Carbonic anhydrase III in neuromuscular disorders

Journal of the Neurological Sciences, 1983, 59:383-388 Elsevier

383

CARBONIC ANHYDRASE III IN NEUROMUSCULAR DISORDERS

ROBERT HEATH 1, MARTIN S. SCHWARTZ 2, IAN R. F. BROWN t and NICHOLAS D. CARTER I

]Departments o! Child Health and Chemical Pathology and 2Department of Neurology, St George's Hospital Medical School, London SWI7 ORE (Great Britain) (Received 2 November, 1982) (Revised, received 8 December, 1982) (Accepted 20 December, 1982)

SUMMARY

Plasma carbonic anhydrase III (CAIII) and creatine kinase (CK) were measured in 44 patients with a variety of neuromuscular disorders. Markedly elevated CK levels were associated with similarly increased levels of CAIII. In 9 patients, only the CAIII was elevated, but in 2 patients only the CK was raised. The determination of plasma CAIII is thus an important index of muscle disorder and is probably more sensitive than CK.

INTRODUCTION

In the investigation of disorders of muscle the most useful laboratory test is measurement of creatine kinase (CK) (Rosalki 1976). In certain disorders serum aldolase, pyruvate kinase, and lactate dehydrogenase (LDH) may be raised but these parameters are less sensitive indicators in muscle disorders. Recently, serum myoglobin measurement has also been found to be of value (Askmark et al. 1981). A radioimmunoassay has been developed for a skeletal muscle specific isoenzyme, carbonic anhydrase III (CAIII) (Heath et al. 1982a) which comprises 1 2% soluble muscle protein (Carter et al. 1979). Plasma CAIII has been found to be elevated in Duchenne muscular dystrophy (Heath et al. 1982a) but is not elevated in acute myocardial infarction (Heath et al. 1982b). In the present study we have compared the levels of plasma CK and CAIII in patients with a variety of neuromuscular disorders.

This study was supported by the Muscular Dystrophy Group. 0022-510X/83/$03.00 (c9 1983 Elsevier Science Publishers B.V.

384 MATERIALS AND METHODS

A series of 44 patients with probable neuromuscular disorders were studied. All patients had nerve conduction studies, needle electromyography and underwent routine biochemical and haematological tests. From a number of patients diagnostic needle muscle biopsies were obtained. Blood samples were taken into lithium-heparin coated tubes and plasma was separated and aliquoted within 2 h and stored at -70 °C prior to assay. Haemolysed samples were excluded from the assays. CK and CAIII determinations were performed on atiquots of the same plasma sample. CAIII was analysed using a radioimmunoassay in which a constant amount of [125I]CAIII competes with sample (unknown) or standard (known) for binding to a constant but limiting amount of anti-CAIII serum. A second antiserum was used in the separation procedure (Heath et al. 1982a). Creatine kinase was analysed spectrophotometrically using the Boehringer creatine kinase (N-acetylcysteine activated) UV-test kit (Cat. No. 126375) adapted to the LKB reaction rate analyser, Ultrolab 8600. Normal plasma levels of creatine kinase were less than the 95th percentile limits of 270 IU/1 in males and 200 IU/1 in females which were obtained from 80 healthy adults in each case. Plasma CAIII levels were considered raised if values greater than 79 ng/ml (mean + 2 SD) were obtained, assuming a normal distribution (values from 40 healthy adults). There is no significant difference between male and female plasma levels of CAIII. RESULTS

In 21 patients both the CK and CAIII were elevated (see Table 1). These comprised 7 patients with polymyositis or dermatomyositis, 4 patients with limbgirdle dystrophy, 3 with hypothyroid myopathy, 2 with McLeod's syndrome (an unusual variant of the Kell blood group) (Marsh et al. 1981) and 1 each with myotonic dystrophy, alcoholic myopathy, toxic myopathy, facioscapulohumerat dystrophy and Duchenne muscular dystrophy. The relationship of the raised levels of CK and CAIII are shown in Fig. 1. The regression coefficient was 0.906 (P <0.005). In 9 patients the CK was normal but the CAIII was elevated (see Table 2). These included 4 patients with motor neurone disease, 1 patient each with proximal muscular weakness of undetermined aetiology, myotonia congenita, post-infection polymyositis (Schwartz et al. 1978) oculopharyngeal muscular dystrophy and hypothyroid myopathy. In these cases the CAIII was only moderately elevated (80-176 ng/ml), with the exception of a patient with motor neurone disease, where the CAIII was 400 ng/ml. All of these patients had diagnostic muscle biopsies. In 2 patients the CK was elevated but the CAIII was normal (see Table 3). One of these patients was a young woman with post-infection polymyositis with a slightly elevated CK (211 IU/1). The other patient had an unusual syndrome of

TABLE 1 P A T I E N T S W I T H R A I S E D CK A N D R A I S E D CAIII LEVELS Patients

Age (yr)

Sex

Diagnosis

CK(IU/I)

CAlll(ng/ml)

I M.R. 2 L.D. 3 D.B. 4 D.H. 5 J.H. 6 B.L. 7 C.P. 8 E.H. 9 A.R. 10 C.B. 11 R.F. 12 J.E. 13 D.P. 14 B.D. 15 D.B. 16 .I.J. 17 F.C. 18 M.C. 19 J.W.

