Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities in early stages of development in dystrophic chickens

Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities were assayed in superficial pectoral muscles of hereditary dystrophic chickens, 1 week, 2 weeks, 4 weeks and 4 months after hatching. In control chickens, activities of G6PDH and 6PGDH were very low at 4 months of age; however, at 1 week of age, they were much higher than those at 4 months of age. Activities of G6PDH and 6PGDH were significantly higher in dystrophic chickens compared with those in the controls at all the stages of development studied. These findings suggest that considerable activities of G6PDH and 6PGDH are present within the pectoral muscle cells at early stages of development, at least in dystrophic chickens. GAPDH activity was significantly lower in dystrophic chickens at 2 weeks, 4 weeks and 4 months of age compared with those in control chicken. These findings together with our previous studies (Mizuno 1984a,b) in which increased activities of superoxide dismutases, catalase, glutathione peroxidase and glutathione reductase were reported in dystrophic chickens, indicate the presence of an increased capacity for the turnover of oxygen-free radicals within muscle cells in dystrophic chickens, and that oxygen-free radicals and the related activated oxygen species may be playing a role in inducing cellular damage.     

Changes in superoxide dismutase,catalase, glutathione peroxidase,and glutathione reductase activities and thiobarbituric acid-reactive products levels in early stages of development in dystrophic chickens

CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and thiobarbituric acid-reactive products were assayed in the superficial pectoral muscles of genetically dystrophic chickens (line 413) and their controls (line 412) 1, 2, and 4 weeks, and 4 months after hatching. In control chickens, all these enzyme activities declined as they grew older. In dystrophic chickens, all these enzyme activities were significantly elevated at all stages of development studied, and their developmental time courses were quite different from those in the controls. Thiobarbituric acid-reactive products were also significantly elevated in dystrophic chickens after 2 weeks of age. Invasion of macrophages and lipid cells were not manifest until 4 weeks after hatching in the dystrophic chickens studied. Therefore, observed abnormalities were considered to represent biochemical pathologies within muscle cells. Increased activities of the enzymes which are responsible for the regulation of active oxygen species and the elevated thiobarbituric acid-reactive products would indicate the presence of increased turnover of those active oxygen species. These findings indicated that active oxygen species were playing a significant role in the pathogenesis of muscular dystrophies. The possible mechanisms of cellular damage by active oxygen species are discussed.     

Enzymatic analysis of individual anterior horn cells in amyotrophic lateral sclerosis and Duchenne muscular dystrophy

Five enzyme activities related to glucose metabolism, i.e. glucose-6-phosphate dehydrogenase (G6PDH), isocitric dehydrogenase (ICDH), citrate synthase (CS), ATP citrate lyase (ACL), and pyruvate dehydrogenase complex (PDC), were estimated in anterior horn cells (AHCs) of spinal cords in amyotrophic lateral sclerosis (ALS) and Duchenne muscular dystrophy (DMD) patients by means of the NADP and CoA cycling methods.In ALS, only CS activity was reduced significantly in AHCs, but that of posterior root ganglion cells (PRGCs) remained within the normal range.In DMD, ICDH, CS, ACL and PDC activities were within normal range. However, G6PDH activities were elevated significantly in 2 out of 3 patients examined.     

Additional biochemical criteria in the differential diagnosis of myositis

Summary Thirty-six biopsy specimens of human biceps and vastus lateralis muscles were examined by histometric analysis and determination of enzyme activities (phosphorylase, triosephosphate dehydrogenase, 3-hydroxyacyl-CoA-dehydrogenase, lactate dehydrogenase, hexose isomerase, citrate synthetase, 6-phosphogluconate dehydrogenase). The series included 13 specimens from patients suffering from a benign form of muscular dystrophy (limb girdle and Becker type of muscular dystrophy) and 12 specimens from patients with an acute (n=5) or chronic (n=7) form of myositis. Muscle fibres were atrophic in myositis and hypertrophic (with an increased variation of fibre diameters) in muscular dystrophies, as has been shown previously. When myositis samples were compared with either normal or dystrophic muscles, a highly significant lowering of glycolytic enzyme activity was found in chronic myositis, while the activity of 6-phosphogluconate dehydrogenase was elevated to highly significant levels. Measurements of the latter enzyme's activity might be of additional value in differentiating chronic forms of myositis from benign muscular dystrophies.     

Anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia

The development of the anti-oxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase was studied in the fetal guinea pig brain at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The activities of these enzymes remained constant during 30-45 days of gestation and increased significantly during the 45-60 day period, with the exception of superoxide dismutase, which remained unchanged throughout the gestational period. The enzyme activities in fetal brain tissue at every gestational age were unaffected by maternal hypoxia (inspired oxygen, 7% for 40 min). The concurrent development of glucose-6-phosphate dehydrogenase, glutathione reductase, and glutathione peroxidase during 45-60 days of gestation indicates an increased ability of the fetal brain to detoxify lipid peroxidation products by reinforcing the glutathione system. The results of this study indicate that the anti-oxidant enzymatic defense mechanisms in the guinea pig brain are fairly mature at birth. However, these mechanisms are underdeveloped during the early stages of gestation and, therefore, during this period the brain might be at potential risk for lipid peroxidative damage under conditions leading to increased formation of oxygen free radicals.     

Oxidative stress in Rett syndrome

The investigation of parameters that might influence the neurological evolution of Rett syndrome might also yield new information about its pathogenic mechanisms. Oxidative stress caused by oxygen free radicals is involved in the neuropathology of several neurodegenerative disorders, as well as in stroke and seizures. To evaluate the free radical metabolism in Rett syndrome, we measured red blood cell antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase) and plasma malondialdehyde, as lipid peroxidation marker in a group of patients with Rett syndrome. No significant differences were observed in erythrocyte glutathione peroxidase, glutathione reductase and catalase activities, between the Rett syndrome patients and the control group. Erythrocyte superoxide dismutase activities were significantly decreased in Rett syndrome patients (P<0.001) compared with the control group. Plasma malondialdehyde concentrations were significantly increased in Rett syndrome patients (P<0.001). An unbalanced nutritional status in Rett syndrome might explain the reduced enzyme activity found in these patients. Our results suggest that free radicals generated from oxidation reactions might contribute to the pathogenesis of Rett syndrome. The high levels of malondialdehyde reflect peroxidative damage of biomembranes that may contribute to progressive dementia, impaired motor function, behavioural changes, and seizures, in Rett syndrome. We found a probable relationship between the degree of oxidative stress and the severity of symptoms, which should be further investigated with a larger number of patients in different disease stages.     

Toxic effects of MPP and MPTP in PC12 cells independent of reactive oxygen species formation

MPTP is a toxin presumed to damage dopamine-secreting neurons by an oxygen free radical-mediated mechanism. Two steps in MPTP metabolism are the primary candidates for oxygen free radical generation: (a) MPTP oxidation to MPP(+) by a monoamine oxidase and (b) NADH dehydrogenase inhibition by MPP(+). In order to test the idea that MPTP toxicity is mediated by oxygen free radicals, we assessed lipid peroxidation and the effects of antioxidants in dopaminergic PC12 cells treated with MPTP or MPP(+). For comparison purposes, we also examined the effects of the pro-oxidant tert-butyl-hydroperoxide (TBHP) and of the dopaminergic toxin 6-hydroxydopamine (6-OHDA) in PC12 cells. MPTP and MPP(+), unlike TBHP, failed to induce lipid peroxidation in PC12 cells after a 4-h exposure. All toxins tested (MPTP, MPP(+), TBHP and 6-OHDA) caused a dose-dependent decrease in [(3)H]dopamine ((3)H-DA) uptake in PC12 cultures. The hydroperoxide scavengers glutathione and N-acetyl-cysteine and the superoxide and peroxide scavenger EUK-134 protected PC12 cells from TBHP- and 6-OHDA-induced decrease in (3)H-DA uptake. However, no protection by these antioxidants at various concentrations and time regimens was observed against MPTP- or MPP(+)-induced decreases in (3)H-DA uptake in PC12 cells. In addition, incubation of PC12 cells with the energy-rich substrate, NADH, attenuated MPP(+)-induced decrease in (3)H-DA uptake. These results suggest that MPTP-induced toxicity in dopaminergic PC12 cell cultures, does not involve oxygen free radical production, but rather may be caused by impairment in energy metabolism.     

