Serum CPK in muscular dystrophy and myotonia dystrophica

Serum

CYK

in Muscular

Myotonia ByT.

Dystrophy

and

Dystrophica

S. DANOWSKI,G. SABEH,J. W. VESTER,R. A. ALLEY, T. J. ROBBINS, C. T. TSAI, M. PAZIRANDEH AND K. SEKARAN

Creatine phosphokinase (CPK) activity in serum was measured in control subjects and in patients with various forms of muscular dystrophy or with myotonia dystrophica by the one enzyme backward reaction (C + ATP s CP + ADP) without and with the addition of a sulfhydryl compound (mercaptoethanol). In healthy subjects and in patients without myopathy or neuropathy the addition of the sulfhydryl compound to such assays based on one enzyme did not raise the mean serum CPK value (differences recorded without or with mercaptoethanol were not statistically significant), nor did it alter the range of minimum-mazimum values. In terms of reference standards serum CPK values lie between zero and 16.9 I.U. in 99 per cent of control subjects and the remainder is between 17 and 40.9 I.U. The addition of mercaptoethanol to the one enzyme backward reaction, judging from our studies, increases the mean value of

CPK in patients with pseudohypertrophic and limb girdle muscular dystrophy and decreases the overlap of these groups with control values. Similar trends in FSH dystrophy and myotonia dystrophica were not statistically significant, but our observations are limited in number. Comparisons of our data based on the one enzyme backward reaction with results from laboratories employing this plus two other enzyme reactions indicate that both methods can yield serum CPK values of the same order of magnitude. Despite differences in the magnitude of serum CPK activity reported by various laboratories employing a variety of assay methods, there is general agreement concerning the frequency and the course of increased serum CPK activity in the various primary myopathies, myotonia dystrophica, and in neurologic disorders. (Metabolism 17: No. 9, September, 808817, 1968)

From

the University of Pittsburgh School of Medicine, Pittsburgh, Pa., and the Medical Oakland Veterans Administration, and Shadyside Hospitals, Pittsburgh, Pa. Aided by grants from the Muscular Dystrophy Associations of America, Inc., the Western Pennsylvania Arthritis Foundation, and the Health Research and Services Foundation. Received for publication February 13, 1968. GEORGE SABEH, M.D.: Research Associate in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. JOHN W. VESTER, M.D.: Assistant Professor of Biochemistry and Medicine, University of Pittsburgh School of Medicine. RICHARD A. ALLEY, M.D.: Research Fellow in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. TERRENCE J.

Center,

ROBBIXS, M.D.: Research Fellow in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. CONRAD T. TSAI, M.D.: Research Fellow in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. MAHMOOD PAZIRANDEH, h4.D.: Research Fellow in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. KAMALESH SEKARAN, M.D.: Research Fellow in Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine. T. S. DANOWSKI, M.D.: Professor of Medicine, University of Pittsburgh School of Medicine. 808

SERUM

809

CPK

I

N HEALTH LEVELS of creatine phosphokinase or creatine kinase (CPK or CK) in serum are low or zero and they may be increased slightly or markedly in myopathy or in neurologic disorders. However, the variety of assay technics, units of enzyme activity, and control values is bewildering.1-2i Our data consist of serum CPK levels measured without or with added sulfhydryl by the one enzyme ‘backward reaction“ in healthy control subjects, in patients with ailments unaccompanied by neurological disorder, and in individuals tonia dystrophica. A review of the assay provides reference standards for equating MATERIALS

symptoms or signs of a muscle or with muscular dystrophy or myotechnics and calculations of others clinical and laboratory experiences.

