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γ-Glutamyl transpeptidase
Journal of the Neurological Sciences, 1976, 28:225-231 ,~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
225
7-GLUTAMYL TRANSPEPTIDASE Elevated Activity in Myotonic Dystrophy
BASIL ALEVIZOS*, MICHAEL SPENGOS, DEMETRIS VASSILOPOULOS and COSTAS STEFANIS Department of Neurology and Psychiatry, University of Athens, Aeginition Hospital, Athens (Greece)
(Received 7 October, 1975)
SUMMARY 7-Glutamyl transpeptidase, a membrane-bound enzyme playing an important role in the active amino acid transport across cellular membranes, is shown to be elevated in the serum of patients with myotonic muscular dystrophy. No increase of AP, LAP, G O T and G P T activities in the sera of some of the patients studied is observed. Possible interpretations in relation to the pathogenesis of myotonic dystrophy are discussed.
INTRODUCTION Myotonic muscular dystrophy is an autosomal dominant disease with clinical manifestations in many organ systems. Cataract, frontal baldness, testicular atrophy, muscular dystrophy, myotonia, skeletal changes, cardiac abnormalities and mental deterioration are the more common symptoms of the disease. Myotonic dystrophy as well as other muscular dystrophies, was believed to be a primary disease of muscle but it has been suggested that it may be neurogenic (McComas and Mossawy 1965; McComas, Sica and Campbell 1971). The evidence for and against a neurogenic component in muscular dystrophies has since been the subject of considerable controversy (Bradley 1971; Emery and Gosden 1974). Lately the possibility that myotonic dystrophy is a generalized metabolic disorder has been raised and evidence of a systematic defect in certain cellular membranes * Present address: Institute of Psychiatry, Department of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, Great Britain.
226 has been presented (Roses and Appel 1973; Roses and Appel i974: ~taterfieki. Chesnut, Roses and Appel 1974). The basic metabolic error in the disease has not been elucidated, We assumed that the metabolic defect in myotonic dystrophy might lie in the 7-glutamyl cycle (Orlowski and Meister 1970) since many clinical manifestations ¢~1~ the disease are expressed in organ systems where the enzyme y-glutamyt transpeptidase (7-GT) is localized in high concentrations. Furthermore, 7-GT is a membrane-bound enzyme and this would be consistent with the evidence of systematic defect in cell membranes in myotonic dystrophy. ?,-GT is associated with cell membranes of different tissues, parJ!cularly in cells, where high rates of amino acid transport are observed. The proposed function in the kidney is resorption of amino acids with glutathione serving as a carrier. Thus, the enzyme is concentrated in the brush border m the proximal convoluted tubules of the kidney, the apical portion of the epithelial cells covering the jejunal villi, the glandular epithelium of the seminal ve~icles, epididymus, Fallopian tubes, prostate, endometrium, lactating breast, bronchi~ thyroid, hypophysis, the endothelial cells of brain capillaries and choroid plexus and the tens. 7-GT plays a key role in the/-glutamyl cycle, proposed ab a system f,w amino acid transport (Orlowski and Meister 1970) and catalyses transfer of the !,-glulamyi residue of glutathione to amino acid as follows: Glutathione } amino acid :: 7-glutamyl amino acid i cysteinytgtycine. It is proposed that this reaction functions in amino acid transport Raised v-GT activity is fotmd in cholestasis (Ideo, Morganti and l)ioguardi 1972), in liver damage (Rutenberg, Goldbarg and Pineda 1963), in aicohol consumption (Rosalki and Rau 1972), in cases of symptomatic porphyria cumnea tarda (Adjarov and Iwanow 1973), in myocardial infarction (Szczeklik, Ori0wski and Dyczkowska 1969), in epilepsy (Ewen and Griffiths 1973) and after iaking antiepileptic drugs (Rosalki, Tarlow and Rau i971). METHODS
Subjects were selected on the basis of the clinical criteria and posture results of laboratory tests for myotonic dystrophy, so diagnosed by two neurologists. Myotonia was a prominent symptom in all subjects. Cataract, testicular atrophy, frontal baldness, muscular weakness and atrophy were all presentl Cataract was detected by slit lamp. Potential subjects suffering from alcoholism, li~er disease or recent myocardial infarction were excluded. No subject had received barbiturates or phenytoin within 2 months or other drugs within I month of the examination period. Blood was taken as soon as possible after admission and 7-GT activity wa~ measured at 25 °C on the basis of the Szasz method (Szasz 1969) using ),-glutamylp-nitroanilide as substrate. The increase in optical density at 405 nm due to tile formation of p-nitroaniline was directly proportional to the y-GT activity. The reagents used were supplied by Boehringer Mannheim GmbH in the form of a test
227 TABLE 1 AGE, SEX, DURATION, 7-GT, AP, LAP, GOT, GPT AND CPK ACTIVITIES IN SUBJECTS WITH MUSCULAR MYOTONIC DYSTROPHY Case No.
