thiobarbituric-acid and Hemolysis

Sulfaguanidine (SG), belongs to the class of sulfonamide drug used as an effective antibiotic. In the present work, using crystal engineering approach two novel cocrystals of SG were synthesized (SG-TBA and SG-PT) with thiobarbutaric acid (TBA) and 1,10-phenanthroline (PT), characterized by solid state techniques viz., powder X-ray diffraction (PXRD), fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and the crystal structures were determined by single crystal X-ray diffraction studies. A comparative antibacterial activity and hemolytic potential was done on SG drug, coformers and their cocrystals. The tested cocrystals formulations showed almost two fold higher antibacterial activity against the tested strains of bacteria Gram-positive bacteria (S. mutans and E. faecalis) and Gram-negative bacteria (E. coli, K. pneumonia and E. clocae) over SG alone and their coformers. Cocrystal SG-TBA showed better antibacterial activity and reduced hemolysis, thereby, reduced cytotoxicity than SG-PT.

Topics: Anti-Bacterial Agents; Bacteria; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Crystallization; Crystallography, X-Ray; Drug Compounding; Healthy Volunteers; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Conformation; Phenanthrolines; Powder Diffraction; Spectroscopy, Fourier Transform Infrared; Structure-Activity Relationship; Sulfaguanidine; Thiobarbiturates

Oxygen free radicals produced during normal aerobic metabolism have been implicated in several pathophysiological mammalian processes. The importance of free radical-mediated fatty acid oxidation has received much attention. The generation of active oxygen species may lead to lipid peroxidation and formation of reactive products, which may be involved in severe damage of cell molecules and structures. Free radical metabolism in pregnancy and in diabetes mellitus is still unclear. To add new insights to the question, changes in lipid peroxidation products and activities of three antioxidant enzymes: catalase (CAT), glutathione peroxidase (GPX) and superoxide dismutase (SOD) in maternal red blood cells haemolysates were evaluated in pregnant women with insulin-dependent diabetes mellitus (IDDM-PW) and in healthy pregnant women (HPW). Healthy non-pregnant women were the control group for IDDM-PW and HPW, respectively. Pregnancy provoked an increase of lipoperoxidation products and an high SOD activity since early pregnancy, while CAT and GPX activities did not change during gestation. IDDM-PW showed higher content of lipoperoxidation breakdown products and lower SOD activity at each trimester, if compared with HPW; moreover, a slight increase of CAT and SOD activity is reported during late diabetic pregnancy. IDDM-PW were in very good metabolic control at time of sampling. The variations reported suggest an easier membrane lipoperoxidability and, consequently, an easier membrane damage during diabetic gestation.

Topics: Adult; Catalase; Erythrocytes; Female; Glutathione Peroxidase; Hemolysis; Humans; Lipid Peroxidation; Longitudinal Studies; Oxidation-Reduction; Pregnancy; Pregnancy in Diabetics; Superoxide Dismutase; Thiobarbiturates

PD 117596 is a novel quinolone compound that is being investigated for use as an antibacterial agent. Early investigations demonstrated a significant phototoxic liability associated with this compound. These studies were undertaken to investigate the mechanism of phototoxicity using an in vitro model. In the UVA region, PD 117596 was found to be a more efficient producer of singlet oxygen than rose bengal, ciprofloxacin, nalidixic acid, or PD 118879, another quinolone under investigation. The quantum yield of photoreaction for PD 117596 was relatively low (phi = 0.021); however, it was approximately 10-fold higher than other tested quinolones. In vitro studies using a mouse erythrocyte model were used to further investigate the mechanism of phototoxicity. PD 117596-induced photohemolysis was found to be oxygen dependent with a relatively rapid onset that progressed even after removal of light. Preirradiation of the compound prevented subsequent hemolytic or photohemolytic action. BHA, BHT, alpha-tocopherol, and the iron chelator DTPA were all found to be effective at ameliorating the photohemolytic response. The photohemolytic response was markedly enhanced when D2O was substituted for H2O in the incubation medium, indicating a singlet oxygen-mediated mechanism of action. A rise in thiobarbituric acid products was noted within 1 hr of irradiation and was maximal at the time of onset of overt photohemolysis. These data suggest that singlet oxygen production by irradiated PD 117596 is responsible for secondary changes in mouse red blood cells including lipid peroxidation and ultimately results in cellular lysis.