AND METHODS

The diagnosis of pseudohypertrophic (PHMD), limb girdle (LG), or facie-scapulohumeral (FSH) muscular dystrophy, or myotonia dystrophica was based on the history, physical findings, neurological examination, estimates of muscle performance by a physical therapist, measurement of force exerted during a single maximal contraction of each hand in making a fist, ergometric recording of IO5 successive contractions of the right hand and left hand at one-second intervals,28 electromyography, mean nerve conduction velocity, and examination of drill biopsy specimens of skeletal muscle under light and electron microscopy.ssJc CPK was measured in sera of healthy children and adults, in patients without muscle weakness, and in persons with muscular dystrophy or myotonia dystrophica following an overnight fast and after sitting or reclining for one-half hour or longer. The samples of venous blood were refrigerated at 0” to 4” C. immediately after withdrawal. CPK activity

was measured

in serum

at pH 9.0

and 37.5”

C. by the method

of Noda,

Kuby, and Lardys.9 with only slight modifications. This assay is based on the one enzyme backward reaction: Creatine + ATP $ Creature P + ADP. We found that increasing the amount of ATP in the incubation mixture by a factor of four gave rates that were first order with respect to enzyme in serum. Measurements were made without and with an added sulfhydryl compound (mercaptoethanol at 7 X 10-4 molar concentration) in the assay mixture. Levels of CPK are expressed in International Units (I.U.),sl namely, micromoles of creatine phosphate measured as phosphates2 produced per liter of serum per minute (um./L./min.). It is to be noted that data in the literature expressed as pm./ml./hour can be converted to International Units by multiplying by 16.67. RESULTS

Eflects of Added

Sulfhydyl

Compound

on Actioity of CPK in Control Sera

The addition of a sulfhydryl compound to control sera at assay appeared to raise CPK activity. Thus, mean values of 0.38 and 1.03 pm./L./min. (I.U.) Table

l.--fZreatine Phosphokinase Actidty in Duplicate Aliquots of Sera from Control Subjects Assayed Without and With Added Mercaotoethanol Without X +S.D.

Healthy Female

Adult Females Patients*

Male Patients*

Serum CPK added -SH (No.) Min.-Max.

(nm./L./min.) With added -SH X +S.D. (No.) Min.-Max.

0.33 + 1.2

(70)

O-16.7

1.03 +2.9

(70)

O-16.7

0.64 f 1.6

(25)

O-36.7

2.54 f5.2

(25)

o-22

1.46i4.7

(42)

O-24.7

14.51 k7.1

(42)

O-26.7

*Without history or physical findings indicative of myopathy or neurologic disorder. tDifference between this mean value and that obtained in sera from male patients assayed without added sulfhydryl (-SH) compound is statistically significant (t test).

132 36

23 6

40 11

26 9

PHhID without -SH with -SH

Limb Girdle without -SH with -SH

FSH without -SH with -SK

Alyotonia Dystrophica without - SH with -SH 7.0 * 7.6 2.1 f 6.2

30.5 f 47 68.2 it 101

40.1+ 62 *126.1 k 152

158.8 + 140 $287.7 + 142

0.94 f 3.2 1.61+ 3.7

( 31) ( 9)

( 53) ( 11)

( 6)

( 30

( 36)

(187)

O-28 O-19

O-150 O-327

O-164 O-359

o-737 O-501

O-36.7 O-21.4

Serum CPK (pm.lL./min.) (No.) Min.-Max. X+S.D.

*Based on analyses in 245 healthy adult females and 52 healthy children. tBased on analyses in 100 healthy adult females and 13 healthy children. $Difference between this mean value obtained on assay with added -SH statistically significant.

297 113

Number of Subjects

Controls without - SH* with -SHt

Subjects

7.7 11.1

22.6 18.2

13.0

11.8 2.7

1.0 0.8

9.4 9.1

8.7

18.2 10.8

9.4 18.2

21.7 16.6

25.7 10.8

1.9

16.6

16.6 13.5

Percent Distribution of Serum CPK Values 17-40.9 1.L. 41-100 I.U. 101..2OOI.U. 201-3001.U.

9.1

16.6

15.0 59.5

is

301-7001.U.

and that observed in sera assayed without the addition of mercaptoethanol

88.9

92.3

56.6 45.5

56.5 50

12.8 2.7

99.2

99

O-16.9 I.U.