Age (years)
Sex
Duration ~-GT AP LAP GOT GPT CPK of disease activity (years) (rnU/ml) (mU/ml) (mU/ml) (mU/ml) (mU/ml) (mU/ml)
1 2 3 4 5 6 7 8 9 10 II 12 13
31 30 34 29 37 35 43 23 33 34 14 17 38
M M M M M M F M F F F F F
1 I 6 2 1 5 25 1 8 5 2 4 4
32.32 10.23 22.14 17.13 25.06 63.59 20.01 9.69 23.43 10.50 16.16 14.55 51.87
21 18.5 23 17 19
24
8.8 1l 6.5 4.3 9.2
7.5
6.2 7.2 I1 7.2 4.6
16
7.8 9.6 4.5 6.5 14
10 59 16 105 45
9.2
kit for 7 - G T d e t e r m i n a t i o n ( ~ - G T Monotest Cat. No. 15885). Solutions were prepared as r e c o m m e n d e d a n d the reaction started by the a u t o m a t i c a d d i t i o n of 0.2 ml serum. By d e t e r m i n i n g the m e a n optical density difference per m i n 7 - G T activity in m U / m l was calculated. N o r m a l values were established on 13 healthy volunteers (6 males, 7 females) a n d f o u n d to be 6.06-11.44 m U / m l . In some subjects (Cases 1, 2, 3, 4, 5 a n d 9) alkaline phosphatase (AP) and leucina m i n o p e p t i d a s e (LAP) activity were estimated as cholestatic markers a n d aspartate aminotransferase ( G O T ) a n d alanine aminotransferase ( G P T ) activity, the increase of which is indicative of liver cellular injury were determined as well as creatine phosphokinase (CPK) activity (Cases No. 1,2, 3, 4 a n d 5). RESULTS The findings are shown in Table 1 which gives each subject's age, sex, d u r a t i o n of the disease, 7 - G T activity a n d AP, LAP, G O T , G P T a n d C P K activities.
TABLE 2 VALUES OF SERUM 7-GT ACTIVITY IN MYOTONIC DYSTROPHY AND NORMALS
No. of cases Mean ± SD Range
Normals
Patients
13 8.97 ± 1.86 6.06-11.44
13 24.36 ~_ 16.35 9.69-63.59
Significance of difference
P < 0.01 (t = 3.372)
228 Table 2 compares 7-GT activity in normal subjects with that in sut!iect,~ ~.ho have myotonic dystrophy. The results show that the serum ?,-GT activity is ,.-aised in myotonic dystrophic cases compared to controls. The mean value for the tormer was 24.36 ~ SD 16.35. The mean for the latter was 8.97 i: SD 1.86 (P i).ul). No increase of the activity was shown in I case of Thomsen's disease (8.S,'~, mU/ml)o 1 of Welander's disease (5.92 mI.J/ml) and t of non-specific muscular Wa~illg (7.54 mU/ml). The activities of AP, LAP, GOT, GPT and CPK were normal. In our i:tborator3 the normal values for AP are up to 30 mU/ml and for LAP, GOT and GP-I up t~ 20 mU/ml. No significant correlation was found between ),-GT and CPK Cataract was present in 8 out of 13 subjects. EMG showed myotonic discharges in all subjects and slight myopathic changes in some of them (Cases 3, 6, 7.9 and 12). DISCUSSION
Ten out of 13 subjects with myotonic dystrophy showed an increase m >,-G] activity. In those patients with normal activity no special clinical characteristics were observed. it could be argued that the raised v-GT activity is connected with the pathogenesis of myotonic dystrophy as an abnormal function of the 7-glutamyl cycle. This would be in agreement with the clinical manifestations of the disea.~e. As mentioned above, many symptoms of myotonic dystrophy are manifested in organs with high concentrations of 7-GT. The kidney contains very high levels of T-GT and other enzymes of the 7-gtutamyl cycle (7-glutamyl cyclotransferase, 7-glutamyl cysteine synthetase) and this is consistent with the specialized function of the kidney in the reabsorption of amino acids, thus avoiding the loss of these metabolites (Meister 1973). Furthermore, the abovementioned enzymes are localized in the brush border of the proximal convoluted tubule which is believed to be the main site of the reabsorption of amino acids. Aminoaciduria has been demonstrated in myotonic dystrophy (Emery and Burt 1972). They found that the excretion of glutamine is more excessive and this agrees with the finding that 7-GT is more active toward glutamine. In addition, competitive phenomena for the enzyme may occur, as infusion of any individual amino acid leads to generalized aminoaciduria. 7-GT is found in high levels in seminal vesicles, epididymus, Fallopian tubes and prostate, and hypogonadism is one of the main clinical manifestations of the disease. In the lens (bovine and rabbit), },-GT and cyclotransferase was found in high concentrations in the capsule epithelium and the cortical fraction as well as extremely high levels of glutathione, which has a high turnover rate (Rathbun and Wicker 1973; Reddy and Unakar 1973). Epstein and Kinoshita (1970)demonstrated glutathione to be necessary for maintaining normal lens membrane functiom Kern and Ho (1973) have further demonstrated transport of glutamic acid and glutamine and
