Journal of the neurological Sciences, 1975, 25:389-396


~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Serum Factors Influencing Creatine Phosphokinase In Vitro Studies using Diffusates M. MASCREEN, B. BISWAKUMAR AYD K. VALMIKINATHAN Institute of Neurology, Madras Medical College, Madras 600 003 (India) (Received 6 January, 1975)


Dialysis-oriented serum creatine phosphokinase (CPK) studies initiated in this department have clearly indicated the diverse functional nature of serum factor(s) influencing CPK activity (Valmikinathan 1973; Snehalatha, Valmikinathan, Srinivas and Jagannathan 1973; Snehalatha, Mascreen, Valmikinathan and Jagannathan 1974; Snehalatha and Valmikinathan 1974; Srinivas, Mascreen and Valmikinathan 1974). This has prompted us to look for these functional factor(s) in the diffusates obtained following dialysis of serum samples as such and during serial CPK estimations carried out after electromyography (EMG) in some cases of neuromuscular disease. In this communication, we present confirmatory evidences of the functional characteristics of these serum factor(s) based on the in vitro influence of diffusates on serum CPK. MATERIAL AND METHODS

The subjects of the present study included a few normal control subjects without neurological disease, 1 case of dermatomyositis, 1 case of Duchenne muscular dystrophy (DMD), 1 case of hypothyroid neuromyopathy (HTNM), 2 cases of spinal muscular atrophy (SMA) and 3 cases of neurogenic atrophy (NA), the diagnosis being based on clinical, electromyographic (EMG) and histological criteria. Serum samples were routinely analysed, in duplicate, for certain biochemical parameters such as creatine phosphokinase (E.C. CPK), serum aspartic aminotransferase (glutamic-oxalacetic transaminase {E.C. .GOT) and serum alanine aminotransferase- (glutamic-pyruvic transaminase (E. C. GPT). Assay of serum CPK was carried out both before and after dialysis following the procedure detailed earlier (Snehalatha et al. 1973). Similar CPK studies were carried out on serum samples obtained before and at varying time intervals ranging between 0 and 240 hr after EMG in a case of DMD and 3 cases of NA.



Dialysis studies Usually, 0.5 ml of serum was subjected to dialysis overnight at 4~'C against 10 ml cold glass-distilled water using cellophane dialysis tubing. After the period of dialysis, the protein concentration of the material left inside the bag was estimated following Lowry's method as detailed earlier (Snehalatha et al. 1973) and this incidentally helped us to make necessary corrections for evaluation of serum C PK. Similar dialysis studies were carried out against cold glass-distilled water containing 0.1 M cysteine and the CPK results were compared with the corresponding values obtained following dialysis of the serum samples against cold glass-distilled water without added cysteine. Preparation of diffusates The material left outside the bag (diffusate) following dialysis of serum samples was concentrated to 1 mt approximately by perevaporation at 20°C. Usually, it required 16-18 hr to bring it to the desired volume. In most of the cases, as in those of dermatomyositis, SMA and HTNM, the diffusates were obtained following dialysis of random serum samples. However, in the case of DMD and in the 3 cases of NA, diffusates were prepared with appropriate serum samples during serial CPK estimation after EMG. In DMD, the diffusates were obtained at 96 and 160 hr and in NA 25 hours after the EMG. The diffusates referred to above were marked as: Diffusate I (D I --- Dermatomyositis) Diffusate IV(D IV .... HTNM) Diffusate II(D II - - 96 hr DMD) Diffusate V (D Va, b ..... SMA) Diffusate III(D III - - 160 hr DMD) Diffusate VI(D VIa, b,c NA) These terms will be frequently used in the subsequent discussion. Influence of diffusates on serum CPK This was assessed with the appropriate serum samples from subjects under study as well as with those from normal controls. The serum CPK was re-assayed after 1 in 10 dilution with distilled water and was compared with corresponding C PK figures after the addition of the diffusate individually. The diffusate added in these in t,itro studies corresponded to 0.1 ml of original serum. Paper chromatography of D II and D III was carried out with Whatman No. 1 paper in a butanol: acetic acid: water (40:7:5) system. After the chromatographic run, the dried paper was sprayed with ninhydrin in order to locate the spots. EMGs were carried out with a Medela Model MS.4 using 25 gauge concentric needle electrodes. Both affected groups of muscle as well as a few proximal and distal muscle groups not clinically affected were examined. RESULTS

