Methemoglobin Formation In Erythrocytes Research Articles

Potential Antioxidative Activity of Homocysteine in Erythrocytes under Oxidative Stress.

Homocysteine is an amino acid containing a free sulfhydryl group, making it probably contribute to the antioxidative capacity in the body. We recently found that plasma total homocysteine (total-Hcy) concentration increased with time when whole blood samples were kept at room temperature. The present study was to elucidate how increased plasma total-Hcy is produced and explore the potential physiological role of homocysteine. Erythrocytes and leukocytes were separated and incubated in vitro; the amount of total-Hcy released by these two kinds of cells was then determined by HPLC-MS. The effects of homocysteine and methionine on reactive oxygen species (ROS) production, osmotic fragility, and methemoglobin formation in erythrocytes under oxidative stress were studied. The reducing activities of homocysteine and methionine were tested by ferryl hemoglobin (Hb) decay assay. As a result, it was discovered that erythrocytes metabolized methionine to homocysteine, which was then oxidized within the cells and released to the plasma. Homocysteine and its precursor methionine could significantly decrease Rosup-induced ROS production in erythrocytes and inhibit Rosup-induced erythrocyte's osmotic fragility increase and methemoglobin formation. Homocysteine (but not methionine) was demonstrated to enhance ferryl Hb reduction. In conclusion, erythrocytes metabolize methionine to homocysteine, which contributes to the antioxidative capability under oxidative stress and might be a supplementary protective factor for erythrocytes against ROS damage.

The effect of dopamine on in vitro methemoglobin formation in erythrocytes of patients with Parkinson’s disease under oxidative stress

Using electron paramagnetic resonance, the dose-dependence effect of dopamine on methemoglobin formation in erythrocytes of patients with Parkinson’s disease under the activation of oxidative stress induced by acrolein and the possibilities for the correction of this pathological process using carnosine in vitro experiments have been examined. It was shown that incubation of erythrocytes with 1.5 mM dopamine did not change the methemoglobin content, while incubation with 15 mM dopamine caused a two fold increase in the methemoglobin content compared to its initial level; 10 μM acrolein increased methemoglobin formation threefold. Administration of 15 mM dopamine and, after 1 h, 10 μM acrolein to the incubation system increased methemoglobin formation tenfold compared to its initial level. Preincubation of erythrocytes with 5 mM carnosine followed by acrolein addition prevented the increase in the methemoglobin content, while carnosine had no effect on methemoglobin formation induced by dopamine.

Methemoglobin Formation in Erythrocytes Stored for Different Time Periods during Extracorporeal Circulation

Methemoglobin Formation in Erythrocytes Stored for Different Time Periods during Extracorporeal Circulation

The effect of antioxidants on in vivo and in vitro methemoglobin formation in erythrocytes of patients with Parkinson’s disease

Methemoglobin formation was examined in erythrocytes of 16 patients with Parkinson’s disease (PD) (stage 3–4 by the Hoehn and Yahr scale). The patients receiving levodopa-containing drugs (madopar, nakom) were also treated with intramuscular injections of mexidol (daily dose 100 mg/day) for 14 days. Control group included 12 clinically healthy persons. The erythrocyte methemoglobin content was determined by electronic paramagnetic resonance (EPR) using the EPR signal intensity with the g-factor 6.0. The methemoglobin content was significantly higher in erythrocytes of PD patients than in healthy donors. The complex therapy with mexidol normalized the methemoglobin content in erythrocytes of PD patients. Incubation in vitro of erythrocytes of donors and PD patients with acrolein increased the methemoglobin content, while incubation with carnosine normalized the methemoglobin content in erythrocytes of PD patients. Prophylactic (i.e. before acrolein addition) and therapeutic administration of carnosine to the incubation system with acrolein decreased the methemoglobin content to its initial level. Results of this study suggest that inclusion of the antioxidants mexidol and carnosine in the scheme of basic therapy of PD may reduce side effects associated with methemoglobinemia.

Fenugreek Supplementation Imparts Erythrocyte Resistance to Cypermethrin Induced Oxidative Changes In Vivo

Erythrocytes are excellent model to study the xenobiotic induced oxidative changes. Pyrethroid pesticides are increasingly being used in insecticidal preparations from the simple mosquito coils to house hold aerosols to sophisticated ultra low volume foggers and sprays. Cypermethrin a Type II pyrethroid pesticide is used widely in pest control. Fenugreek is a potent antioxidant. We have evaluated the potential of aqueous extract of germinated fenugreek seeds in counteracting cypermethrin induced oxidative changes in erythrocytes of male Wistar rats. Male Wistar rats were treated with 1/10 LD50 (25mg/kg body weight) of cypermethrin and 10 percent aqueous extract of germinated fenugreek for 60 days. Cypermethrin treatment caused significant decrease in non enzymatic antioxidants, glutathione (GSH), vitamin E, vitamin C, increased methemoglobin formation in erythrocytes and increased their mechanical fragility. Treatment with fenugreek reversed the cypermethrin induced oxidative changes in erythrocytes and restored all the parameters to near normal levels. The overall results reveal the ameliorating effect of aqueous extract of germinated fenugreek on cypermethrin induced toxicity in erythrocytes.