229 incorporation into lenticular glutathione. This transport process is located in the single layer of epithelium across the anterior surface. Furthermore glutathione concentration decreases in the lens with the course of cataract formation in man (Friedburg and Manthey 1973). ~-GT activity has also been demonstrated in the thyroid gland. Thyroid abnormalities have been described in myotonic dystrophy, particularly thyroid enlargement without clinical evidence of abnormal thyroid formation (Lee and Hughes 1964). A number of cases of myxoedema have been reported as well. In the bronchi, the enzyme is bound in the epithelial membrane and its presence in high concentrations in purulent sputum of patients with chronic bronchitis suggests abnormal release due to destruction of epithelial cells (Barton, Powers and lurenco 1974). Chest infections are common in myotonic dystrophy and they have been attributed to paralysis of respiratory muscles. However, bronchitis, which usually occurs, cannot be sufficiently explained by muscular weakness. Kilburn, Eagan, Sieker and Heyman (1959) carried out lung function tests on two groups of patients, one with and one without myotonia, and found mechanical function to be impaired in both groups but impairment was more marked in the group without myotonia, although carbon dioxide retention and anoxaemia occurred more severely in the myotonic group. Whether chest infections in myotonic dystrophy result from paralysis of respiratory muscles or a defective ~-glutamyl cycle participates, remains to be determined. Assuming that the v-GT is primarily involved in myotonic dystrophy, the problem arises as to why the increase was not seen in all subjects studied. The possibility that there may be different forms of myotonic dystrophy is unlikely as no difference in the clinical profile of patients with normal v-GT activity has been observed. A second explanation could be that the abnormality is not yet apparent in the serum. Study of the enzyme in other tissues or body fluids (white blood cells, urine, CSF, etc.) would be necessary to clarify this point. Another possibility for the increased v-GT activity in myotonic dystrophy could be that the membrane-bound enzyme is shedding from the damaged membrane, originally affected, and the clinical expression in the above-mentioned organ systems is due to the reduced functioning of these organs as a consequence of the disturbance in amino acid transport. A third possibility might be that the increased activity results from cholestasis or liver damage in patients with myotonic dystrophy, but no increase of AP, LAP, GOT and GPT activities was found in the sera of 6 patients studied. The enzyme is absent from skeletal muscle and in spite of the suggestion that in patients with Duchenne's muscular dystrophy enzymes are released into the serum not only from skeletal muscle and the heart but also from the liver (Kleine 1970), no increased v-GT activity was revealed in patients with Duchenne muscular dystrophy (Rosalki and Thomson 1971). However, more detailed testing of liver functioning could be necessary for this possibility to be tested. A fourth possibility is that microsomal enzyme induction, obviously genetic in its cause, is responsible for the increased 7'-GT activity. Martin, Martin and
230 G o l b e r g (1975) found a significant positive correlation between serum 7-(i¢[ activit) and serum triglyceride c o n c e n t r a t i o n and suggested that this may rellect hepatic microsomal enzyme i n d u c t i o n in hyperlipidaemic subjects. L a m b and F:allon (19721 have shown that carbohydrates increase triglyceride levels and evidence ha~ been presented that this is due to microsomal enzyme induction. Increased t~g~yceride levels have been d e m o n s t r a t e d in myotonic dystrophy ( A n d i m a n , Dhopeshwarkar~ C a m p i o n and Peter 1974), and a b n o r m a l i t y in carbohydrate metabolism has been shown as well. Diabetes mellitus occurs occasionally and i m p a i r m e n t ~,f glucose tolerance is quite c o m m o n in this disease (Lee and Hughes 1964: Caughey and P a c h o m o v 1961). It is also of interest to note that antiepileptic drugs, in particula~ h y d a n t o i n and p h e n o b a r b i t o n e , which are k n o w n to induce microsomai enzymes (Dent, Richens, Rowe and Stamp 1970), have a beneficial effect in myotonic dystrophy. Finally, the nature of the increased ),-GT activity in subjects with myt~tonic dystrophy is not evident in this study. F u r t h e r work is p l a n n e d to clarify ,~!w role of ?,-GT in the pathogenesis of m y o t o n i c dystrophy.