All the biochemical parameters measured in the present study showed little or no variation in control subjects. There was a moderate elevation of GOT and GPT levels in DMD and in dermatomyositis which did not change substantially during the study period. However, the serum CPK showed some interesting variations. This was observed in random serum samples obtained from the subjects as well as in the post-



E M G serum samples of 1 case of D M D and 3 cases of NA. Hence we focussed our attention upon the serum C P K changes in the subjects with neuromuscular disease. These CPK variations were not related to any experimental shortcomings such as dialysis effect, concentration of thiols, etc. This conclusion was based on the similar CPK activities found in serum dialysed against distilled water with and without cysteine. At the same time, these results could be interpreted in relation to serum factor(s) influencing CPK activity. Similar factors seem to hold good for the observed results during serial CPK studies after EMG. It seems likely that the stress of E M G needling, in some unknown manner, alters intrinsic muscle metabolism leading to an elaboration of serum factors with diverse influences on C P K activity (Valmikinathan 1973). These factor(s) were found to be either inhibitory or activating in character. Inhibitory influences (Table 1) These were observed in a case ofdermatomyositis and a case of D M D at some stage (96 hr) following EMG. The elevated C PK figure of 65 IU/I in a case of dermatomyositis rose to 181 IU/I following dialysis of the sample, suggesting the presence of an in vivo inhibitor, removal of which by dialysis, resulted in elevation of C P K activity similar to that reported earlier in dermatomyositis (Snehalatha et al. 1973). In vitro studies with the diffusate (D I) of this serum confirmed the in vivo properties as judged by the results obtained with sera of normal controls and subjects under study (Table 1). In DMD, at 96 hr following EMG, the serum C P K activity estimated by the conventional method was 30 IU/I and rose to 488 IU/I after dialysis, suggesting the presence of an in vivo inhibitor. In vitro studies using diffusate D II of the corresponding serum


CPK in IU/1 without any diJfusate

with difJusates DI


Normal controls 1 2

22 16

12 8

10 10

D M D (160 hr) SMA




1 2 HTNM Dermatomyositis~ NA 1 2 3

111 101 101 181

50 15 6 45

60 25 16 90

165 180 120

50 120 40

70 100 54

Study subjects a


Dialysed serum sample was used



O - - O Non-dialysed ser'urr! O - - O Dialysed sePum 70O

D-60O -L ~ ~_400

. ~ . . /3. ///




Dj " //





96 Time in hour's



Fig. 1. Pattern of CPK changes following E M G in a case of Duchenne muscular dystrophy.

sample (96 hr) elaborated the functional inhibitory property as judged by the results obtained with sera of normal controls and subjects under study (Table 1). The pattern of CPK changes in D M D under the present experimental situation is more variable with time and hence necessitates a brief description at this stage. As indicated in Fig. 1, the pre-EMG CPK value of 100 IU/I dropped to 26 IU/I following dialysis, a finding similar to our earlier report in D M D (Snehalatha et al. 1973). Following the EMG, with time, there was an oscillatory type of change in CPK activity characterised by both inhibitory as well as activating influences at 96 and 160 hr respectively. TABLE 2 ACTIVATING INFLUENCE

(in t,itro) OF DIFFUSATES ON SERUM CPK C P K in I U / t

Without ~llly diffusate Normal controls 1 2 3 Study subjects D M D (96 hr) a SMA" 1 2 HTNM ~ Dermatomyositis NA l 2 3

D Ill

22 16 5

7(/ 56 35



0 0 5 65

100 II0


D I I"

45 3S 30




47 32 30

36 28 40










72 86 74

68 70 54

80 68 60

310 100 80

101 70

111 84




92 50 46

86 75 64

47 3O 26

~Dialysed serum samples of DMD, SMA and H T N M were used.