Topics: 4-Quinolones; Animals; Anti-Infective Agents; Antioxidants; Ciprofloxacin; Dose-Response Relationship, Drug; Fluoroquinolones; Free Radical Scavengers; Hemolysis; Male; Mice; Mice, Inbred Strains; Nalidixic Acid; Nitroso Compounds; Oxygen; Photochemistry; Photolysis; Quinolones; Thiobarbiturates

Cinnarizine has antivasoconstrictor properties and improves red-cell deformability. Its major side-effects are the induction of extrapyramidal reactions. It is a calcium antagonist, but it was suggested that its effects may depend on other mechanisms, namely on antiperoxidant properties. We have studied these properties in different biological systems, intact red-cells included. The occurrence of lipid peroxidation was determined by the formation of 2-thiobarbituric acid reactive products. Cinnarizine was found to inhibit spontaneous lipid peroxidation in rat liver homogenates, copper-induced lipid peroxidation in human plasma and copper-induced and hydrogen peroxide-induced lipid peroxidation in human red-cells. In red-cells, the inhibition of lipid peroxidation is accompanied by the inhibition of hemolysis. Copper-induced red-cell lipid peroxidation is 85% inhibited by as little as 5 microM cinnarizine. The antioxidant activity of cinnarizine may contribute to explain some of the effects of this drug.

Topics: Animals; Antioxidants; Cinnarizine; Copper; Dose-Response Relationship, Drug; Erythrocytes; Female; Hemolysis; Humans; Hydrogen Peroxide; Lipid Peroxidation; Liver; Rats; Rats, Inbred Strains; Thiobarbiturates

We have recently modified the dialysis tubing osmotic lysis and resealing method to examine the role of intracellular red blood cell (RBC) antioxidants. However, the potential effect of resealing on the RBC was not fully investigated. This study examined a number of cellular characteristics to determine the effects of physical lysis and resealing on the RBC. Following resealing, RBC exhibited normal morphology and at most only slight alterations in mean cell volume and mean cell hemoglobin concentration. RBC density distribution was significantly affected by resealing with increased populations of both light and dense cells, though the mean cell density was similar to that of control cells. Endogenous enzyme activities and adenosine triphosphate (ATP) concentration were unaffected by the resealing procedure. While reduced glutathione (GSH) concentration was decreased by 15%, RBC oxidant sensitivity was found to be unaltered. Cellular deformability of the resealed RBC was 80% to 90% that of the control cells. Membrane phospholipid and fatty acyl composition of the resealed RBC were unaffected when compared with matched control samples. Membrane transport, permeability, and Ca2(+)-mediated cellular vesiculation were minimally altered by resealing. Finally, entrapment of fluorescent compounds demonstrated that greater than 95% of the resealed RBC had incorporated exogenous agents. In summary, the osmotic lysis and resealing method described resulted in only minor changes in cellular characteristics while allowing for the efficient loading of compounds to which the RBC membrane is normally impermeable. Consequently, this method provides great potential for the selective modification of erythrocyte constituents in order to further define their roles within the RBC.

Topics: Adenosine Triphosphate; Cell Membrane Permeability; Dextrans; Erythrocyte Deformability; Erythrocyte Indices; Erythrocyte Membrane; Erythrocytes; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Glutathione; Hemolysis; Humans; Osmotic Pressure; Phospholipids; Potassium; Sodium; Thiobarbiturates

The oxidation of vitamin E-deficient rat red blood cells (RBCs) induced by the hypoxanthine-xanthine oxidase (HX-XOD) system has been performed in an aqueous suspension. The generation of chemiluminescence and the accumulation of thiobarbituric acid-reactive substances (TBARS) were observed initially and were followed by hemolysis. Interestingly, the total counts of chemiluminescence were closely related to the amount of TBARS. The predominant change of membrane proteins induced by the reaction was the depletion of spectrin bands in gel electrophoresis. When RBC ghosts were oxidized with HX-XOD, the sulfhydryl (SH) groups of membrane proteins decreased at an early stage of the incubation, which was coincident with the above protein alteration. Membrane alpha-tocopherol suppressed not only the formation of TBARS but also chemiluminescence and hemolysis; nevertheless, it did not inhibit the protein damage and the loss of SH groups. Moreover, it was concluded that the chemiluminescence observed during the oxidation of RBC membranes was associated mainly with the peroxidation of lipids and only to a minor extent with the oxidation of proteins.