REFERENCES Adjarov, D. and A. D. lwanow (19731 Neue Aspekte der klinischen Bedeutung der ?~-Glutamyltranspeptidasebestimmung in Serurn, Acta hepato-gastroenterol., 20:315 324. Andiman, R. M., G. Dhopeshwarkar, D. C. Campion and J. B. Peter (1974) Myotonic dystrophy and myotonia congenita - - ATPase and lipid composition of erythrocyte membranes and serum lipids with special reference to desmosterol. In: W. G. Bradley, D. Gardner-Medwin and J. N. Walton (Eds.), Recent Advances in Myolog3, (Proceedings of the 3rd International Congress on Muscle Diseases, Newcastle upon Tyne, September, 19741 (International Congres~ Series No. 360), Excerpta Medica, Amsterdam, 1975, p. 346. Barton, A., J. L. Powers and R. V. Lurenco (1974) Gamma-glutamyl transpeptida,se m chronic obstructive pulmonary disease, Proc. Soc. exp. Biol. Med., 149: 99-103. Bradley, W. G. (1971) Nerve, muscle and muscular dystrophy; Develop. Med. (Vlihl Nero:o/., 13: 528-531. Butterfield, D. A., D. B. Chesnut, A. D. Roses and S. H. Appel (19741 Electron spin resonance studies of erythrocytes from patients with myotonic muscular dystrophy, Proc. nat_ Acad. Sci. (Wash.), 71 : 90% 913. Caughey, J. E. and N. Pachomov (19611 Carbohydrate metabolism in patients with dystrophia myotonica, N.Z. reed. J., 60:376 382. Dent, C. E., A. Richens, D. J. F, Rowe and T. C. B. Stamp (197010steomalacia with long-term anticonvulsant therapy in epilepsy, Brit. reed. J., 4:69 72. Emery, A. E. H.and D. Burt (1972) Amino acid, creatine and creatinine studies in myotonic dystrophy, Clin. chim. Acta, 39: 361-36L Emery A. E. H. and C. Gosden (1974) A neurogenic component in muscular dystro!3hy, .1. reed. Genet., 11:76 79. Epstein, D. L. and J. H. Kinoshita (1970) The effect of diamide on lens glutathione and lens membrane function, Invest. Ophthal., 9: 629-638. Ewen, L. M. and J. Griffiths (1973) F-Glutamyl transpeptidase . Elevated activities in certain neurologic diseases, Amer. J. clot, Path., 59: 2-9. Friedburg, D. and K.-F. Manthey (1973) Glutathione and NADP linked enzymes in human senile cataract, Exp. Eye Res., 15:173 177~ ld6o, G., A. Morganti and N. Dioguardi (1972)?,-Glutamyl transpeptidase- A clinicai and experimental study, Digestion, 5:326 336. Kern, H. L. and C.-K. Ho (1973) Transport of L-glutamic acid. L-glutamine and their incorporation into lenticular glutathione, Exp. Eye Res., 17: 455-462.
