

Io3_-

• !

+I •

•

=

....~I~-.

¢¢t-~

t

|

l

l

l

l

l

l

oQQQqQqQ

~

,

l

Q

o o o o o o o o Jnoq / ItU / IOUj'd

I

o

I

o

I

q

~ 44p

,~

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

E

o

~-

E

.~

I 9±0.32

2.4+037

!

73+3.9

1.9-+0.48

2.9±0.33

62±0.4

144 ±3.2

161-+4.7

46.6±25.9

CPK 12S+39

21.1 + 6 . 5

1500.0

1.0-

2.0-

3.0-

4.0-

I O.O-

!

:

DERMATOMYOSITIS

;

°

°

(3)

110)

MYOTONIC KUGIrLBIrRG DYSTROPHY WIrLANDIER ANO S. II.A.

POLYMYOSITI$ {7)

(3)

(21)

UNKNOWN MYOPATHIES (61)

NORMAL CONTROLS (ADULTS) (8)

110)

MYOTONIC KUGELBERG DYSTROPHY WELANOI[R ANO $.U.A,

,.

° g

°

(7)

POLY MYOSITIS

13)

DI[RMATOMYOSITIS

(21)

UNKNOWN MYOPATHIES

. ;,

~

o

Fig. 2. Serum P K and CPK activities from patients affected with different types of neuromuscular disorders.

n - (61)

NORMAL CONTROLS (ADULTS)

-,..

I

: t

-.

!

•

•

o

- I0.0

- 200

- 30.0

- 40.0

100.0

-300.0

50.0-

o

- 500.0

70.0-

•

- 700.0

90.0 °

30.0-

I I00.0

900.0

I I 0.0-

o

1300.0

1.2:1:005

PK

130.0-

150.0-

o)

'-I (I)

c

]>

t~

356 A

B

BECKER (17 patients)

65-

65-

5,5-

55-

45-

45-

35-

35-

*---*

~" .* .".

LIMB GIRDLE SYNDROME (14 patients) FACIO-SCAPULO HUMERAL (20 patients)

,---.

25-

"~ .~LGMD

i5-

t

_

e

SH

5I

0

I

I

20

: 40

;

: 60

SERUM

;

I

80

I

I

,'

I00

; : : : 120 140

I

0

: 4

:

PK{pmol/ml/hour)

; 8

:

I t2

I I I I l i I I 16 20 24 20

PK(prnol/ml/hour)

SERUM

D

I

DUCHENNE (26 patients)

2016-

tu 12(.9