Activating influences (Table 2)

This type of change was commonly encountered in most of the myopathies such as DMD, SMA and HTNM. The same picture was observed in NA after EMG. Serum CPK activity at 160 hr after E M G in the case of DMD, estimated by the conventional method was 673 IU/I which on dialysis dropped to 205 IU/I, suggestive of the presence of an in ~ivo activator. In vitro studies of diffusate D I I I showed activation of CPK in normal controls and study subjects. Similar results were obtained with diffusates of H T N M (D IV) and SMA (D Va, b). Following dialysis the elevated CPK activity (101 IU/I) in H T N M dropped to 5 IU/I, as in our earlier report (Srinivas et al. 1974). Similarly, the elevated C P K figures of 111 IU/I and 101 IU/I in 2 cases of SMA dropped to zero after dialysis, a finding in accordance with our earlier observation in SMA (Snehalatha et al. 1973). Dialysis was found to have practically no influence on the initial moderately elevated serum CPK activities (47, 30 and 26 IU/I, respectively) in 3 cases of NA. This picture persisted until 5 hr following EMG. However, at 25 hr after EMG, there was a sharp rise in serum CPK level (165, 180 and 120 IU/I, respectively) with a return to pre-EMG levels of activity following dialysis (Fig. 2). This incidentally suggests involvement of an in vivo activator at this stage (25 hr). In vitro studies of the corresponding diffusates (D Via, b, c) have confirmed the activating phenomenon. Based on the in vitro studies with diffusates, the percentage inhibition or activation was found to be of the order of 37-94 and 45-700, respectively. In estimating percentages of activation or inhibition, we preferred to take the re-assayed CPK activities of serum samples, which were slightly lower when compared to previous corresponding figures. This was done in order to account for possible slight loss of enzyme activity on storage of samples. At the same time, it was interesting to note that the pattern of influence of the diffusates was well maintained irrespective of the CPK activities used for comparison and evaluation. Basically, these diffusates showed a broad degree of functional specificity and diversity. This, perhaps, is related to some underlying differences in the composition of the diffusates. This to some extent was brought out by routine mapping of ninhydrinpositive materials of D II and D I I I on paper chromatography (Fig. 3). The massive 0--0




diolysi$ diolysis

.7. 160-


> o D_ 8 0 (2


in hours

Fig. 2. Pattern of CPK changes followingEMG in a case of neurogenic atrophy.



Fig. 3. Paper chromatography of the diffusates. Spots located after ninhydrin spray, lxqt: D 11, rigtht: D III.

ninhydrin-positive spot of DIII with an R.r-value 0.38 (Spot 5, Fig. 3) was repeatedly absent in the paper chromatogram of D If. Similarly, a minor ninhydrin-staining spot (Spot 2, Fig. 3) was missing in the paper chromatogram of D II1. DISCUSSION

The results of the present in vitro studies (Tables 1 and 2), using diffusates of serum samples, support our earlier in vivo observation of serum factor(s) influencing CPK activity (Valmikinathan 1973; Snehalatha et al. 1973; Snehalatha et al. 1974, Srinivas et al. 1974; Snehalatha and Valmikinathan 1974). Broadly speaking, these factor (s)are either inhibitory or activating in character (Tables i and 2). In most of the myopathies like DMD, HTNM, and SMA, there is an activating factor in serum; when this is removed by dialysis, the original elevated serum CPK activity drops considerably. Similarly, dermatomyositis is characterised by the presence of inhibitory factor(s); when these are removed by dialysis, the serum CPK rises rapidly. These characteristic functional variations in serum factor(s) influencing CPK activity are further elaborated by the serial CPK studies carried out after EMG in