231 Kilburn, K. H., J. T. Eagan, H. O. Sieker and A. Heyman (1959) Cardiopulmonary insufficiency associated with myotonic dystrophy, New Engl. J. Med., 261 : 1089-1096. Kleine, T. O. (1970) Evidence for the release of enzymes from different organs in Duchenne's muscular dystrophy, Clin. chim. Acta, 29: 227-231. Lamb, R. G. and H. J. Fallon (1972) An enzymatic explanation for increased hepatic triglyceride formation in rats fed high sugar diets, J. clin. Invest., 51 : 53a. Lee, F. I. and D. T. D. Hughes (1964) Systematic effects in dystrophia myotonica, Brain, 87: 521-536. McComas, A. J. and S. J. Mossawy (1965) Research in Muscular Dystrophy, (Proceedings of 3rd Symposium of the Muscular Dystrophy Group), Pitman, London, p. 317. McComas, A. J., R. E. P. Sica and M. J. Campbell (1971) "Sick" mononeurones - - A unifying concept of muscle disease, Lancet, 1 : 321-325. Martin, P. J., J. V. Martin and D. M. Golberg (1975) 7-Glutamyl transpeptidase, triglycerides and enzyme induction, Brit. med. J., 1 : 17-19. Meister, A. (1973) On the enzymology of amino acid transport, Science, 180:33 39. Orlowski, M. and A. Meister (1970) The 7-glutamyl cycle - - A possible transport system for amino acids, Proc. nat. Acad. Sci. (Wash.), 67: 1248-1255. Rathbun, W. B. and K. Wicker (1973) Bovine lens 7-glutamyl transpeptidase, Exp. Eye Res., 15: 161 171. Reddy, V. N. and N. J. Unakar (1973) Localization of 7-glutamyl transpeptidase in rabbit lens, ciliary process and cornea, Exp. Eye Res., 17: 405-408. Rosalki, S. B. and D. Rau (1972) Serum 7-glutamyl transpeptidase activity in alcoholism, Clin. chim. Acta, 39: 4147. Rosalki, S. B. and W. H. S. Thomson (1971) Serum gamma-glutamyl transpeptidase in muscle disease, Clin. chim. Acta, 33: 264. Rosalki, S. B., D. Tarlow and D. Rau (1971) Plasma gamma-glutamyl transpeptidase elevation in patients receiving enzyme-inducing drugs, Lancet, 2:376 377. Roses, A. D. and S. H. Appel (1973) Protein kinase activity in erythrocyte ghosts of patients with myotonic muscular dystrophy, Proc. Nat. Acad. Sci. (Wash.), 70: 1855-1859. Roses, A. D. and S. H. Appel (1974) Muscle membrane protein kinase in myotonic muscular dystrophy, Nature (Lond.), 250: 245-247. Rutenberg, A. M., J. A. Goldbarg and E. R. Pineda (1963) Serum 7-glutamyl transpeptidase activity in hepatobiliary pancreatic disease, Gastroenterology, 45: 4348. Szasz, G. (1969) A kinetic photometric method for serum 9J-glutamyl transpeptidase, Clin. Chem., 15:124 136. Szczeklik, E., M. Orlowski and M. Dyczkowska (1969) The late enzymic test of myocardial infarction, Pol. reed. J., 8: 14-20.
225
7-GLUTAMYL TRANSPEPTIDASE Elevated Activity in Myotonic Dystrophy
BASIL ALEVIZOS*, MICHAEL SPENGOS, DEMETRIS VASSILOPOULOS and COSTAS STEFANIS Department of Neurology and Psychiatry, University of Athens, Aeginition Hospital, Athens (Greece)
(Received 7 October, 1975)
SUMMARY 7-Glutamyl transpeptidase, a membrane-bound enzyme playing an important role in the active amino acid transport across cellular membranes, is shown to be elevated in the serum of patients with myotonic muscular dystrophy. No increase of AP, LAP, G O T and G P T activities in the sera of some of the patients studied is observed. Possible interpretations in relation to the pathogenesis of myotonic dystrophy are discussed.