Topics: Animals; Erythrocyte Membrane; Hemolysis; Hydrogen Peroxide; Lipid Peroxidation; Luminescent Measurements; Male; Membrane Lipids; Membrane Proteins; Oxidation-Reduction; Rats; Rats, Inbred Strains; Spectrin; Sulfhydryl Compounds; Superoxides; Thiobarbiturates; Vitamin E; Vitamin E Deficiency; Xanthine; Xanthine Oxidase; Xanthines

Weanling male Sprague Dawley rats were fed a vitamin E and C-free basal diet with or without supplementation of 100 IU vitamin E per kg diet. After 20 weeks, the vitamin E-deficient rats were divided into four groups, six in each group, and received supplemental ascorbic acid and/or vitamin E by tube feeding daily for 7 days: Group I, 30 mg ascorbic acid/100 g body wt.; Group II, 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; Group III, 30 mg ascorbic acid and 0.03 mg RRR-alpha-tocopheryl acetate/100 g body wt.; and Group IV, placebo. The six control rats (Group V) received placebo. The rats were sacrificed, blood and liver samples were collected for biochemical determinations. Vitamin E deficiency significantly increased erythrocyte (RBC) spontaneous hemolysis, liver thiobarbituric acid (TBA) value, activities of glutamateoxaloacetate transaminase (GOT), pyruvate kinase (PK), and creatine phosphokinase (CPK) in plasma, and significantly lowered plasma vitamin E levels and glutathione peroxidase (GPX) activities. Tube-feeding ascorbic acid for 7 days produced partial reversal effect on liver TBA values, activities of plasma PK, GOT, CPK, and plasma vitamin E levels but not on RBC hemolysis and plasma GPX activity. Tube feeding both ascorbic acid and vitamin E showed similar partial reversal effect as feeding vitamin E alone on all the parameters stated above. The results suggest that ascorbic acid may spare the metabolism of vitamin E and partially reverse the changes in some of the biochemical parameters characteristic of vitamin E deficiency.

Topics: alpha-Tocopherol; Animals; Ascorbic Acid; Aspartate Aminotransferases; Creatine Kinase; Drug Therapy, Combination; Glutathione Peroxidase; Hemolysis; Lipid Peroxides; Liver; Male; Pyruvate Kinase; Rats; Rats, Inbred Strains; Thiobarbiturates; Tocopherols; Vitamin E; Vitamin E Deficiency

Topics: Animals; Erythrocytes; Free Radicals; Hemolysis; Lipid Peroxides; Luminescent Measurements; Male; Membrane Lipids; Rats; Rats, Inbred Strains; Superoxides; Thiobarbiturates; Vitamin E; Vitamin E Deficiency; Xanthine Oxidase

The relationship between haemoglobin and membrane oxidation was studied using liposomes containing haemoglobin (haemosomes) as a red cell model. Rapid oxidation occurred in haemosomes formed from purified haemoglobin and unsaturated lipid (egg phosphatidylcholines). After 3 h at 37 degrees C most of the haemoglobin was oxidized, predominantly to methaemoglobin with some haemichrome formation. The oxidation of haemoglobin was paralleled by membrane lipid peroxidation as measured by thiobarbituric acid reactivity. These changes were largely abolished by using freshly prepared haemolysate instead of purified haemoglobin, or when haemosomes were prepared with saturated phosphatidylcholines. In haemosomes consisting of fresh haemolysate and saturated phosphatidylcholine, the rate of haemoglobin oxidation at 37 degrees C corresponded to that of non-encapsulated haemolysate, and after 4 months storage at 4 degrees C 45% of oxyhaemoglobin was oxidized. In haemosomes prepared from purified haemoglobin and egg lecithin, alpha-tocopherol, catalase and ascorbate each protected against both haemoglobin oxidation and lipid peroxidation. Superoxide dismutase or reduced glutathione had no effect. In unsaturated-lipid haemosomes containing haemolysate, the rate of haemoglobin oxidation increased when catalase was inhibited or reduced glutathione was depleted, but after long term incubation only concurrent catalase-inhibition and glutathione depletion could increase thiobarbituric acid reactivity. These results demonstrate a close interdependence between haemoglobin oxidation and lipid peroxidation, and show that constituents of haemolysate strongly protect against both processes. H2O2 appears to be an important mediator, with its removal by either catalase or the glutathione/glutathione peroxidase system protecting against both oxidative changes.

Topics: Catalase; Glutathione; Hemoglobins; Hemolysis; Liposomes; Membrane Lipids; Models, Biological; Oxidation-Reduction; Spectrophotometry; Thiobarbiturates; Time Factors

Lipid peroxidation of human erythrocyte hemolysates was evaluated by determining thiobarbituric acid reactive substance by fluorospectrophotometer. Because of the non-linear relationship between malondialdehyde formation and hemoglobin concentration, a constant hemoglobin concentration is required to obtain reproducible results. Disruption of the hemolysate by sonication and the presence of formed elements are necessary to induce the reaction, the optimal pH of which was found to be limited to the region of 7.4. Either ferric or ferrous ions are essential for the reaction to proceed, but if both are present, their catalytic activity is abolished. The present method will be useful in the investigation of the mechanism of oxidative damage to erythrocytes under various pathological conditions.

Topics: Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Lipid Peroxides; Malondialdehyde; Spectrometry, Fluorescence; Thiobarbiturates