Selenium metabolism and supplementation in patients with muscular dystrophy

We studied selenium metabolism in patients with Duchenne muscular dystrophy and in contrast to previous reports found no significant abnormalities in these patients. Supplementation of muscular dystrophy patients and control subjects with sodium selenite (1 mg selenium/day) induced a variable rise in the activity of the selenium-dependent enzyme glutathione peroxidase in plasma and red cells, but no significant change in muscle glutathione peroxidase activities. There was no effect of selenium supplementation on disease activity in the patients with muscular dystrophy. Thiobarbituric acid-reacting substances (an index of free radical-mediated lipid peroxidation) were elevated in the muscle of patients with Duchenne muscular dystrophy in contrast to patients with other forms of muscular dystrophy and control subjects. This elevation was unaffected by selenium supplementation.     

Enzymatic analysis of individual posterior root ganglion cells in olivopontocerebellar atrophy, amyotrophic lateral sclerosis and Duchenne muscular dystrophy

Four enzyme activities related to glucose metabolism, i.e. those of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49), lactic dehydrogenase (LDH; EC 1.1.1.27), pyruvate dehydrogenase complex (PDC) and citrate synthase (CS; EC 4.1.3.7) were estimated in posterior root ganglion cells (PRGCs) of the spinal cord in patients suffering from olivopontocerebellar atrophy (OPCA), amyotrophic lateral sclerosis (ALS), and Duchenne muscular dystrophy (DMD) by means of the NAD, NADP and CoA cycling methods. In ALS and DMD, the enzyme activities examined were within normal ranges. In OPCA, PDC activity was significantly reduced and LDH activity tended to be lower than that in controls.     

Comparison of the intermediary metabolism of fatty acids in denervated and dystrophic murine skeletal muscle.

Certain aspects of lipid metabolism have been examined in denervated muscle from normal mice and in dystrophic muscle from mice of the Bar Harbor strain 129. A number of parameters show no change or similar changes. For example, the utilization of palmitate-[1-14C] and palmitylcarnitine by mitochondria from denervated and dystrophic hind leg skeletal muscle showed parallel decreased in the oxidation of palmitate (30-42%) and palmitylcarnite (37-66%). A comparable study with acetylcarnitine showed a striking difference with no change evident in mitochondria from denervated muscle and 80-85% decrease in dystrophic muscle. The study of succinate dehydrogenase and the enzymes of beta-oxidation in the above mitochondrial preparation showed similar findings except for acyl CoA dehydrogenase activity (an enzyme with a regulatory role in beta-oxidation) which was significantly diminished (29%) in denervated muscle, whereas no change was observed in dystrophic muscle. The findings show a close parallel in a number of parameters but distinct differences were observed in denervated as compared with dystrophic muscle. It is unlikely that the muscular disorder in murine muscular dystrophy can be explained solely on the basis of denervation or the loss of a neural trophic factor.     