Table Z.--Serum CPK Measured Without and With Added SuZfhydryZ in Healthy Controls and in Patients with Muscular Dystrophy or Myotonia Dystrophica

SEHUhI

CPK

811

were obtained without and with the addition of mercaptoethanol to sera of healthy adult female controls. In female patients without history or physical findings indicative of myopathy or neurologic disease the mean values proved to be 0.64 and 2.54 pm./L./min. without and with the addition of the sulfhydryl compound, respectively. In sera from male patients without myopathy or neuropathy the average CPK activity was 1.46 and 4.51 I.U. without and with mercaptoethanol. However, the difference in only the last of these three sets of values proved to be statistically significant by t test (Table I).

Our experience suggests that the addition of a sulfhydryl agent to sera from healthy adults or from patients without myopathy or neuropathy tends to raise CPK activity but such increases may or may not be statistically significant. Assay of Serum CPK in Muscular Dystrophy Without and With Added Sulfhydyl

and in Myotoniu Dystrophica

The control data in Table 2 are based on sera from healthy adults and children. Suggested differences between the serum CPK values in the adults and children in these control groups were not statistically significant and hence these data have been pooled. In those studied prior to recognition of the sulfhydryl effect and hence assayed without this constituent, the mean serum CPK activity was 0.94 + 3.2 pm./L./min. Sera from a subsequent group of control subjects (consisting, again, of healthy adults and children) assayed with added mercaptoethanol yielded a mean serum CPK value of 1.61 t 3.7 I.U. The difference between these two sets of control values is not statistically significant. Moreover, the minimum-maximum ranges in the two groups were of the same order of magnitude. Thus, in each set, some 99 per cent of the values proved to be 16.9 I.U. or lower. The remainder, approximately, 1 per cent, fell between 17 and 40.9 I.U. (Table 2). On the other hand, sera from patients with pseudohypertrophic muscular dystrophy (PHMD) assayed without and with added sulflhydryl yielded mean values of 159 * 140 and 288 + 142 ,um./L./min., respectively. The difference is statistically significant by t test. The serum CPK values determined without addition of a sulfhydryl compound were between 41 and 700 pm./L./min. or higher in 78 per cent of the assays. However, a higher proportion of values in this range, about 96 per cent, was recorded in PHMD when the sera were measured with added sulfhydryl. In other words, when sulfhydryl was added the number of values within the normal range of 0 to 40.9 I.U. was reduced markedly, from 24.6 per cent to 5.4 per cent, by the addition of the sulfhydryl compound. Similarly, mean CPK values recorded in sera from patients with limb girdle muscular dystrophy assayed with the addition of the sulfhydryl compound were higher: 126 + 152 with and only 40 + 62 I.U. without mercaptoethanol. Also, the overlap of the serum CPK values of this group of dystrophy patients with the healthy controls was apparently reduced by use of the sulfhydryl compound, 68 versus 50 per cent (Table 2).

DANOWSKI ET AL.

812 Table

3.2PK

in Paired

Serum CPK (I.U. =pm./L./min.) With added -SH Without added -SH X+S.D. (No.) Min.-Max. X +S.D. (No.) Min.-Max.

Group

157.9 + 102

PHMD

11.7 +21

FSH Myotonia

Measurements Without and With Added Suljhydryl Sera from Patients With Myopathy

Dystrophica

0.9 k2.6

(36)

O-387

*287.7 +I 142

(36)

S-501

(11)

O-327

(9)

O-18.7

(11)

O-64

68.2+101

(9)

o-7.7

2.lk6.2

*The difference between this value obtained with an added sulfhydryl (-SH) and that recorded in the same sera with addition of a sulfhydryl compound (mercaptoethanol) is statistically significant (t test). Apparently similar differences in the FSH and myotonia dystrophica groups are not statistically significant.