3 cases of NA and l case of D M D (Figs. 1 and 2). In NA, there is an involvement of activator which is responsible for the elevated CPK level at 25 hr following EMG. In vitro studies using diffusates of these serum samples have supported this view (Table 2). The most interesting type of variation in serum CPK was observed in a case of DMD following EMG (Fig. 2). Basically, there was an oscillatory type of CPK change characterised by evidence of the presence of inhibitory as well as activating serum factor(s) at 96 and 160 hr respectively after EMG. These findings were confirmed by in vitro studies using the respective diffusates (D II and III). as indicated in Tables 1 and 2. The presence of both an inhibitory and an activating influence of serum factor(s) in the same case of DMD at different time intervals following EMG, has led us to speculate that these diffusates with differences in function may also have differences in composition. Paper chromatographic studies of these diffusates (Fig. 3) have given some support to this view, but these isolated observations must clearly be confirmed by further work. This is, perhaps, the first detailed study of the serum tactor(s) which have a considerable influence on serum CPK activity and our results are likely to have considerable implications in relation to studies of muscle physiology and of neuromuscular disorders in general. From our results, it is possible to speculate that these factor(s) are not only different functionally but vary in type, constitution and number from one disease to another. This finding warrants further detailed investigations towards the characterisation of these factor(s). ACKNOWLEDGEMENTS

We are grateful to Prof. B. Ramamurthi, Head of the Institute of Neurology, Madras, for his keen interest in this study. Our thanks are also due to Prof. K. Jagannathan, Professor of Neurology, Institute of Neurology, Madras, for helpful discussion and to the Principal, Madras Medical College, Madras and the Superintendent, Government General Hospital, Madras for providing the necessary facilities.


In vitro studies with diffusates obtained following dialysis of serum samples have

enabled us to confirm the presence of inhibiting and activating factors influencing CPK activity. Activating factors are commonly found in many neuromuscular disorders like Duchenne muscular dystrophy (DMD), spinal muscular atrophy and hypothyroid neuromyopathy, and inhibiting factors in dermatomyositis. These findings are further elaborated during serial serum CPK studies after E M G in 3 cases of neurogenic atrophy and 1 case of DMD. The in vivo inhibitory and activating influence of serum factor(s) at 96 and 160 hr respectively after E M G was confirmed by in vitro studies with the diffusates of these serum samples. The functional variations of these diffusates are shown to be related to differences in the composition of these diffusates suggested by paper chromatography.



SNEHALATHA,C. AND K. VALMIKINATHAN(1974) Creatine phosphokinase level in limb-girdle muscular dystrophy - - Effect of dialysis, Clin. chim. Acta, 51: 315. SNEHALATHA,C., M. MASCREEN,K. VALMIKINATHANAND K. JAGANNATHAN(1974) Dialysis effect on serum creatine phosphokinase during steroid therapy in dermatomyositis, Clin. chim. Acta, 53: 217. SNEHALATHA, C., K. VALMIKINATHAN,K. SRINIVASAND K. JAGANNATHAN(1973) Creatine phosphokinase level in neuromuscular disorders - - Effect of dilution and dialysis, Clin. chim. Aeta, 44 : 229. SRINIVAS, K., M. MASCREEN AND K. VALMIKINATHAN(1974) Neuromyopathy of hypothyroidism A preliminary study (Abstract No. 411 ). In: D. GARDNER-MEDWIN (Ed.), Abstracts of Papers presented at the 3rd International Congress on Muscle Diseases, Newcastle upon Tyne, 1974 (International Congress Series, No. 334), Excerpta Medica, Amsterdam. VAL~IKINA~dAN,K. (1973 ) Biochemical concepts of neuromuscular disorders, Prac. lnst, Neurol. (Madras), 3: 75.

Serum factors influencing creatine phosphokinase. In vitro studies using diffusates.

In vitro studies with diffusates obtained following dialysis of serum samples have enabled us to confirm the presence of inhibiting and activating fac...
567KB Sizes 0 Downloads 0 Views