Properties of muscles from chickens with inherited muscular dystrophy

Inherited muscular dystrophy of the chicken is an abnormality affecting the normal development and function of fast-twitch skeletal muscles. Several different strains of dystrophic chickens have been developed by selection for high lipid content in the pectoralis muscle and early onset of the disorder or by outcrossing the original New Hampshire stock into an inbred White Leghorn breed. The purpose of this study was to determine whether fast-twitch dystrophic muscles differ in expressed properties within the same bird and to examine the differences in gene expression between dystrophic New Hampshire and White Leghorn breeds. The biochemical and physiological properties examined were lactate dehydrogenase and acetylcholinesterase activities, total lipid content, muscle fiber diameter and electromyographic insertion activity.Results showed that fiber diameter and lipid levels were different in muscles within individual birds of two dystrophic lines and that the dystrophic gene causes rapid fiber atrophy and high lipid content in the White Leghorn breed. In addition, differences in lactate dehydrogenase activity and electromyographic patterns were found between two dystrophic lines. The results suggest that the expressed properties differ within each muscle of the dystrophic bird and that the expression of the dystrophic gene is dependent upon the nature of the genetic background of the breed.     

Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage

Catalpol, an iridoid glycoside abundant in the roots of Rehmannia glutinosa, has been previously found to prevent the loss of CA1 hippocampal neurons and to reduce working errors in gerbils after ischemia-reperfusion injury. In the present study, we investigated the effects of catalpol on astrocytes in an ischemic model to further characterize its neuroprotective mechanisms. Primary cultured astrocytes exposed to oxygen-glucose deprivation (OGD) followed by reperfusion (adding back oxygen and glucose, OGD-R), were used as an in vitro ischemic model. Treatment of the astrocytes with catalpol during ischemia-reperfusion increased astrocyte survival significantly in a concentration-dependent manner, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release and morphological observation. In addition, catalpol prevented the decrease in mitochondrial membrane potential, inhibited the formation of reactive oxygen species (ROS) and the production of nitric oxide (NO), decreased the level of lipid peroxide and the activity of inducible nitric oxide synthase (iNOS), and elevated the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and the content of glutathione (GSH). Our results suggest that catalpol exerts the most significant cytoprotective effect on astrocytes by suppressing the production of free radicals and elevating antioxidant capacity.     

Iron overload, oxidative stress, and axonal dystrophy in brain disorders

Hallervorden-Spatz syndrome is an autosomal-recessive brain disorder with signs of extrapyramidal dysfunction and mental deterioration, which associate with iron accumulation in globus pallidus and substantia nigra pars reticulata. Studies of oxidant stress in parkinsonian animal models suggest a linkage of iron overload to axonal dystrophy. Redox cycling of iron complexes (i.e., ferrous citrate and hemoglobin) increases hydroxyl radicals, lipid peroxidation, axonal dystrophy, and necrotic or apoptotic cell death. An increase of oxidative stress in the basal ganglia because of redox cycling of iron complexes leads to dopamine overflow and psychomotor dysfunction. Iron overload-induced axonal dystrophy has been demonstrated consistently using in vitro and in vivo models with a prominent feature of lipid peroxidation. This iron-induced oxidative stress is often accentuated by ascorbate and oxidized glutathione, although it is suppressed by the following antioxidants: S-nitrosoglutathione or nitric oxide, MnSOD mimics, manganese, U-78517F, Trolox, and deferoxamine. Preconditioning induction of stress proteins (i.e., hemeoxygenase-1 and neuronal nitric oxide synthase) and hypothermia therapy suppress the generation of toxic reactive oxygen, lipid, and thiol species evoked by bioactive iron complexes in the brain. Finally, combined antioxidative therapeutics and gene induction procedures may prove to be useful for slowing progressive neurodegeneration caused by iron overload in the brain.     

Serum carbonic anhydrase III in progressive muscular dystrophy

Serum carbonic anhydrase III (CA-III) levels were determined by means of an enzyme immunoassay method and compared with serum creatine kinase (CK) and muscle-specific enolase (MSE) levels in 143 patients with four types of progressive muscular dystrophy (PMD), namely, Duchenne muscular dystrophy (DMD), limb-girdle dystrophy, facioscapulohumeral dystrophy and congenital dystrophy. Serum CA-III levels were raised in the majority of patients, especially in those with DMD. In DMD patients, the gradual decline in the CA-III level was observed with age. High correlations were found between CA-III, CK and MSE levels. The frequency of cases with elevated CA-III levels was the same as or greater than that of elevated CK or MSE levels in four types of PMD. These results suggest that serum CA-III may be a useful marker of muscle disease.     