INTRODUCTION Myotonic muscular dystrophy is an autosomal dominant disease with clinical manifestations in many organ systems. Cataract, frontal baldness, testicular atrophy, muscular dystrophy, myotonia, skeletal changes, cardiac abnormalities and mental deterioration are the more common symptoms of the disease. Myotonic dystrophy as well as other muscular dystrophies, was believed to be a primary disease of muscle but it has been suggested that it may be neurogenic (McComas and Mossawy 1965; McComas, Sica and Campbell 1971). The evidence for and against a neurogenic component in muscular dystrophies has since been the subject of considerable controversy (Bradley 1971; Emery and Gosden 1974). Lately the possibility that myotonic dystrophy is a generalized metabolic disorder has been raised and evidence of a systematic defect in certain cellular membranes * Present address: Institute of Psychiatry, Department of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, Great Britain.
226 has been presented (Roses and Appel 1973; Roses and Appel i974: ~taterfieki. Chesnut, Roses and Appel 1974). The basic metabolic error in the disease has not been elucidated, We assumed that the metabolic defect in myotonic dystrophy might lie in the 7-glutamyl cycle (Orlowski and Meister 1970) since many clinical manifestations ¢~1~ the disease are expressed in organ systems where the enzyme y-glutamyt transpeptidase (7-GT) is localized in high concentrations. Furthermore, 7-GT is a membrane-bound enzyme and this would be consistent with the evidence of systematic defect in cell membranes in myotonic dystrophy. ?,-GT is associated with cell membranes of different tissues, parJ!cularly in cells, where high rates of amino acid transport are observed. The proposed function in the kidney is resorption of amino acids with glutathione serving as a carrier. Thus, the enzyme is concentrated in the brush border m the proximal convoluted tubules of the kidney, the apical portion of the epithelial cells covering the jejunal villi, the glandular epithelium of the seminal ve~icles, epididymus, Fallopian tubes, prostate, endometrium, lactating breast, bronchi~ thyroid, hypophysis, the endothelial cells of brain capillaries and choroid plexus and the tens. 7-GT plays a key role in the/-glutamyl cycle, proposed ab a system f,w amino acid transport (Orlowski and Meister 1970) and catalyses transfer of the !,-glulamyi residue of glutathione to amino acid as follows: Glutathione } amino acid :: 7-glutamyl amino acid i cysteinytgtycine. It is proposed that this reaction functions in amino acid transport Raised v-GT activity is fotmd in cholestasis (Ideo, Morganti and l)ioguardi 1972), in liver damage (Rutenberg, Goldbarg and Pineda 1963), in aicohol consumption (Rosalki and Rau 1972), in cases of symptomatic porphyria cumnea tarda (Adjarov and Iwanow 1973), in myocardial infarction (Szczeklik, Ori0wski and Dyczkowska 1969), in epilepsy (Ewen and Griffiths 1973) and after iaking antiepileptic drugs (Rosalki, Tarlow and Rau i971). METHODS
Subjects were selected on the basis of the clinical criteria and posture results of laboratory tests for myotonic dystrophy, so diagnosed by two neurologists. Myotonia was a prominent symptom in all subjects. Cataract, testicular atrophy, frontal baldness, muscular weakness and atrophy were all presentl Cataract was detected by slit lamp. Potential subjects suffering from alcoholism, li~er disease or recent myocardial infarction were excluded. No subject had received barbiturates or phenytoin within 2 months or other drugs within I month of the examination period. Blood was taken as soon as possible after admission and 7-GT activity wa~ measured at 25 °C on the basis of the Szasz method (Szasz 1969) using ),-glutamylp-nitroanilide as substrate. The increase in optical density at 405 nm due to tile formation of p-nitroaniline was directly proportional to the y-GT activity. The reagents used were supplied by Boehringer Mannheim GmbH in the form of a test
227 TABLE 1 AGE, SEX, DURATION, 7-GT, AP, LAP, GOT, GPT AND CPK ACTIVITIES IN SUBJECTS WITH MUSCULAR MYOTONIC DYSTROPHY Case No.
