Species differences in methemoglobin reductase activity☆
Abstract
Sodium nitrite induced equivalent levels of methemoglobin in washed erythrocytes from cat, dog, and man, all suspended in Krebs-Ringer phosphate-glucose (pH 7·4). The same levels occurred in human cells with or without aded substrate (glucose or lactate). In all these incubations, reduction of methemoglobin was minimal or absent over a 2-hr period. When 10−5M methylene blue was added with glucose, equivalent increases in rates of methemoglobin reduction occurred in the cells of all three species. Similar rates were seen in rabbit and mouse red cells even without added methylene blue, as long as lactate or glucose was present. Methylene blue further enhanced reductase activity in mouse cells but only in the presence of glucose. Rabbit cells responded much less dramatically, if at all, to methylene blue. Lysates of human, rabbit, and mouse cells were equally sensitive to nitrite, and no spontaneous reduction occurred. These findings suggest that the high reductase activity of rabbit and mouse erythrocytes is NADH-dependent. The mouse but not the rabbit appears to possess also a NADPH-dependent reductase like man, dog and cat.
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Cited by (51)
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HPLC analysis of human erythrocytic glutathione forms using OPA and N-acetyl-cysteine ethyl ester: Evidence for nitrite-induced GSH oxidation to GSSG
2009, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesGlutathione exists in biological samples in the reduced form (GSH), as its disulfide (GSSG) and as a mixed disulfide (GSSR) with thiols (RSH). GSH is the most abundant low-molecular-mass thiol and plays important roles as a cofactor and as a main constituent of the intracellular redox status. Due to its own sulfhydryl (SH) group, GSH reacts readily with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative (GSH-OPA), which allows for sensitive and specific quantitative determination of GSH in biological systems by HPLC with fluorescence (FL) detection. In the present article we report on the utility of the novel, strongly disulfide bond-reducing thiol N-acetyl-cysteine ethyl ester (NACET) for the specific quantitative analysis of GSH and GSSG in the cytosol of red blood cells (RBC) as GSH-OPA derivative with FL (excitation/emission 338/458 nm) or UV absorbance (338 nm) detection. Unlike in aqueous solution, the derivatization of GSH in RBC cytosol yielded two closely related derivatives in the absence of NACET and only the GSH-OPA derivative in the presence of NACET. The HPLC method was optimized and validated for human RBC and applied to measure GSH and GSSG in RBC of healthy subjects. Basal GSH and GSSG concentrations were determined to be 2340 ± 350 μM and 11.4 ± 3.2 μM, respectively, in RBC of 12 healthy young volunteers (aged 23–38 years). The method was also applied to study the effects of nitrite on the glutathione status in intact and lysed human RBC. Nitrite at mM-concentrations caused instantaneous and considerable GSSG formation in lysed but much less pronounced in intact RBC. GSH externally added to lysed RBC inhibited nitrite-induced methemoglobin formation. Our findings suggest that nitric oxide/nitrite-related consumption rate of GSH, and presumably that of NADH and NADPH, could be of the order of 600 μmol/day in RBC of healthy subjects.
Clinical Pathology of the Rat
2006, The Laboratory RatThis chapter provides a basis for how clinical pathology parameters are used to correctly diagnose toxicity and naturally occurring disease in the rat. Hematology data are generally collected by highly automated instrumentation that differentiates and quantifies the formed elements in blood based on their size, internal structures, staining, and/or biochemical characteristics. Such analyses then use species-specific algorithms to classify cells according to type. An evaluation of bone marrow cells is generally not necessary for the interpretation of peripheral blood changes. However, it is important to prepare bone marrow smears at the time of sacrifice for potential future examination. An examination of bone marrow alterations may provide additional information to the interpretation made from peripheral blood analyses, particularly in cases of unexplained cytopenias or malignancies. Routine clinical chemistry tests are run on automated clinical chemistry analyzers. These instruments use a variety of reagents to determine concentrations or activities of analytes in serum or plasma.
Hemoglobin autoxidation and regulation of endogenous H<inf>2</inf>O <inf>2</inf> levels in erythrocytes
2005, Free Radical Biology and MedicineRed cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 × 10−7 s−1 and an endogenous H2O2 and superoxide concentrations of 5 × 10−11 and 5 × 10−13 M, respectively, lower than previous estimates.
Clinical Pathology of the Rat
2005, The Laboratory Rat, