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  IN THIS Article
 ::  Introduction
 ::  Material and methods
 ::  Results
 ::  Discussion
 ::  Acknowledgement
 ::  References

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Year : 1980  |  Volume : 26  |  Issue : 1  |  Page : 63-7

Creatine phosphokinase activity and its isozymes in normal, fetal and Duchenne dystrophic muscle.







How to cite this article:
Karandikar P V, Varadkar A M, Desai A D, Potnis A V. Creatine phosphokinase activity and its isozymes in normal, fetal and Duchenne dystrophic muscle. J Postgrad Med 1980;26:63


How to cite this URL:
Karandikar P V, Varadkar A M, Desai A D, Potnis A V. Creatine phosphokinase activity and its isozymes in normal, fetal and Duchenne dystrophic muscle. J Postgrad Med [serial online] 1980 [cited 2019 Nov 14];26:63. Available from: http://www.jpgmonline.com/text.asp?1980/26/1/63/988




  ::   Introduction Top

There is a preponderance of the enzyme Creatine phosphokinase (CPK, E.C.2.7.3.2) in the muscle tissue and its assessment in the serum has proved to be an extremely useful tool for the differential diagnosis of muscle disorders and for the detection of carriers in Duchenne muscular dystrophy (DMD). The enzyme has been known to exist in dimeric structures in three isozymic forms depicted as 'BB'-the brain type or CPK-1, the `MM'-the muscle type or CPK-3 and an intermediate type-the `MB' or CPK-2, which is a mixture of M and B sub-units. Each of these sub-units is under two separate genetic controls3 and accordingly during ontogeny of the muscle tissue, these genes continue to be expressed or shut off according to a programme.[4] Electrophoretically, the `BB' type is anodic, the `MM' type is cathodic while the `MB' type has an intermediate mobility.
There are conflicting observations on the muscle CPK isozyme profiles in muscular disorders. Thus, while Kar and Pearson[6] observed no overt abnormalities in the CPK isozymes in some myopathies including (DMD), several others. [1], 4],[10],[12] noted a high MB: MM ratio or a fetal pattern in several neuromuscular diseases and myopathies. Reports on the DMD muscle are also at variance with one another. Thus, where as Goto et al[4] found no change, Schapira et al[8] and recently Somer et al[9] found a fetal pattern of the isozymes.
In view of these contradictions, it was felt worthwhile to study the CPK isozyme profiles of muscles obtained from DMD patients and to compare them with those of fetal and normal individuals and also to assess muscle CPK activity as a comparative parameter.

  ::   Material and methods Top

Patients admitted to the Department of Neurosurgery of the King Edward VII Memorial Hospital, Parel, Bombay were investigated clinically (including genetic history), electrically (ECG, EEG and EMG), histologically, histochemically (muscle, ATPase, phosphorylase and NADH-tetrazolium reductase) and biochemically [serum GOT, GPT and creatine phosphokinase (CPK) ]. Only those patients in whom the diagnosis of DMD was established beyond doubt were included in this study. The carrier status of the mother and other female siblings was established wherever possible by ECG, quantitative EMG and by estimation of pre-and post-exercise serum CPK levels.
The quadriceps muscle was biopsied in 20 patients with DMD (age 5-11 years) and 20 normal individuals (age 7-42 years) undergoing orthopaedic surgery. The muscles from the quadriceps area from 15 foetuses (12-24 weeks old) were taken for this study. These foetuses were obtained from the clinic of medical termination of pregnancy of this hospital.
The muscle specimen was washed with chilled saline to remove blood. Connective tissue and visible fat were dissected out and the muscle was quickly transferred to a glass tube frozen at-70 C with a solid CO2 (cardice) and acetone mixture. The muscle was next weighed rapidly in a monopan balance and homogenized with 20 volumes of 0.1 M Tris buffer, pH, 7.4 (containing 50 mg. of cysteine hydrochloride and 48 mg. of MgS04, 7 H2O per 10 ml. buffer). The homogenate was centrifuged at 16,000 x g for 15 minutes at 4 C. The clear supernatant was aspirated and used for the study.
Enzyme assay, isoenzyme separation and detection
The muscle extract was analysed for the total CPK content by the method of Hughes.5 The enzyme activity was expressed as u moles of creatine formed/ hr/mg of wet weight of tissue. Aliquots of muscle extracts were electrophoresed on 7% acrylamide gels in the cold by the method of Davis2 and the CPK isozymes on the gels were detected by the method as outlined by Nielsen and Ludvigsen.7 The gels were preserved in 7% acetic acid and scanned with a yellow filter on a Densicord-255 densitometer and the ratio MB:MM of the isozymes was subsequently determined.

  ::   Results Top

[Fig. 1], [Fig. 2], [Fig. 3] represent CPK isozyme patterns of normal, dystrophic and normal (16-18 weeks) fetal muscles analysed and one may note that only two isozymes of -CPK namely MM and MB are detectable in all of them. Of the three, the 16-18 weeks old foetal muscle showed a conspicuously larger MB band relative to the MM band which distinguished it from the normal pattern. This is reflected in the high MB: MM ratio (2.0) of the fetal muscle [Table - 1].
Though the pattern of the DMD muscle did not differ strikingly from that of the normal, the MB: MM ratio of the pattern revealed significant deviation from it. The CPK activity of the muscles of 12-24 week old foetuses and of DMD patients were not only strikingly lower than the values obtained for normal muscles but in fact were similar [Table - 2].