Age (years)
Sex
Duration ~-GT AP LAP GOT GPT CPK of disease activity (years) (rnU/ml) (mU/ml) (mU/ml) (mU/ml) (mU/ml) (mU/ml)
1 2 3 4 5 6 7 8 9 10 II 12 13
31 30 34 29 37 35 43 23 33 34 14 17 38
M M M M M M F M F F F F F
1 I 6 2 1 5 25 1 8 5 2 4 4
32.32 10.23 22.14 17.13 25.06 63.59 20.01 9.69 23.43 10.50 16.16 14.55 51.87
21 18.5 23 17 19
24
8.8 1l 6.5 4.3 9.2
7.5
6.2 7.2 I1 7.2 4.6
16
7.8 9.6 4.5 6.5 14
10 59 16 105 45
9.2
kit for 7 - G T d e t e r m i n a t i o n ( ~ - G T Monotest Cat. No. 15885). Solutions were prepared as r e c o m m e n d e d a n d the reaction started by the a u t o m a t i c a d d i t i o n of 0.2 ml serum. By d e t e r m i n i n g the m e a n optical density difference per m i n 7 - G T activity in m U / m l was calculated. N o r m a l values were established on 13 healthy volunteers (6 males, 7 females) a n d f o u n d to be 6.06-11.44 m U / m l . In some subjects (Cases 1, 2, 3, 4, 5 a n d 9) alkaline phosphatase (AP) and leucina m i n o p e p t i d a s e (LAP) activity were estimated as cholestatic markers a n d aspartate aminotransferase ( G O T ) a n d alanine aminotransferase ( G P T ) activity, the increase of which is indicative of liver cellular injury were determined as well as creatine phosphokinase (CPK) activity (Cases No. 1,2, 3, 4 a n d 5). RESULTS The findings are shown in Table 1 which gives each subject's age, sex, d u r a t i o n of the disease, 7 - G T activity a n d AP, LAP, G O T , G P T a n d C P K activities.
TABLE 2 VALUES OF SERUM 7-GT ACTIVITY IN MYOTONIC DYSTROPHY AND NORMALS
No. of cases Mean ± SD Range
Normals
Patients
13 8.97 ± 1.86 6.06-11.44
13 24.36 ~_ 16.35 9.69-63.59
Significance of difference
P < 0.01 (t = 3.372)
228 Table 2 compares 7-GT activity in normal subjects with that in sut!iect,~ ~.ho have myotonic dystrophy. The results show that the serum ?,-GT activity is ,.-aised in myotonic dystrophic cases compared to controls. The mean value for the tormer was 24.36 ~ SD 16.35. The mean for the latter was 8.97 i: SD 1.86 (P i).ul). No increase of the activity was shown in I case of Thomsen's disease (8.S,'~, mU/ml)o 1 of Welander's disease (5.92 mI.J/ml) and t of non-specific muscular Wa~illg (7.54 mU/ml). The activities of AP, LAP, GOT, GPT and CPK were normal. In our i:tborator3 the normal values for AP are up to 30 mU/ml and for LAP, GOT and GP-I up t~ 20 mU/ml. No significant correlation was found between ),-GT and CPK Cataract was present in 8 out of 13 subjects. EMG showed myotonic discharges in all subjects and slight myopathic changes in some of them (Cases 3, 6, 7.9 and 12). DISCUSSION
Ten out of 13 subjects with myotonic dystrophy showed an increase m >,-G] activity. In those patients with normal activity no special clinical characteristics were observed. it could be argued that the raised v-GT activity is connected with the pathogenesis of myotonic dystrophy as an abnormal function of the 7-glutamyl cycle. This would be in agreement with the clinical manifestations of the disea.~e. As mentioned above, many symptoms of myotonic dystrophy are manifested in organs with high concentrations of 7-GT. The kidney contains very high levels of T-GT and other enzymes of the 7-gtutamyl cycle (7-glutamyl cyclotransferase, 7-glutamyl cysteine synthetase) and this is consistent with the specialized function of the kidney in the reabsorption of amino acids, thus avoiding the loss of these metabolites (Meister 1973). Furthermore, the abovementioned enzymes are localized in the brush border of the proximal convoluted tubule which is believed to be the main site of the reabsorption of amino acids. Aminoaciduria has been demonstrated in myotonic dystrophy (Emery and Burt 1972). They found that the excretion of glutamine is more excessive and this agrees with the finding that 7-GT is more active toward glutamine. In addition, competitive phenomena for the enzyme may occur, as infusion of any individual amino acid leads to generalized aminoaciduria. 7-GT is found in high levels in seminal vesicles, epididymus, Fallopian tubes and prostate, and hypogonadism is one of the main clinical manifestations of the disease. In the lens (bovine and rabbit), },-GT and cyclotransferase was found in high concentrations in the capsule epithelium and the cortical fraction as well as extremely high levels of glutathione, which has a high turnover rate (Rathbun and Wicker 1973; Reddy and Unakar 1973). Epstein and Kinoshita (1970)demonstrated glutathione to be necessary for maintaining normal lens membrane functiom Kern and Ho (1973) have further demonstrated transport of glutamic acid and glutamine and