Use of in vitro methaemoglobin generation to study antioxidant status in the diabetic erythrocyte

Poor glycaemic control in diabetes and a combination of oxidative, metabolic, and carbonyl stresses are thought to lead to widespread non-enzymatic glycation and eventually to diabetic complications. Diabetic tissues can suffer both restriction in their supply of reducing power and excessive demand for reducing power. This contributes to compromised antioxidant status, particularly in the essential glutathione maintenance system. To study and ultimately correct deficiencies in diabetic glutathione maintenance, an experimental model would be desirable, which would provide in vitro a rapid, convenient, and dynamic reflection of the performance of diabetic GSH antioxidant capacity compared with that of non-diabetics. Xenobiotic-mediated in vitro methaemoglobin formation in erythrocytes drawn from diabetic volunteers is significantly lower than that in erythrocytes of non-diabetics. Aromatic hydroxylamine-mediated methaemoglobin formation is GSH-dependent and is indicative of the ability of an erythrocyte to maintain GSH levels during rapid thiol consumption. Although nitrite forms methaemoglobin through a complex GSH-independent pathway, it also reveals deficiencies in diabetic detoxification and antioxidant performance compared with non-diabetics. Together with efficient glycaemic monitoring, future therapy of diabetes may include trials of different antiglycation agents and antioxidant combinations. Equalization in vitro of diabetic methaemoglobin generation with that of age/sex-matched non-diabetic subjects might provide an early indication of diabetic antioxidant status improvement in these studies.

A Study on the Interaction between Hydroxylamine Analogues and Oxyhemoglobin in Intact Erythrocytes

ABSTRACTThe oxidative potency of hydroxylamine (HYAM) and its O-derivatives (O-methyl- and O-ethyl hydroxylamine) is generally larger than the effects of the N-derivatives (N-methyl-, N-dimethyl-, and N,O-dimethyl hydroxylamine). The effects of the two groups of hydroxylamines also differ in a qualitative sense. To elucidate this difference in toxicity profiles we investigated the hemoglobin dependence of the toxicity, the occurrence of cell-damaging products like superoxide and H2O2, and the cellular kinetics of the hydroxylamine analogues. All hydroxylamines were found to depend on the presence and accessibility of oxyhemoglobin to exert their toxicity. This did not provide an explanation for the different toxicity profiles. The interaction of some hydroxylamines with oxyhemoglobin is known to lead to the formation of radical intermediates. Differences in the stability of these radical products are known to occur, and in some cases secondary products are formed. This can contribute to the differences in toxicity. In this respect, production of superoxide radicals was demonstrated for all hydroxylamines in the reaction with oxyhemoglobin. Evidence for H2O2 generation during the reaction of HYAM, O-methyl, O-ethyl-, and N-dimethyl hydroxylamine with oxyhemoglobin was also found. Next to variations in the products formed, differences in cellular kinetics are likely to be among the most important factors that explain the different toxicity patterns seen for the hydroxylamines in erythrocytes. Indeed, differences were found to exist for the kinetics of methemoglobin formation in erythrocytes. Not only was the final level of methemoglobin formed much lower for the N-derivatives, but also the reaction rate with oxyhemoglobin was slower than with HYAM and its O-derivatives. Except for N,O-dimethyl hydroxylamine (NODMH), the same pattern was seen in hemolysates. NODMH tripled its effect on hemoglobin in hemolysate compared with incubations in erythrocytes. This implies that cellular uptake is a limiting factor for NODMH. Since formation of H2O2 is most likely a result of an interaction with hemoglobin, differences in kinetics of methemoglobin formation can be an explanation for the fact that NMH and NODMH did not produce H2O2 to a detectable level. These results indicate that (a) the toxicity of all hydroxylamines depends on an interaction with oxyhemoglobin; (b) the interaction with hemoglobin produces radical intermediates and concomitantly superoxide radicals and H2O2; and (c) differences in uptake, reaction rate with hemoglobin, and stability of the intermediates formed do exist for the different hydroxylamines and contribute to their differences in toxicity.

Studies on the Metabolism of Borate (V) : Effects of Borate on Anaerobic Glycolysis and Methemoglobin Formation in Erythrocytes in vivo and in vitro

For the purpose of studying the toxicity of borate on the blood, the effects on erythrocytes of guinea pigs were investigated. From the results obtained, the boron levels in erythrocytes were elevated by addition of borate to blood, and it was found that boron can enter into erythrocyte at the considerable rate according to the amount of borate added. The enzyme system of anaerobic glycolysis in erythrocyte was found to be markedly inhibited by addition of borate. It was also found that the levels of methemoglobin in erythrocyte revealed a remarkable tendency to increase after boron was added to erythrocyte. Similar results were also obtained in vivo experiments.

Biochemical effects of aromatic amines-I. Methaemoglobinaemia, haemolysis and heinz-body formation induced by 4,4′-diaminodiphenylsulphone

This investigation was made to clarify the mechanism of the increased methaemoglobin formation in erythrocytes that follows the administration of 4,4′diaminodiphenylsulphone. The drug itself does not induce the formation of methaemoglobin, but the results of incubation of erythrocytes with plasma obtained during administration of the drug indicate that a metabolite is responsible for this complication. The metabolite activates the pentose-phosphate shunt of the erythrocytes. A hypothesis is put forward according to which the aromatic amino compound is converted to a nitroso- or hydroxylamino-compound. A coupled oxidation involving the hydroxylamino compound and haemoglobin results in the formation of a nitroso-compound and methaemoglobin, respectively. The hydroxylamino-compound is regenerated by means of a pyridine-nucleotide-dependent enzyme. Rapid methaemoglobin formation after addition of the drug to a system of erythrocytes, microsomes, and NADPH 2 supports the theory.