On the other hand, in our two groups of patients with FSH muscular dystrophy, the apparent increase in serum CPK activity when sera were treated with mercaptoethanol was not statistically significant, though the overlap with normal controls may or may not have decreased (Table 2). CPK in sera from patients with myotonia dystrophica measured without added mercaptoethanol yielded values which were within the control range. However, the proportion of patients in the upper range of control values for healthy persons, 17-40.9 I.U., appears greater but the number of observations is limited. Also, addition of mercaptoethanol to nine sera from patients with myotonia dystrophica did not in any instance result in CPK values above the control range. Simultaneous Measurements of Serum CPK Without and With Added hydryl in Patients with Myopathy

Sulf-

The data in Table 2 described

above were accumulated in part prior to the advent of the sulfhydryl modification. In Table 3, data derived from duplicate aliquots of the same sera which were analyzed simultaneously without and with mercaptoethanol are summarized. In each category the findings are in keeping with those in Table 2. Thus, addition of mercaptoethanol to the assay preparation did increase, judging by t tests, the mean value for serum CPK only in patients with pseudohypertrophic muscular dystrophy. The apparent enhancement of CPK activity in sera from individuals with FSH dystrophy and in myotonia dystrophica is not statistically significant. DISCUSSION

Assays of creatine phosphokinase have been based upon either the forward or backward reaction. In the forward reaction, CP + ADP * C + ATP, either the creatine or the ATP1m7is measured. In the backward reaction, C + ATP = CP + ADP, the creatine phosphate is measured as phosphate or as creatine in a one-enzyme systems-l’ or the ADP is estimated by means of two additional enzyme systems:14-‘7

SERUM

813

CPK

Pyruvic ADP + Phosphoenolpyruvate

’ Pyruvate

,

+ ATP

Kinase Lactic Pyruvate -t DPNH

’

,

DPN + Lactate

Dehydrogenase The end point of this three enzyme reaction is based on the oxidation of DPNH as indicated by the decrease in the optical density of the assay solution at 340 pm. Selected published data from other laboratories on serum CPK in control subjects and in patients obtained by the three enzyme backward reaction of Tanzer and Gilvarg,l* including those based on assay kits,33 are shown in Table 4.16J8,20*21,25-27 These published data and ours, obtained by the one enzyme backward reaction, are in general agreement. This is true when results of analyses from other laboratories performed without or with the addition of a sulfhydryl compound are compared with ours, irrespective of the method used. Also, the data of Hughes,4 Okinawa,12 Swaiman,13 Kar,27 and their colleagues and ours are in agreement in that the addition of a sulfhydry1 compound tended to raise the CPK values. Data are probably still inadequate to establish the superiority of any of the various assay procedures for CPK. Rotthauwe et ali5 and Vassella and colleagues l8 have spoken against the forward reaction based on the formation of creatine. It has also been pointed out that the forward reaction with ATP production as the end point may yield false low values as a result of ATP’ase present in serum. la On the other hand, the one-stage backward reaction has been deemed to be insensitive.ls Our data indicate that the latter need not be true, but further comparisons are needed. Despite differences in methodology and in control values among the workers cited and others,34-60 there is general agreement concerning serum CPK values in primary myopathy and in neurologic disorders. Thus, the various laboratories concur that in muscular dystrophy serum CPK activity is highest in the earlier phases of the pseudohypertrophic (Duchenne) type and decreases with the duration of this disease, that increases in serum CPK are less frequent and less marked in the limb girdle and the facie-scapula-humeral forms, and that only occasional patients with myotonia dystrophica develop slight increases in serum CPK (Table 4). On the other hand, in most instances of primary neuropathy serum CPK is normal. Finally, it is clear from the collected data that myositis, collagen disorders, etc., may or may not be accompanied by increased activity of CPK in serum. Even though an understanding of the frequency and the magnitude of serum CPK changes in a variety of clinical disorders has been attained by these diverse methods, communication has been difficult and the validity and significance of minor changes in serum CPK, such as those reported in carriers of the dystrophy trait,6J3,37-4Qor those observed in occasional patients who

*Sulfhydryl added to assay system.

