SERUM ENZYMES—Variations of Activity in Disease of Muscle

In a study of 58 patients with various diseases of muscle or of the neuromuscular system, the serum activity of various enzymes was measured. Abnormal elevation of serum activities of aldolase, lactic dehydrogenase and, to a lesser extent, glutamic-oxalacetic transaminase and phosphohexose isomerase, was an almost constant feature in patients with progressive muscular dystrophy. These elevations were very frequent in dermatomyositis, common in acute cerebral vascular accidents, and rarely seen in other neurological disorders. Abnormal serum activity of iso-citric dehydrogenase was not observed in the course of the present study.Supplementary protein feeding of patients with muscular dystrophy had no effect on serum enzyme activity, no consistent effect on urinary creatine excretion and no effect on the strength of the patient or the course of the disease.Dystrophic muscles from a dystrophic strain of mice showed a decrease in activity of lactic dehydrogenase and aldolase below that of control muscle and an increase of iso-citric dehydrogenase activity. These findings, taken with the differences in serum activities of lactic dehydrogenase, aldolase and isocitric dehydrogenase in the dystrophic animals, support the conclusion that dystrophic animals handle these soluble enzymes in quite different ways.     

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in "fast-twitch" muscle fibers but has little effect on enzyme activities in "slow-twitch" muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

Content and synthesis of several abundant glycolytic enzymes in skeletal muscles of normal and dystrophic mice

• 1.1. In both 2- and 3-month-old 129 ReJ mice, the catalytic activity levels of three enzymes involved in glycogen breakdown (phosphorylase, enolase, and aldolase) were found to be 35–50% lower in hind limb muscles of dystrophic mice as compared with normal mice.
• 2.2. The reduced activities of these enzymes in the diseased tissue was directly due to corresponcling reductions in the number of enzyme molecules rather than being due to inactivation of the enzymes in the dystrophic muscle.
• 3.3. Results of short term double isotope incorporation experiments conducted with muscle expiants in vitro suggested that the rates of synthesis of these enzymes, and of most other abundant cytosolic proteins, relative to each other, were similar in hind limb muscles of normal and dystrophic mice.
• 4.4. The present work on murine muscular dystrophy is discussed in terms of our previous studies into the influence of avian muscular dystrophy on the content and synthesis of abundant glycolytic enzymes in chicken skeletal muscles.

     

Avian muscular dystrophy: Thyroidal influence on pectoralis muscle growth and glucose-6-phosphate dehydrogenase activity

The pectoralis muscles of dystrophic chickens (line 413) were hypertrophic on the basis of fresh weight and fat-free dry weight. They also had greater DNA content and greater glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities. Of the parameters measured, the largest differences between pectoralis muscles from dystrophic and normal (line 412) chickens were for DNA content and G6PD activity. These parameters were 4.3- and 6.7-fold, respectively, the values for control pectoralis at 5 wk of age. The average number of nuclei per unit length of isolated muscle fiber was also greater (approximately 3-fold) for the dystrophic pectoralis. Body weight and pectoralis fresh weight, fat-free dry weight, DNA content, G6PD activity and 6PGD activity were reduced significantly in propylthiouracil (PTU)-treated normal and dystrophic chickens. Moreover, the effects of PTU were more pronounced in the dystrophic strain. Thyroid deprivation significantly improved the righting ability of the dystrophic chickens, in addition to its influence on muscle hypertrophy and body growth. Thyroxine (T₄) replacement reversed the PTU effects in both strains. Of all the variables measured, total G6PD activity was the most affected by PTU treatment of dystrophic chickens and was only 16% of the control dystrophic value.In addition to the effects of thyroid deprivation on the expression of avian muscular dystrophy, we observed significant differences in thyroid-related variables in the two strains. The average thyroid weight at 4 wk and serum triiodothyronine level at 5 wk for dystrophic chickens were 65 and 76%, respectively, of the normal values. The results that we report here indicate that altered thyroid function affects the expression of avian muscular dystrophy.     

The influence of fasting and pancreatectomy on some glycolytic enzymes in the muscle of garden lizard, Calotes versicolor.

