Lipids and proteins of erythrocyte membrane in Duchenne muscular dystrophy

The lipid and protein composition of erythrocyte membranes from patients with Duchenne muscular dystrophy was studied. When compared with age and sex-matched controls, no significant change was observed in the content of cholesterol and phospholipid, phospholipid components and their fatty acid composition, though the lipid content and fatty acid distributions were different between adult and child controls. The protein distribution on SDS-acrylamide gel electrophoresis was identical for Duchenne patients and controls.     

In vitro studies on calcium activated phosphatidylinositol phosphodiesterase of erythrocyte ghosts from normal individuals and those with myotonic muscular dystrophy

Myotonic muscular dystrophy is an inherited disorder affecting many organs, though the underlying biochemical defect is unknown. A recent publication [1] suggested that the metabolic lesion may be associated with defective phospholipid metabolism. These workers observed impaired calcium-stimulated phosphatidic acid accumulation in red cell ghosts from individuals with myotonic dystrophy compared with normal controls. The present study investigated some points of calcium-activated phosphatidylinositol metabolism in red cell ghosts from patients with myotonic dystrophy, those at risk of developing the disease and normal individuals. No differences between the three groups could be found in the incorporation of 32P into endogenous phosphatidylinositol nor in the distribution of label between the various phosphatidylinositols. Additionally, no differences were observed in either basal or calcium-activated phosphatidylinositol phosphate breakdown by phosphodiesterase. This would suggest that the observed decreased phosphatidate accumulation [1] may be due to impaired diacylglycerol kinase activity.     

Carnitine metabolism in early stages of Duchenne muscular dystrophy

Muscle carnitine deficiency was found in 12 children affected with Duchenne muscular dystrophy (DMD), the diagnosis being made at a preclinical stage or at the beginning of the clinical symptoms. Enzymatic activities related to fatty acid transport and carnitine metabolism were studied in these patients and normal subjects: palmitoyl carnitine transferase was increased, palmitoyl carnitine hydrolase was not found in the muscle, palmitoyl coenzyme A synthetase was normal and palmitoyl coenzyme A hydrolase was increased.     

A possible circulating plasma factor in Duchenne muscular dystrophy

Basal (Na+, K+)AtPase activity was found to be significantly reduced in erythrocyte ghosts from patients with Duchenne muscular dystrophy, and, whereas ouabain inhibited enzyme activity in controls, it stimulated activity in patients. Prior incubation of normal erythrocyte ghosts in Duchenne plasma resulted in an increase in enzyme activity on subsequent exposure to ouabain. This suggests there may be a "circulating plasma factor" in Duchenne muscular dystrophy responsible for the abnormal response to ouabain. This "factor" was rendered inactive by dialysis or deproteination of the plasma.     

Duchenne muscular dystrophy: 45Ca exchange in cultured skin fibroblasts and the effect of calcium ionophore A23187.

Calcium exchange was studied in skin fibroblasts cultured from eight subjects with Duchenne muscular dystrophy, four with Limb Girdle dystrophy and eight normal controls using 45Ca. No difference was found in the time course of calcium exchange between the groups, nor in the level of 45Ca when maximal exchange had occurred. Treatment of the cultures with the calcium ionophore A23187 resulted in higher levels of calcium exchange over a 2-H period. The increase was similar in the cultures from the 3 patient groups studied.     

Adenylate cyclase and ATPase activities in red cell membranes of patients and genetic carriers of Duchenne muscular dystrophy.

Basal adenylate cyclase activity was increased in red cell ghosts from both patients with Duchenne muscular dystrophy and their mothers when the activities were compared to proper age-matched controls. The activity of ATPase measured in the presence of Na+, K+, and Mg2+ was not found to be different in erythrocyte ghosts from Duchenne dystrophic patients, age-matched controls, or the mothers of Duchenne patients, and ouabain inhibited ATPase in ghosts to the same extent in all membrane preparations.     

Activator-associated Ca2+-ATPase in erythrocyte membranes from cystic fibrosis patients

Erythrocyte membranes were prepared by a method which should ensure binding of an activator protein (calmodulin) to the calcium dependent membrane ATPase involved in calcium transport. The level of enzyme activity, assayed at optimum conditions, was 5-400 times higher than that found in previous investigations on cystic fibrosis patients. The Ca2+-ATPase activity of the cystic fibrosis patients was reduced by 15% compared to control subjects, whereas patients suffering from chronic pulmonary diseases did not deviate from controls. Even if a reduction of Ca2+ pumping activity occurs in other cells, a 15% decrease could hardly be the only cause of the changed calcium concentrations in secretions from cystic fibrosis patients.     