(10)

(7)

2-8.5

O-l.6

(11)

LU. Min -Max. (No.)

Controls O-1 Duchenne FSH Limb Girdle Jlyotonia Dystrophica Polymyositis Dermatomyositia Myastbenia gravis Steroid myopathy Werdnig-Hoffmann Amyo. Lat. Scler. Neural. Atrophy IS10 Floppy Infant

Kuhna:

LU. Min.-Max. (No.)

(4) (3) (3) (9) (13)

O-13.5 O-1980 15-40 O-62 O-61 O-62 O-26

(97) (50)

I.U. x 10-4 Min.-Max. (No.)

H+ZSSlO

O-3.5 3-200 1-15 0.4-30 0.4-6 0. I-20 0.2-7 0.1-3 0.1-3 0.1-3 0.1-2.5 0.1-7 0.1-2.8 (20) (23)

(6) (7) (5) (2) (5) (11) (5:

(55) (3) (30)

“‘burn. jmin.-1/L-l” (No.) Min.-Max.

\‘nssella’R

4.3

360

1.1-9.3 90-624

(1)

(1)

(11) (13)

I.[‘.* Min.-Max. (No.)

Iiarl7

Reports of Serum CPK Measured by Three Enzyme Reaction in Myopathy and in Neuropathy

Forster’”

Table 4.--Published

O-5.96

O-o.8 0.24.9

hlin-Max.

I.U.

(10)

(4)

(No.)

l&char’*

Backward

18.4

21.7 2070

Mean

I.U.

(9)

(39) (11)

(No.1

PrestoIll

SERUM

CPK

815

do not have symptoms or signs of myopathy or a neurological disorder, must still be tested. In addition to the desirability of greater uniformity and reproducibility of assay technics, there is need for standardization of conditions of blood sampling and serum storage. Also, at present it is not entirely clear that sex and age of the subject are or are not important in setting up reference standards.5J3JsB34s35 It appears that food intake does not affect the value. Severe exercise certainly does, but the role of ordinary activity is not clear.36p37Physical activity before sampling may be critical in search for carriers in whom only slight elevations may be present. 5~23,37-4g Muscle trauma such as intramuscular injections, muscle biopsy, etc., should probably be avoided prior to or during the collection of samples of blood. REFERENCES 1. Ennor, A. H., and Rosenberg, H.: Some properties of creatine phosphokinase, Biothem. J. 57:203, 1954. 2. Dreyfus, J.-C., Schapira, G., and Demos, J.: Etude de la creatine-kinase serique chez les myopathes et leurs familles. Rev. Franc. d’Etudes Clin. et Biol. 5384, 1960. 3. -, and Schapira, G.: Technique de dosage de la creatine phosphokinase du serum. Rev. Franc. d’Etudes Clin. et Biol. 6700, 1961. 4. 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, 1962. 5. Pearce, J. M., Pennington, R. J., and Walton, J. N.: Serum enzyme studies in muscle disease. Part I. Variations in serum creature kinase activity in normal individuals. J. Neural. Neurosurg. Psychiat. 27:1, 1964. 6. Oliver, I. T.: A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochem. J. 61: 116, 1955. 7. Nielson, L., and Ludvigsen, B.: Improved method for determination of creatine kinase. J. Lab. Clin. Med. 62:159, 1963. 8. Kuby, S. A., Noda, L., and Lardy, H. A.: Adenosinetriphosphate-creatine transphophorylase. I. Isolation of the crystalline enzyme from rabbit muscle. J. Biol. Chem. 209:191, 1954. 9. Noda, L., Kuby, S. A., and Lardy, H.: ATP-creatine transphosphorylase. In: Methods in Enzymology, Vol II. New York, Academic Press, 1955, p. 605.