The leg muscle of Calotes versicolor consists mainly of white fibers. They can be classified into small, intermediate and broad fibers according to their diameter and staining reaction. The histochemical study of some of the glycolytic enzymes such as aldolase, phosphorylase, lactic dehydrogenase (LDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH) is described in the muscle of normal, pancreactomized and insulin injected animals. Highest activity of all enzymes was observed in the small fibers, though they occur in negligiable amount (3%). The intermediate and broad fibers exhibited medial and lowest activities respectively. 24 h after the extirpation of the pancreas a considerable inhibition is noted in the activities of phosphorylase, LDH and alpha-GPDH. Induced inanition for 21 days ensued insignificant but gradual decrease and increase in the alpha-GPDH and phosphorylase activities respectively in both unoperated and operated animals. The aldolase shows its mere presence in normal and trace activity in pancreatectomized animals. The injection of insulin (40 i.u./kg) at the interval of 30 min, 60 min, 4 h, 12 h and 24 h is given. The activities of all enzymes is stimulated at 30 min and depleted 4 h after the injection. The level of activity was remaintained at 12 h to 24 h stage. The possible physiological role and significance of enzymes in the synthesis and breakdown of glycogen is discussed.     

Enzyme alteration in skeletal muscle of mice with muscular dystrophy.

1. Developmental enzyme alterations were investigated in skeletal muscle of the hereditary progressive muscular dystrophy (PMD) mice of C57BL/6J strain. 2. Enzymes examined were classified into three groups according to changes of activities in dystrophy muscle during ageing. Activities of creatine kinase (EC 2.7.3.2), pyruvate kinase (EC 2.7.1.40), glycogen phosphorylase (EC 2.4.1.1), and fructose-biphosphate aldolase (EC 4.1.2.13), each of which had the respective muscle specific isoenzyme of extremely high activity in normal adult skeletal muscle, decreased rapidly in dystrophy muscle from the early stage of the disease with ageing. Activities of glycogen synthase (EC 2.4.1.11) and hexokinase (EC 2.7.1.1) were higher in dystrophy muscle in the early stage but decreased gradually to lower levels than those in the control with ageing. Activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were always much higher in dystrophy muscle than in the control, with no relation to ageing. 3. Isoenzymes of creatine kinase, pyruvate kinase and phosphorylase in dystrophy muscle were mainly the muscle types, indicating that muscle differentiation was not blocked profoundly even in dystrophy muscle. In limited cases, especially in the early stage of the disease, very weak activities of the non-muscle fetal type isoenzymes of creatine kinase and phosphorylase were detected, apparently associated with partial muscle regeneration in dystrophy muscle.     

A multivariate study on enzymatic changes in limb muscles and heart muscle of dystrophic mice

The present study was undertaken to elucidate further the enzymatic changes in dystrophic muscle using multivariate analysis. The activities of 14 kinds of enzymes, including 6 exopeptidases, 4 endopeptidases, beta-N-acetyl-D-glucosaminidase, phosphatase, esterase, and ribonuclease, were examined in forelimb and hindlimb muscles as well as in cardiac muscle of dystrophic mice and their controls. Two principal components identified from the enzymatic spectrum proved to be related especially to aminopeptidases and to serine proteinases, respectively. The enzymatic changes in forelimb muscle were very similar to those in hindlimb muscle when both were compared to those in cardiac muscle. The changes in aminopeptidases were unique to the limb muscles, whereas those of serine proteinases were unique to cardiac muscle of dystrophic mice. In the future, more attention should be focused on the role of exopeptidases in pathogenetic mechanisms of muscular dystrophy, because of the possibility that they play a major role in the initial stage of muscular dystrophy.     

Enzymological studies on hereditary avian muscular dystrophy.