229 incorporation into lenticular glutathione. This transport process is located in the single layer of epithelium across the anterior surface. Furthermore glutathione concentration decreases in the lens with the course of cataract formation in man (Friedburg and Manthey 1973). ~-GT activity has also been demonstrated in the thyroid gland. Thyroid abnormalities have been described in myotonic dystrophy, particularly thyroid enlargement without clinical evidence of abnormal thyroid formation (Lee and Hughes 1964). A number of cases of myxoedema have been reported as well. In the bronchi, the enzyme is bound in the epithelial membrane and its presence in high concentrations in purulent sputum of patients with chronic bronchitis suggests abnormal release due to destruction of epithelial cells (Barton, Powers and lurenco 1974). Chest infections are common in myotonic dystrophy and they have been attributed to paralysis of respiratory muscles. However, bronchitis, which usually occurs, cannot be sufficiently explained by muscular weakness. Kilburn, Eagan, Sieker and Heyman (1959) carried out lung function tests on two groups of patients, one with and one without myotonia, and found mechanical function to be impaired in both groups but impairment was more marked in the group without myotonia, although carbon dioxide retention and anoxaemia occurred more severely in the myotonic group. Whether chest infections in myotonic dystrophy result from paralysis of respiratory muscles or a defective ~-glutamyl cycle participates, remains to be determined. Assuming that the v-GT is primarily involved in myotonic dystrophy, the problem arises as to why the increase was not seen in all subjects studied. The possibility that there may be different forms of myotonic dystrophy is unlikely as no difference in the clinical profile of patients with normal v-GT activity has been observed. A second explanation could be that the abnormality is not yet apparent in the serum. Study of the enzyme in other tissues or body fluids (white blood cells, urine, CSF, etc.) would be necessary to clarify this point. Another possibility for the increased v-GT activity in myotonic dystrophy could be that the membrane-bound enzyme is shedding from the damaged membrane, originally affected, and the clinical expression in the above-mentioned organ systems is due to the reduced functioning of these organs as a consequence of the disturbance in amino acid transport. A third possibility might be that the increased activity results from cholestasis or liver damage in patients with myotonic dystrophy, but no increase of AP, LAP, GOT and GPT activities was found in the sera of 6 patients studied. The enzyme is absent from skeletal muscle and in spite of the suggestion that in patients with Duchenne's muscular dystrophy enzymes are released into the serum not only from skeletal muscle and the heart but also from the liver (Kleine 1970), no increased v-GT activity was revealed in patients with Duchenne muscular dystrophy (Rosalki and Thomson 1971). However, more detailed testing of liver functioning could be necessary for this possibility to be tested. A fourth possibility is that microsomal enzyme induction, obviously genetic in its cause, is responsible for the increased 7'-GT activity. Martin, Martin and
230 G o l b e r g (1975) found a significant positive correlation between serum 7-(i¢[ activit) and serum triglyceride c o n c e n t r a t i o n and suggested that this may rellect hepatic microsomal enzyme i n d u c t i o n in hyperlipidaemic subjects. L a m b and F:allon (19721 have shown that carbohydrates increase triglyceride levels and evidence ha~ been presented that this is due to microsomal enzyme induction. Increased t~g~yceride levels have been d e m o n s t r a t e d in myotonic dystrophy ( A n d i m a n , Dhopeshwarkar~ C a m p i o n and Peter 1974), and a b n o r m a l i t y in carbohydrate metabolism has been shown as well. Diabetes mellitus occurs occasionally and i m p a i r m e n t ~,f glucose tolerance is quite c o m m o n in this disease (Lee and Hughes 1964: Caughey and P a c h o m o v 1961). It is also of interest to note that antiepileptic drugs, in particula~ h y d a n t o i n and p h e n o b a r b i t o n e , which are k n o w n to induce microsomai enzymes (Dent, Richens, Rowe and Stamp 1970), have a beneficial effect in myotonic dystrophy. Finally, the nature of the increased ),-GT activity in subjects with myt~tonic dystrophy is not evident in this study. F u r t h e r work is p l a n n e d to clarify ,~!w role of ?,-GT in the pathogenesis of m y o t o n i c dystrophy.
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