10. Ebashi, S., Toyokura, Y., Mimoi, II., and Sugita, H.: High creatine phosphokinase activity of sera of progressive muscular dystrophy. J. Biochem. 46:103, 1959. 11. Okinaka, S., Kumagai, H., Ebashi, S., Sugita, H., Momoi, H., Toyokura, Y., and Fujie, Y.: Serum creatine phosphokinase: activity in progressive muscular dystrophy and neuromuscular disease. Arch. Neural. 4:520, 1961. 12. -, Sugita, H., Momoi, H., Toyokura, Y., Watanabe, T., Ebashi, F., and Ebashi, S.: Cysteine-stimulated serum creatine kinase in health and disease. J. Lab. & Clin. Med. 64:299, 1964. 13. Swaiman, K. F., and Sandler, B.: The use of serum creatine phosphokinase and other serum enzymes in the diagnosis of progressive muscular dystrophy. J. Pediat. 63:116, 1963. 14. Tanzer, M. L., and Gilvarg, C.: Creatine and creature-kinase measurement. J. Biol. Chem. 234:3201, 1959. 15. Rotthauwe, H.-W., Zurukzoglu-Srlavounou, S., and Hammann, H.: Untersuchungen zur Methodik der Aktivitatsbestimmung der Kreatinphosphokinase. Klin. Wschr. 39: 1269, 1961. 16. Forster, V. G., and Escher, J.: Kreatinphosphokinase in der Diagnostik von Herzinfarkt und Myopathien. Helv. Med. Acta 4:513, 1961. 17. Aebi, V. U., Richterich, R., Stillhart, H., Colombo, J. P., and Rossi, E.: Progressive Muskeldystrophie. III. Serumenzyme bei der Muskeldystrophie im Kindesalter. Helv. Pedat. Acta 16:425, 1961. 18. Vassella, F., Richterich, R., and Rossi, E.: The diagnostic value of serum creatine

816 kinase in neuromuscular and muscular dis. . ease. Pedratrrcs 35:322, 1965. 19. Colombo, J. P., Richterich, R., and Rossi, E.: Serum-Kreatin-Phosphokinase: Bestimmung und diagnostische Bedeutung. Klin. Wchnschr. 40:37, 1962. 20. Hess, J. W., MacDonald, R. P., Frederick, R. J., Jones, R. N., Neely, J., and Gross, D.: Serum creatine phosphokinase (CPK) activity in disorders of heart and skeletal muscle. Ann. Int. Med. 61:1016, 1964. 21. Eshchar, J., and Zimmerman, H. J.: Creatine phosphokinase in disease. Am. J. Med. Sci. 253:272, 1967. 22. Schneider, K. W., and Heise, E. R.: The diagnostic significance of increased serum creatine-phosphokinase activity. German Med. Monthly 8:397,1963. 23. Kelly, S., Kelly, W. D., and Swift, H. L.: Serum CPK in four generations of a muscular dystrophy family. Amer. J. Clin. Path. 45~377, 1966. 24. Stich, W., and Tsirimbas, A.: Die Enzymdiagnostik des Herzinfarktest. Internist 2:502, 1961. 25. Kuhn, E., Stehlin, H.-G., and Stein, W.: Kreatinphosphokinase (CPK) in serum bei myotonischer dystrophie. Klin. Wchnschr. 40~744, 1962. 26. Preston, J. A., Batsakis, J. G., Briere, R. O., and Taylor, R. V.: Serum creatine phosphokinase. Amer. J. Clin. Path. 44:71, 1965. 27. Kar, N. C., and Pearson, C. M.: Activation of creatine phosphokinase by suIfhydryl compounds in normal and muscular dystrophy sera. Proc. Sot. Exp. Biol. Med. 118:662, 1965. 28. Greene, R., Rideout, D. F., and Shaw, M. L.: Ergometry in the diagnosis of myasthenia gravis. Lancet 2:281, 1961. 29. Fisher, E. R., Cohn, R. E., and Danowski, T. S.: UltrastructuraI observations of skeletal muscle in myopathy and neuropathy with special reference to muscular dystrophy. Lab. Invest. 15:778, 1966. 30. Sabeh, G., Fisher, E. R., and Danowski, T. S.: A speed drill technique for rapid muscle biopsy. J. Lab. Clin. Med. 65:523, 1965. 31. Report of the Commission on Enzymes of the International Union of Biochemistry. Symposium Series. Vol. 20. New York, Pergamon Press 1961.