Superoxide dismutase, glutathione peroxidase and lipoperoxidation in Down's syndrome fetal brain

Certain aspects of the metabolism of oxygen derivatives were investigated in the cerebral cortex from Down's syndrome (trisomy 21) fetuses. In comparison with controls of similar gestational age, the specific activity of the cytosolic Cu/Zn-dependent superoxide dismutase (SOD-I) was significantly elevated by 60 +/- 5%. This is consistent with a gene dosage effect, as the gene coding for SOD-I is on chromosome 21. In order to determine whether the increase in SOD-I activity is associated with an adaptive rise in glutathione peroxidase (GSHPx), as has been observed in other tissues, the activity of this enzyme was also estimated but was found not to be altered in the Down's syndrome brain. In addition, in vitro lipoperoxidation, estimated by the formation of malondialdehyde (MDA) on incubation of homogenates fortified with ascorbate and Fe2+, was significantly elevated (36 +/- 4%) in cerebral cortex of the Down's syndrome fetuses. The concentration of total combined polyunsaturated fatty acids (PUFA) was not significantly altered in the tissue, although there is evidence for differences in the composition of certain phospholipids. It is proposed that, on account of the evidence for a potential perturbation of oxygen free radical metabolism (notably an increased SOD-I activity not compensated by a rise in GSHPx) and for enhanced in vitro peroxidizability of PUFA, there may be increased lipoperoxidative damage in the Down's syndrome brain prenatally.     

Some factors affecting spontaneous transmitter release in dystrophic mice

Neuromuscular junctions of slow- and fast-twitch skeletal muscles from dystrophic (dy2J/dy2J) and control mice of the C57BL/6J strain were used to investigate the effect of muscular dystrophy on nerve-terminal regulation of their intracellular concentration of free calcium ions. The frequency of spontaneous miniature endplate potentials (MEPPs) was taken as an indicator of the intraterminal free calcium ion concentration. Dicoumarol, 2,4-dinitrophenol, ruthenium red, and the calcium ionophore A-23187 all potentiated the MEPP frequency in dystrophic muscles at concentrations which had negligible effects on normal muscles. Dystrophic muscle preparations were also more sensitive to an increased extracellular calcium concentration. Usually, these manipulations had more effect on the nerve terminals of dystrophic slow muscle than on those of dystrophic fast muscle. We conclude that muscular dystrophy alters the nerve terminal's ability to regulate the concentration of intracellular free calcium ions.     

Mechanisms of hippocampal reoxygenation injury

Mechanisms of 12 min of hypoxia and subsequent reoxygenation were studied in rat hippocampal slices. General cell injury in reoxygenation was indicated by increased lactate dehydrogenase (LDH). Increase in conjugated dienes (CD) showed that oxygen radical burst induced lipid peroxidation (LPO). ATP increase was also involved in reoxygenation injury, since cyanide, an inhibitor of ATP synthesis, decreased this damage. The results obtained on using inhibitors of oxygen radicals generation, i.e., allopurinol, indomethacin, rotenone, and antimycin A, strongly suggest that the sources of oxygen radicals were the xanthine/xanthine oxidase system, prostaglandin synthesis, and mitochondrial respiratory chain. The involvement of oxygen radicals in oxidative stress was confirmed also by using chain-breaking antioxidants, trolox α-tocopherol and stobadine, [(-)-cis-2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-1H-pyrido (4,3b)indole]. Stobadine added at the onset of reoxygenation was most effective, acting in a dose-dependent manner and found to be without effect when applied in hypoxia. Cytochrome-c oxidase was decreased in reoxygenated hippocampal slices treated with stobadine.     