50 65 38 54 42 39 17 46 46 55 48 40 54 31 16 44 27 39 67

Female Male Female Male Female Female Female Female Male Male Male Male Male Male Female Male Female Male Female

1964 1(t84 400 663 1309 1065 318 1144 367 3278 1051 2068 1535 1293 3850 580 285 560 3850

740 800 141 33(1 470 109 225 305 475 16{10 130 335 580 199 980 335 197 395 24(1(I

20

C.A.

35

Female

32(1

118

21

T.W.

6

Polymyositis Polymyositis Polymyositis Polymyositis Polymyositis Polymyositis Dermatomyositis Hypothyroid myopathy Hypothyroid myopathy Hypothyroid myopathy McLeod Syndrome McLeod Syndrome Limb-girdle dystrophy Limb girdle dystrophy Limb girdle dystrophy Limb girdle dystrophy Myotonic d.'cstrophy Alcoholic myopathy Toxic (hypokalaemic) myopathy Eacioscapulohumeral dystrophy Duchenne muscular dystrophy

7810

2888

Male

(K (IU/[) 10,000

..f."

1,000

~ o ° eo

• y =2"15x÷2/+8.19 r=0.91 n=21 P<0.005

100 -

10-

i

i

i

I

10

100

1,000

10,000 (A-Ill (ng/mr)

Fig, 1. G r a p h showing the relationship between plasma CK and CAIlI levels in patients where levels of both of these enzymes were raised.

386 TABLE 2 PATIENTS WITH NORMAL CK AND RAISED CAIII LEVELS Patients

Age (yr)

Sex

Diagnosis

1 2 3 4 5 6 7 8

49 52 58 66 26 37 39 64

Male Male Female Male Female Male Male Female

Motor neurone disease 70 Motor neurone disease 126 Motor neurone disease 192 Motor neurone disease 161 Proximal myopathy 34 Myotonia congenita 92 Post-infection polymyositis 23 Oculopharyngeal muscular dystrophy 79 Hypothyroid myopathy 21

R.F. W.K. T.W. F.M. C.P. P.W. W.D. J.V.

9 W.H.

Male

CK(tU/1)

CAltl(ng/ml) 80 109 112 400 100 121 112 176 84

TABLE 3 PATIENTS WITH RAISED CK AND NORMAL CAIII LEVELS Patients

Age (yr)

Sex

Diagnosis

CK(IU/1)

CAIIl(ng/ml)

1 P.P.

42

Female

211

40

2 R.H.

44

Male

Post-infection polymyositis Paresthesiae

434

43

persistent paresthesia with n o r m a l sensory nerve c o n d u c t i o n velocities, n o r m a l muscle biopsy but a persistently elevated CK. Electrophoresis showed this to be the C K - M M isoenzyme. In 12 patients the C K and C A I I I levels were both n o r m a l (see Table 4). These included 5 patients with muscle pain, 2 with m y o t o n i a congenita, 2 with h y p e r t h y r o i d m y o p a t h y a n d 1 each with a possible metabolic m y o p a t h y , nonspecific proximal muscle weakness and osteomalacia. DISCUSSION In the evaluation o f patients with suspected n e u r o m u s c u l a r disorders an elevated plasma C K usually indicates an a b n o r m a l i t y o f skeletal muscle, a l t h o u g h elevated C K levels have been reported in n o r m a l subjects following strenuous exercise (Rosalki et al. 1976). In the present study nearly all the patients with elevated C K had similarly increased levels o f C A I I I . O n l y 2 o f o u r patients showed an increased C K alone and in 1 o f these this elevation was slight. In 8 o f our patients only the C A I I I was elevated. This g r o u p included 4 patients with m o t o r n e u r o n e disease o f less than 1 year duration. A n increased level o f C K is occasionally f o u n d in patients with

387 TABLE 4 PATIENTS WITH N O R M A L CK A N D N O R M A L CAIII LEVELS Patients

Age (yr)

Sex

Diagnosis

CK(IU/I)

CAlll(ng/ml)

1 H.M. 2 J.M. 3 V.S. 4 R.S. 5 R:C. 6 M.H. 7 E.H. 8 P.R. 9 A.S. 10 J.H. It G.H. 12 E.L.

38 40 40 23 55 17 26 45 27 46 38 36

Male Male Male Male Male Male Female Female Female Male Female Female

Muscle pain Muscle pain Muscle pain Muscle pain Muscle pain Myotonia congenita Myotonia congenita Hyperthyroid myopathy Hyperthyroid myopathy Metabolic myopathy Proximal muscle weakness Osteomalacia