DANOWSKI

ET AL.

32. Fiske, C. H., and Subbarow, Y.: Phosphocreatine. J. Biol. Chem. 81639, 1929. 33. Determination of the activity of creatine phosphokinase in serum (UV-method with DPNH). Brochure prepared by C. F. Boehringer and Soehne, G. H.: Mannheim, Germany, 1963. 34. Cutillo, S., Coletta, A., Rea, F., and Bemi-Carani, M.: The behavior of creatine phosphokinase activity in the serum of subjects with progressive muscular dystrophy. Boll. Sot. Ital. Biol. Sper. 39:769, 1963. 35. Schiavone, J. D., and Kaldor, J.: Creatine phosphokinase levels and cerebral disease. Med. J. Australia 2:790, 1965. 36. Vejjajiva, A., and Teasdale, G. M.: Serum creatine kinase and physical exercise. Brit. Med. J. 1:1653, 1965. 37. Stephens, J., and Lewin, E.: Serum enzyme variations and histological abnormalities in the carrier state in Duchenne dystrophy. J. Neurol. Neurosurg. Psychiat. 28: 104, 1965. 38. Dreyfus, J.-C., Schapira, G., Demos, J., and Rosa, R.: The value of serum enzyme determinations in the identification of dystrophic carriers. Ann. N. Y. Acad. Sci. 138: 304,1966. 39. Rossi, E. R., Richterich, R., and Aebi, U.: Detection des heterozygotes de la dystrophie musculaire progressive. Bull. Mem. Sot. Med. Hop. Paris 113:54, 1962 40. Wilson, K. M., Evans, K. A., and Carter, C. 0.: Creatine kinase levels in women who carry genes for three types of muscular dystrophy. Brit. Med. J. 1:750, 1965. 41. Milhorat, A. T., and Goldstone, L.: The carrier state in muscular dystrophy of the Duchenne type. J.A.M.A. 194:130, 1965. 42. Walton, J. N., and Pennington, R. J. T.: Studies on human muscular dystrophy with particular reference to methods of carrier detection. Ann. N. Y. Acad. Sci. 138: 315,1966. 43. Hughes, B. P.: Serum enzymes in carriers of muscular dystrophy. Brit. Med. J. 2:963, 1962. 44. Aebi, U., Richterich, R., Columbo, J. P., and Rossi, E.: Progressive muscular dystrophy II. Biochemical identification of the carrier state in the recessive sex linked juvenile (Dnchenne) type by serum creatinephosphokinase determinations. Enzymol. Biol. Clin. 1:61, 1961-62. 45. Shaw, R. P.: Serum enzymes in sex-

SERUM

CPK

muscular linked (Duchenne) dystrophy. Arch. Neurol. 16115, 1967. 46. Goto, I., Peters, H. A., and Reese, H. H.: Creatine phosphokinase in neuro-muscular disease. Arch. Neurol. 16529, 1967. 47. Emery, A. E. H.: Muscle histology in carriers of Duchemre muscular dystrophy. J. Med. Genet. 2:1, 1965. 48. Smith, H. L., Amick, L. D., and Johnson, W. U.: Detection of subclinical and car-

817 rier states in Duchenne muscular dystrophy. J. Pediat. 69:67, 1966. 49. Dubowitz, V.: Myopathic changes in muscular dystrophy carriers. Proc. Roy. Sot. Med. 56:810, 1963. 50. Vester, J. W., Sabeh, G., Newton, R. H., Finkelhor, H. B., Fetterman, G. H., and Danowski, T. S.: Muscle creature phosphokinase in primary myopathies. Proc. Sot. Exp. Biol. Med. 128:5, 1968.