White and red muscles of normal and genetically dystrophic chickens were compared with regards to activity levels of three soluble enzymes, glyceraldehyde-3-phosphate dehydrogenase, creatine phosphokinase, and acetyl phosphatase. In dystrophic white muscle (pectoral), activity of the two sulfhydryl enzymes, glyceraldehyde-3-phosphate dehydrogenase and creatine phosphokinase, was preferentially lost from the sarcoplasm resulting in decreased specific activities. By contrast, acetyl phosphatase was preferentially retained and showed increased specific activity. Dystrophic white muscle had decreased sulfhydryl content in the soluble proteins, severe reduction in muscle mass, fatty infiltration, and fragmentation of fibers. Red dystrophic muscles (thigh) were minimally involved in accordance with the known sparing of red fibers. Enzyme activities were correlated with histological observations. The results suggested that the disease process in dystrophic white muscle may be related to alterations in the sulfhydryl groups of proteins. The data are correlated with the beneficial effects of our treatment of hereditary avian dystrophy with the sulfhydryl compound, penicillamine (Chou, T.H., Hill, E.J., Bartle, E., Woolley, K., LeQuire, V., Olson, W., Roelofs, R., and Park, J.H. (1975) J. Clin. Invest. 56, 842-849).     

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.     

Dissociation of creatine kinase activity from hydrolytic enzyme activities in muscle of dystrophic mice

Time course studies were done to reexamine the age-dependency of intramuscular enzymatic changes in dystrophic mice. Most of the activities of hydrolytic enzymes in dystrophic mice were elevated in comparison to the controls throughout the span of 8 weeks which was examined. In contrast, the activity of creatine kinase remained depressed throughout the same period. This tendency was similarly seen in the muscles of forelimb and hindlimb but not in heart muscle. The observations are compatible with the notion that the increased activities of hydrolytic enzymes are causally related to the destruction of muscular tissue, leading to the malfunction of contractile machinery in the dystrophic muscles.     

Enzymuntersuchungen im Serum von schwachsinnigen Anstaltspatienten

Summary A number of enzymes alkaline and acid phosphatase, lactic acid dehydrogenase, glutamic oxalacetate transaminase (GOT), aldolase, glucose-6-phosphat dehydrogenase (G-6-PD) in the serum of 81 patients with Langdon Down's syndrome and mental deficiency (idiocy) were investigated and then compared with a sample of 37 healthy persons. The enzyme activities were determined spectrophotometrically.Statistically significant differences of alkaline and acid phosphatase, lactic acid dehydrogenase, and glutamic oxalacetate transaminase were demonstrated between oligophrenic patients and healthy persons; there was a distinct increase in the serum of patients. The values for aldolase and glucose-6-phosphat dehydrogenase were equal both in the serum of patients and of controls. There was no significant difference between the values of adult oligophrenics on one side and adult patients with Down's syndrome on the other. Until further notice children with serious mental deficiency — oligophrenics of unknown genesis as well as patients with Down's syndrome — demonstrated no definite evidence of enzymologically deviations compared with those of the control group.

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(Direktor: Prof. Dr. med. H. Schade)

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Nach einem am 22. 10. 1967 auf der 10. Tagung der Gesellschaft für Anthropologie und Humangenetik gehaltenen Vortrag.     

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

Increased contractility in vascular smooth muscle of dystrophic hamsters

To investigate the "vascular" hypothesis of muscular dystrophy, the sensitivity and contractility of aortic spiral strips of dystrophic (BIO 14.6) and normal (FIB) hamsters have been determined to various smooth muscle agonists. The results obtained with cumulative dose-response curves show that there is no increase in the sensitivity of the dystrophic compared with the normal aorta to noradrenaline, phenylephrine, isoproterenol, histamine, or 5-hydroxytryptamine. However, there was a significant increase in the force generated by aortic strips of the dystrophic animals to all agonists. Determination of noncollagen and collagen protein showed that there was no difference in the relative proportions of these proteins in the aortas from the two strains. The results show that in this animal model of dystrophy an increased response to vasopressor amines occurs and is in accordance with that expected of the vascular hypothesis.     

Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract.

Duchenne muscular dystrophy is characterized by the absence of dystrophin from muscle cells. Dystrophic muscle cells are susceptible to oxidative stress. We tested the hypothesis that 3 wk of endurance exercise starting at age 21 days in young male mdx mice would blunt oxidative stress and improve dystrophic skeletal muscle function, and these effects would be enhanced by the antioxidant green tea extract (GTE). In mice fed normal diet, average daily running distance increased 300% from week 1 to week 3, and total distance over 3 wk was improved by 128% in mice fed GTE. Running, independent of diet, increased serum antioxidant capacity, extensor digitorum longus tetanic stress, and total contractile protein content, heart citrate synthase, and heart and quadriceps beta-hydroxyacyl-CoA dehydrogenase activities. GTE, independent of running, decreased serum creatine kinase and heart and gastrocnemius lipid peroxidation and increased gastrocnemius citrate synthase activity. These data suggest that both endurance exercise and GTE may be beneficial as therapeutic strategies to improve muscle function in mdx mice.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.     