  ::   Discussion Top

Goto et al[14] studying the ontogenesis of CPK isoenzymes in fetal skeletal muscle observed a predominance of BB and a trace of MB isozyme in very early stage of embryonic life, which gradually shifted to the predominance of AM over MM type in the intermediate phase followed by lack of BB type but a predominance of MM over MB in the late or mature stage of muscle development. The present study confirmed that mature normal human skeletal muscle has higher content of MM rather than MB CPK isozyme, which is evident from the low MB : MM ratio of 0.19 obtained in the present study.
In DMD muscle too, only MM and MB isozymes were detected; however, the ratio of MB : MM (0.4) was notably different from that of the normal muscle. No BB isozyme was detectable in the dystrophic muscle. This is in agreement with the finding of Goto et al[4], Van der Veen and Willebrandsll and Dawson and Fine. [3] On the other hand, Kar and Pearson, [6] Schapira et al[8] and Somer et al[9] found all the isozymic types in the dystrophic muscle.
The status of the CPK isozyme pattern in DMD muscle is far from established. Goto et al[4] and Kar and Pearson6 found no variations from the normal. Schapira et al8 and Somer et al[9] reported higher MB + BB : MM ratio in the dystrophic isozyme pattern which made them suggest that it resembles the fetal type. The MB : MM ratio (0.4) being higher than that of normal muscle (0.19), our study also indicates that the pattern is abnormal and resembles the fetal type. The close correlation between the total CPK activity of the fetal and DMD muscle observed in the present study also is in keeping with such a possibility. However, the change in the CPK isozyme profile of DAM muscle is not as striking as that observed in most neuromuscular disorders or other myopathies where higher MB : MM ratios were also noted.[4],[9] In view of this, one may conclude that alteration in CPK isozyme profile is a phenomenon seen in most muscle diseases and no characteristic pattern can be attributed to DMD alone.

  ::   Acknowledgement Top

This study was supported by the grants from Indian Council of Medical Research, New Delhi. We would like to express our gratitude to Dr. A. P. Banerji for his valuable comments and advice during the preparation of this manuscript. Our thanks are also due to Prof. A. P. Desai, for his continuous support and help and to Mr. K. S. R. Krishnan who has provided excellent secretarial assistance.

  ::   References Top

1.Cao, A.; De Virgiliis, S.; Lippi, C. and Coppa, G.: Serum and muscle creatine kinase isoenzymes and serum aspartate aminotransferase isoenzymes in progressive muscular dystrophy, Enzyme, 12: 49-62, 1971.  Back to cited text no. 1    
2.Davis, B. J.: Disc electrophoresis, Il Methods and applications to human proteins, Annals N.Y. Acad. Sci., 121: 404-427, 1964.  Back to cited text no. 2    
3.Dawson, D. M. and Fine, I. H.: Creative kinase in human tissues, Arch. Neurol., 16: 175-180, 1967.  Back to cited text no. 3    
4.Goto, I., Nagamine, M. and Katsuki, S.: Creatine phosphokinase isoenzymes in muscles-Human fetus and patients: Arch. Neurol. (Chic.), 20: 422-429, 1969.   Back to cited text no. 4    
5.Hughes, B P.: A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera, Clin. Chim. Acta, 7: 597-603, 1962.  Back to cited text no. 5    
6.Kar, N. C. and Pearson, C. M.: Creatine phosphokinase isoenzmes in muscle in human myopathies, Amer. J. Clin. Path., 43: 207-209, 1965.  Back to cited text no. 6    
7.Nielsen, L. and Ludvigsen, B.: Improved method for determination of creatine kinase, J. Lab. Clin. Med., 62: 159-168, 1963.  Back to cited text no. 7    
8.Schapira, F., Dreyfus, J. C. and Allard, D.: Les Isozymes de la creatine kinase et de 1'aldolase du muscle foetal et pathologique, Clin. Chim. Acta, 20: 439-447, 1968 (Eng. Abstract).  Back to cited text no. 8    
9.Somer, H., Dubowitz, V. and Donner, M.: Creatine kinase isoenzymes in neuromuscular diseases, J. Neurol. Sci. , 29: 129-136, 1976.  Back to cited text no. 9    
10.Tzvetnova, E.: Creatine kinase isoenzymes in muscle tissue of patients with neuromuscular diseases and human fetuses, Enzyme, 12: 279-288, 1971.  Back to cited text no. 10    
11.Vander Veen, K. J. and Willebrands, A. F.: Isoenzymes of creatine phosphokinase in tissue extracts and in normal and pathological sera, Clin. Chim. Acta, 13: 312-316, 1966.  Back to cited text no. 11    
12.Zsigmond, E. K. and Starkweather, W. H.: Abnormal serum and muscle creatine phosphokinase (CPK) isoenzyme pattern in a family with malignant hyperthermia, Anaesthesist, 22: 16-22, 1973.  Back to cited text no. 12    

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Online since 12th February '04
2004 - Journal of Postgraduate Medicine
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