186 83 164 109 177 56 49 42 91 168 44 47

78 69 21 33 30 35 22 49 75 64 69 20

motor neurone disease, but usually only in patients with long histories whose biopsies show secondary myopathic changes including increased central nucleation and muscle fibre splitting (Schwartz et al. 1976). This preferential rise of CAIII suggests that mild secondary myopathic changes may occur at an early stage of the disease or may be related to possible catabolic changes in skeletal muscle (Corbett et al. 1982). High levels of CK were found to be associated with high levels of CAlII. A similar relationship between myoglobin and CK levels has been reported in Duchenne muscular dystrophy (Kiessling and Beckmann 1981). In Duchenne muscular dystrophy, elevation of plasma CAIII to at least 10 times normal levels has been found (Heath et al. 1982a). Increased fetal plasma levels of CAIII have also been noted at fetoscopy performed in pregnancies at high risk for Duchenne muscular dystrophy (Carter et al. 1982). There are several explanations for increased levels of CK and CAIII. These include:increased passage across or through the sarcolemma, including functional leaks (Rowland 1980), decreased elimination from serum, initial levels within surviving muscle fibres, and possible genetic factors (Nicholson 1981). In addition, the size of the molecule involved may be important: myoglobin 17,500 MW; CAlII 28,000 MW, CK 80,000 MW. The smaller molecules may pass more readily through "'holes" in the sarcolemmal membrane. This might be important in the early stages of a disorder where intermittent or small leaks may be present. The half life of CAIII in plasma has not yet been determined, but that of CK is 1-2 days (Wilkinson 1976) and of myoglobin 2~, h (Hallgren et al. 1978). The CAIII determination is particularly valuable in neuromuscular disorders where the CK is normal. When the CK is increased the CAIII is nearly always similarly raised. It would be of value to measure the levels of CAII1 in other neuromuscular disorders and to assess the effect of therapy on these levels.

388 REFERENCES Askmark, H., P. O. Ostermann, L. E. Roxin and P. Venge (1981) Radioimmunoassay of serum myoglobin in neuromuscular diseases, Neurol. Neurosurg. Psychiat., 44: 68-72. Carter, N. D., S. Jeffery, A. Shiels, Y. Edwards, T. Tipler and D.A. Hopkinson (1979) Characterization of human carbonic anhydrase III from skeletal muscle, Biochem. Genet., 17: 837-854. Carter, N.D., R. Heath, S. Jeffery and C. Rodeck 0982) Fetal plasma carbonic anhydrase IlI in Duchenne dystrophy, Lancet, i: 39-40. Corbett, A.J., R.C. Griggs and R.T. Moxley, llI (1982) Skeletal muscle catabolism in amyotrophic lateral sclerosis and chronic spinal muscular atrophy, Neurology (Minneap.), 32: 550-552. Hallgren, R.F.A., L.E. Roxin and P. Venge 0978) Myoglobin turnover - Influence of renal and extrarenal factors, J. Lab. clin. Med., 91 : 246-254. Heath, R., I. Brown, S. Jeffery and N.D. Carter (1982a) Radioimmunoassay of carbonic anhydrase III levels in plasma as an index of tissue damage, Biochem. Soc. Trans., 10:109-110. Heath, R., S. Jeffery and N.D. Carter (1982b) Radioimmunoassay of human carbonic anhydrase Ill in dystrophic states, Clin. chim. Acta, 119: 299-305. Kiessling, W. R. and R. Beckmann (1981) Duchenne muscular dystrophy Does serum myoglobin correlate with serum creatine kinase? Muscle & Nerve, 4: 257. Marsh, W.L., N.J. Marsh, A. Moore, W.A. Symmans, C.L. Johnson and C.M. Redman (1981) Elevated serum creatine phosphokinase in subjects with McLeod syndrome, l/ox Sang. (Basel), 40:403-411. Nicholson, L.V.B. (1981) Serum myoglobin in muscular dystrophy and carrier detection, J. neurol. Sci., 51: 411-426. Rosalki, S.B. (1976) Enzymes in diseases of skeletal muscle. In: J.H. Wilkinson (Ed.), The Principles and Practice of Diagnostic Enzymology, Edward Arnold, London, pp. 263-302. Rowland, L.P. (1980) Biochemistry of muscle membranes in Duchenne muscular dystrophy, Muscle & Nerve, 3: 3-20. Schwartz, M.S., M.K. Sargeant and M. Swash (1976) Longitudinal fibre splitting in neurogenic muscular disorders - - Its relation to the pathogenesis of "myopathic" change, Brain, 99: 617-636. Schwartz, M.S., M. Swash and M. Gross (1978) Benign post infection polymyositis, Brit. reed. J., II: 1256-1257. Wilkinson, J.H. (1976) General considerations. In: J. H. Wilkinson (Ed.), The Principles and Practice of Diagnostic Enzymology, Edward Arnold, London, pp. 221-240.