GSH system in relation to redox state in dystrophic skin fibroblasts

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.     

Development of outcome measures according to dystrophic phenotypes in canine X-linked muscular dystrophy in Japan

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder characterized by primary muscle degeneration. Therapeutic strategies for DMD have been extensively explored, and some are in the stage of human clinical trials. Along with the development of new therapies, sensitive outcome measures are needed to monitor the effects of new treatments. Therefore, we investigated outcome measures such as biomarkers and motor function evaluation in a dystrophic model of beagle dogs, canine X-linked muscular dystrophy in Japan (CXMD_J). Osteopontin (OPN), a myogenic inflammatory cytokine, was explored as a potential biomarker in dystrophic dogs over the disease course. The serum OPN levels of CXMD_J dystrophic dogs were elevated, even in the early disease phase, and this could be related to the presence of regenerating muscle fibers; as such, OPN would be a promising biomarker for muscle regeneration. Next, accelerometry, which is an efficient method to quantify performance in validated tasks, was used to evaluate motor function longitudinally in dystrophic dogs. We measured three-axis acceleration and angular velocity with wireless hybrid sensors during gait evaluations. Multiple parameters of acceleration and angular velocity showed notedly lower values in dystrophic dogs compared with wild-type dogs, even at the onset of muscle weakness. These parameters accordingly decreased with exacerbation of clinical manifestations along with the disease course. Multiple parameters also indicated gait abnormalities in dystrophic dogs, such as a waddling gait. These outcome measures could be applicable in clinical trials of patients with DMD or other muscle disorders.     

Free radicals: a potential pathogenic mechanism in inherited muscular dystrophy

Despite years of intensive work, the biochemical defect responsible for the pathogenesis of inherited muscular dystrophy has not been identified either in humans or animal models. This review examines evidence in support of the hypothesis that free radicals may be responsible for muscle degeneration in this disorder. A variety of cellular abnormalities noted in dystrophic muscles can be accounted for by free radical mediated damage. In addition, chemical by-products associated with free radical damage are found in dystrophic muscle tissue from humans and animals with this disease. Various enzymatic antioxidant systems can be enhanced as a normal cellular response to oxidative stress, and such changes are seen both in dystrophic muscle cells and certain other tissues of dystrophic animals. An increased level of free radical damage would follow from either: enhanced production of free radical species, or a deficient component of the cellular antioxidant system, such as vitamin E. The free radical hypothesis of muscular dystrophy can account for data supporting several alternative theories of the pathogenesis of this disease, as well as other observations which have not previously been explained.     

Changes in some cytoplasmic enzymes from red cells fractionated into age groups by centrifugation in Ficoll/Triosil gradients. Comparison of normal humans and patients with Duchenne muscular dystrophy.

1. A procedure is described for the fractionation into age classes of human red cells on a Ficoll/Triosil discontinuous density gradient. The fractionated cells appear minimally affected by the procedure. 2. Individual blood samples show a normal cell distribution, but the mean cell density may vary slightly. To allow for this in comparing samples, cell ages are expressed in terms of the mean and standard deviations from the mean. 3. Reticulocyte number decays exponentially in the gradient. By back extrapolation, a standard deviation can be computed corresponding to the theoretical age at which the sample contains 100% reticulocytes. This value can be used to calculate the theoretical activity of an enzyme in the reticulocytes and the enzyme half-life. 4. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase activities in unfractionated and age-fractionated cells were investigated in adults, children and patients with Duchenne muscular dystrophy. 5. The half-life for adult glucose 6-phosphate dehydrogenase (48 days) compares with that found by other workers. In children the half-life is 24 days, although the initial activity is essentially unaltered. 6. The half-life of 6-phosphogluconate dehydrogenase was found to be 117 days; the activity of glutathione reductase did not alter throughout the life of the cell. There was no difference between adults and children for these enzymes. The same results were obtained with dystrophic patients as for control children for all three enzymes.