Effects of fimbria-fornix transection and ganglioside treatments on histochemical staining for glucose-6-phosphate dehydrogenase in the lateral septum

The present study examined whether ganglioside treatments would affect an enzyme marker (glucose-6-phosphate dehydrogenase; G6PDH) of neural metabolism in an established model system (the hippocamposeptal projection) of deafferentation and sprouting. Rats were subjected to unilateral transections of the fimbria-fornix (FF) in order to (1) interrupt the hippocamposeptal projection, (2) deafferent the lateral septal nucleus (LSN) ipsilaterally, and (3) induce sprouting by the contralateral FF. In untreated rats which were killed at 2-4 days postlesion, histochemical staining for G6PDH was reduced by 35-40% in the deafferented LSN relative to the contralateral side. However, at 6-8 days (i.e., when sprouting begins), staining intensity returned toward contralateral values (i.e., recovered). This pattern of changes in G6PDH staining was not observed in the caudate nucleus adjacent to the LSN. In ganglioside-treated rats which were killed at 4 days, there was a significantly smaller reduction of G6PDH staining in the deafferented LSN (23%; P = .05). This effect was not observed in the LSN of treated rats killed at 2 days, nor in the caudate nucleus at either time point. The present data indicate that (1) FF transection results in a reduction and subsequent recovery of G6PDH staining in the deafferented LSN; and (2) ganglioside treatments may accelerate the onset of the recovery of G6PDH activity. We suggest that gangliosides' effect on G6PDH reflects an acute enhancement of biosynthetic events in deafferented neurons.     

Comparison of gamma-glutamyl transpeptidase activity in tissues of normal and dystrophic hamsters and mice.

The activity of gamma-glutamyl transpeptidase (gamma-GT), a membrane-bound enzyme, was assayed by a sensitive fluorometric method in the brain, heart, kidney, liver, skeletal muscle and serum of normal and dystrophic hamsters and mice. Normal and dystrophic hamsters were of the golden brown and BIO 14.6 line respectively. Both normal and dystrophic mice were of the C57 BL/6J -dy2j strain. Kidneys of both hamsters and mice contained the highest enzyme activity followed by the brain, skeletal muscle, liver and heart. Gamma-GT activity was elevated in dystrophic skeletal muscles but decreased in the dystrophic kidneys and sera. In the brain, heart and liver of dystrophic animals from both species enzyme activity was similar to that in normals.     

Canine X-linked muscular dystrophy studied with in vivo phosphorus magnetic resonance spectroscopy.

Duchenne muscular dystrophy (DMD) is an X-linked disease characterized by progressive muscle weakness and degeneration. Dystrophin is the product of the missing gene in this disorder. However, the cause of the dystrophic process is not understood. Transient muscle injury is normally seen after muscle exercise, and may be a necessary process in muscle growth and preservation. We, therefore, chose to evaluate the role of exercise in Duchenne dystrophy by studying the canine X-linked animal model (CXMD). These dogs also lack dystrophin and have clinical signs similar to humans. Exercise was initiated by electrical stimulation, and muscle metabolism was monitored with phosphorus magnetic resonance spectroscopy (P-MRS). Dogs with CXMD had abnormal muscle pathology and markedly elevated serum CK. The inorganic phosphate (Pi) to phosphocreatine (PCr) ratio was increased in CXMD dogs at rest compared with normal dogs (Pi/(Pi + PCr) = 0.166 +/- 0.054 for CXMD and 0.073 +/- 0.017 for normals, mean +/- SE). No changes in resting ATP, pH, phosphomonoesters (PME), and phosphodiesters (PDE) were seen. The mean Pi/(Pi + PCr) and pH values during stimulation were normal in the CXMD dogs. Two to three days after electrical stimulation, resting Pi/(Pi + PCr) ratios were significantly increased in the CXMD dogs (0.127 +/- 0.029 compared with 0.172 +/- 0.054, mean +/- SD). Normal dogs showed no increase in Pi/(Pi + PCr) following stimulation. There was a 50-fold greater increase in serum CK in CXMD compared with normal dogs following exercise. These results indicate greater muscle injury in CXMD muscle, and suggest that in the absence of dystrophin, exercise-induced muscle injury may play a role in the dystrophic process.