Oxidative stress changes in L-arginine-induced pancreatitis in rats.

The important role of oxygen radicals in acute experimental pancreatitis was demonstrated by study of the changes in the antioxidant system in the blood, liver, kidney, and pancreas of rats after the administration of a large quantity of L-arginine (L-Arg). The changes in lipid peroxidation and in reduced and oxidized glutathione were followed, as well as the activities of peroxide-decomposing enzymes (glutathione peroxidase and catalase) and H2O2-producing superoxide dismutases. The results demonstrated that "oxidative stress" develops and acute pancreatitis appears rapidly after L-Arg treatment. Oxidative stress symptoms are expressed 24 h after the final treatment. Slow restitution of the studied antioxidant system can be demonstrated as early as after 48 h.

Coenzyme production using immobilized enzymes. III. Immobilization of glucose-6-phosphate dehydrogenase from bakers' yeast.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) from Bakers' yeast was immobilized with the highest activity on polyacrylamide beads possessing carboxylic functional groups activated by a water-soluble carbodiimide. The optimal pH values for the catalytic activity of the soluble and the immobilized glucose-6-phosphate dehydrogenase were practically identical, lying between pH 9.0 and 9.2. The optimal temperature for both the soluble and the immobilized enzyme was about 50 degrees C. The apparent Km values of the immobilized enzyme were slightly higher than those of the soluble enzyme. The immobilization improved the stability of the enzyme in the pH range 6.0-9.0 at 45 degrees C. The operational stability of the immobilized glucose-6-phosphate dehydrogenase proved favorable in a column experiment during 37 days of operation.

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparent Km value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55 degrees C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.

Determination of glycolytic intermediates in a flow injection system using immobilized enzymes.

Some glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, enolase and phosphoglyceromutase) were immobilized on a polyacrylamide-type bead polymer containing carboxylic functional groups activated by water-soluble carbodiimide. The immobilized enzymes were used for the determination of pyruvic acid, phosphoenolpyruvic acid, 2-phosphoglyceric acid and 3-phosphoglyceric acid in a flow injection system. The immobilized lactate dehydrogenase column was repeatedly employed for the determination of pyruvic acid in clinical samples. The results of the flow injection method accorded well in accuracy, sensitivity and reproducibility with those of soluble enzyme analysis.

Effects of Ca2+ on catalytic activity and conformation of trypsin and alpha-chymotrypsin in aqueous ethanol.

The effects of calcium ions on the conformation and catalytic activity of trypsin and alpha-chymotrypsin were studied in aqueous ethanol. The activity of alpha-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of alpha-chymotrypsin did not decrease in the presence of 1.2M CaCl2 and 0.6M CaCl2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.

Immobilization of lactate dehydrogenase on polyacrylamide beads.

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.

Structure and activity of alpha-chymotrypsin and trypsin in aqueous organic media.

The effects of different concentrations (20-95%) of organic solvents (ethanol, 1,4-dioxane and acetonitrile) were studied on alpha-chymotrypsin and trypsin from bovine pancreas. The changes in secondary structure were followed by CD measurements, and the apparent Michaelis constants (KMapp) and the stabilities of the enzymes were determined. Significant alterations in the CD spectra were found for both enzymes at the different organic solvent concentrations. The apparent KM values of trypsin and alpha-chymotrypsin decreased as the low solvent concentrations were elevated, but then increased in the presence of higher organic solvent concentrations. The stabilities of the enzymes changed on increase of the organic solvent concentration; trypsin exhibited a higher stability than that of alpha-chymotrypsin in all organic solvents. These results show that at an organic solvent content of 95% the manifestation of an enzyme activity similar to that measured in water can be attributed to the similar compositions of the secondary structural elements.

Effects of polyhydroxy compounds on the structure and activity of alpha-chymotrypsin.

The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of alpha-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of alpha-chymotrypsin.

Oxidative stress in experimental diabetes induced by streptozotocin.

It is known that streptozotocin (STZ) penetrating into the organism generates nitrogen monoxide (NO). Therefore, it is justified to presume, that in beta-cell destruction thereby induced, peroxinitrit resulting from NO and superoxide (O2-) reaction has an important role. It has also been studied how pro- and antioxidant systems change in STZ induced experimental diabetes in rat organs. Beside pro- and antioxidant systems of plasma and red blood cell hemolysates, changes in homogenates of the following organs were studied: liver, kidney, heart, lungs, spleen, brain, muscles and pancreas. We tested and compared antioxidant enzymes (superoxide dismutase-, glutathione peroxidase- and catalase activities) glutathione reductase activity regenerate reduced glutathione (GSH). The oxidized, reduced glutathione values and lipid peroxidation changes were measured. From our studies it has appeared that STZ treatment generally induces an oxidative predominance in tissues. Changes in this model thereby, can be compared to changes occurring in type 1 human diabetic patients.

Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent.

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of alpha-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this alpha-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.

Effects of synthetic pyrethroids and methidation on activities of some digestive enzymes in carp (Cyprinus carpio L.).

The effects of pyrethroid pesticides (deltamethrin, permethrin and cypermethrin) and an organophosphate ester (methidation) on the activities of carp trypsin, alpha-chymotrypsin, carboxypeptidase A and lipase were studied. The enzymes were isolated from the gastrointestinal tract and the effects of the pesticides were investigated during incubation for 5 min. The activity of trypsin was influenced only slightly by the presence of deltamethrin and methidation, whereas permethrin and cypermethrin caused significant inhibition. The pyrethroid pesticides at lower concentrations resulted in a slight activation of alpha-chymotrypsin. Methidation inhibited the alpha-chymotrypsin activity by about 20%. These pesticides modified the lipase activity to a lesser extent; the highest inhibition was measured with cypermethrin. The carboxypeptidase A activity was inhibited by both pyrethroid pesticides and methidation. The results suggest that these pesticides might interact with the active conformation of the studied hydrolytic enzymes, resulting in changes in their activities.

Pro-, antioxidant and rheologic studies in the blood of type 2 diabetic patients.

This study was conducted on type 2 non-insulin-dependent diabetes mellitus (NIDDM) cases and healthy blood donors. Lipid peroxidation (LP) products in plasma and red blood cell (RBC) hemolysates were estimated as total thiobarbituric acid reactive substances (TTBARS). The plasma and hemolysate reduced and oxidized glutathione (GSH and GSSG) levels are compared. In the hemolysates the antioxidant enzymes namely superoxide dismutase (SOD), glutathione peroxidase (GPx-ase), glutathione reductase (GR-ase) and catalase (C-ase) are also compared. The RBC filtration characteristics are determined and compared with controls: 1. LP and GSH in diabetic plasma were significantly higher, but in the hemolysate the GSH raised but the LP was significantly lower in diabetics than in healthy controls. 2. Superoxide dismutase and C-ase were significantly higher in NIDDM hemolysate. Contrary the GPx-ase activity was significantly lower in diabetics. 3. The diabetic RBCs filtration characteristics are changed in respects significantly namely the Fi was lower, the Tc and CR were higher. It means higher rigidity and oxidative damage of the membrane of diabetic RBCs.

Pro-, antioxidant and filtration changes in the blood of type 1 diabetic patients.

In our present work we attempt to clarify the pro-, antioxidant status (redox status) of blood and the red blood cell (RBC) filtration changes in type 1 (insulin dependent diabetes mellitus = IDDM) diabetic patients, broadening our biochemical knowledge about the mechanism of disease. Further on we try to apply our observations in therapy. Our studies on enzymes and the pro- and antioxidant status in type 1 diabetes are closely related to earlier works. Our studies on antioxidants have been extended deeper on redox conditions for example on the reduced and oxidized glutathione (GSH and GSSG) and glutathione reductase activity. The properties and changes of antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) as well as lipid peroxidation (LP) have been studied earlier without selecting the different type of human diabetics. At the same time the red blood cell filtration characteristics are compared also with normal values. The results of our studies confirmed the earlier findings that human diabetes is accompanied by a strong oxidative predominance (oxidative stress) in blood.

Lipid peroxidation and antioxidant system changes in acute L-arginine pancreatitis in rats.

The important role of oxygen radicals in acute experimental pancreatitis was demonstrated by study of the changes in the antioxidant system in the blood, liver, kidney and pancreas of rats after the administration of a large quantity of L-arginine (L-Arg). The changes in lipid peroxidation and in reduced and oxidized glutathione were followed as well as the activities of peroxide-decomposing enzymes (glutathione peroxidase and catalase) and H2O2-producing superoxide dismutases. The results demonstrated that acute pancreatitis and "oxidative stress" develop rapidly after L-Arg treatment. "Oxidative stress" symptoms are expressed 24 hours after the final treatment. Slow restitution of the studied antioxidant system can be demonstrated as early as after 48 hours.

Further prove on oxidative stress in alloxan diabetic rat tissues.

After intravenous administration of alloxan monohydrate (AL) diabetes developed in rats. Forty-eight hours after the injection the animals were sacrificed, their blood was collected in heparin containing tubes and the tissues were dissected and frozen (-70 degrees C) until their homogenization for pro- and antioxidant testing. Our results can be summarised as follows: (i) In the blood hemolysate the lipid peroxidation slightly elevated and the activity of antioxidant enzymes and reduced glutathione decreased. (ii) Similar phenomena could be observed in the different examined organ homogenates. The organs tested for pro- and antioxidant system were as follows: the liver, heart, skeletal muscle, kidney and pancreas. In our present work we attempt to confirm the data in support of the oxidative predominance over antioxidants in oxidative stress of AL diabetic rats.