A radiochemical method for the determination of transketolase activity in erythrocyte hemolysates

A radiochemical method for the determination of transketolase activity is reported. It is based on incubation of erythrocyte hemolysates with radioactive ribose 5-phosphate followed by isolation of sedoheptulose 7-phosphate on anion-exchange columns. The experimental conditions are discussed and the presence of phosphatase activity in the incubation mixture is demonstrated. The proposed method is compared with a current colorimetric method. Reference values are given for the transketolase activity and the thiamine pyrophosphate effect.     

Influence of the redox state of glutathione upon pyruvate kinase in the intact erythrocyte

We have adapted the hexokinase glucose procedure to an immobilized enzyme stirrer for the determination of glucose concentrations in human blood plasma. The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity.     

Serum pyruvate kinase in carriers of duchenne muscular dystrophy

Independent studies by two different groups (Madras and Edinburgh) have failed to confirm the suggestion that measurement of the serum level of pyruvate kinase (EC 2.7.1.40, PK) may be superior to measurement of the serum level of creatine kinase (EC 2.7.3.2, CK) for detecting female carriers of X-linked Duchenne muscular dystrophy (DMD). At present the serum level of creatine kinase remains the best test for this purpose.     

Abnormalities of erythrocyte membranes in biliary atresia: ultrastructure and lipid composition.

Biochemical and ultrastructural studies of red blood cell membranes from seven patients with congenital biliary atresia have been performed. Scanning electron microscopic observations revealed that more than half of these cells were of abnormal shapes, such as target, spur and cup-formed cells. By freeze-fracture electron microscopy, membrane particle-free smooth areas were noted in the fracture faces. In addition, the number of membrane-associated particles was 20% less than those in control subjects. The lipid analysis of red blood cells showed a significant increase in phospholipid and a marked increase in cholesterol content. The increase of phospholipid content was primarily caused by the increase of lecithin. The acyl chain analysis of erythrocyte lecithin demonstrated an increase in palmitic, palmitoleic and oleic acid, and a decrease in stearic and linoleic acid. These observations are similar to those of acquired biliary obstruction. The fluidity of erythrocyte membrane lipid, studied by a fluorescence technique using fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), was found to be less in an individual with congenital biliary atresia than in the control subject.     

Determination of erythrocyte uroporphyrinogen I synthetase activity in chronic renal failure

To obtain some information on porphyrin metabolism in uraemic patients, the activity of erythrocyte uroporphyrinogen I synthetase was measured in patients with chronic renal failure. The results indicate a decreased enzymatic activity which is not due to urea interference, in the hemolysates of these patients.     

Human erythrocyte thiopurine methyltransferase: Radiochemical microassay and biochemical properties

A radiochemical micromethod for the determination of thiopurine methyltransferase (TPMT) activity in human red blood cells (RBC) is described. Both 6-mercaptopurine and 6-thioguanine were substrates for the TPMT activity in the human RBC: Apparent Michaelis-Menten (K_M) values for 6-mercaptopurine and 6-thioguanine were 3.2 × 10^?4 M and 2.0 × 10^?4 M, respectively. The apparent K_M value for S-adenosyl-l-methionine, a co-substrate for the reaction, was 1.7 × 10^?6 M. The pH optimum for the reaction was approximately 7.5. Blood samples from 73 randomly selected adult subjects had a mean activity of 10.2 ± 2.4 (mean ± S.D.) units/ml packed red blood cells. The range of activities was from 4.6 to 14.2 units/ml. The results of experiments in which partially purified human kidney TPMT was added to RBC lysates and of experiments in which “low” and “high” activity lysates were mixed gave no indication that individual variations in RBC TPMT activity were due to endogenous inhibitors or activators of the enzyme.     

An improved method for the determination of human erythrocyte pyrimidine 5'-nucleotidase activity

A radiometric method is described for measuring pyrimidine 5'-nucleotidase activity in human peripheral blood haemolysate. The substrate [14C]uridine monophosphate is converted to [14C]uridine. Two simple and rapid methods to isolate the product of the reaction employing either descending chromatography or elution from DEAE cellulose paper have been developed. With the described methods the specific activity of pyrimidine 5'-nucleotidase of human erythrocytes is 6.7 +/- 2.7 (+/- 1 S.D.) nmol/h/mg protein and this activity appears to be widely distributed in human tissue.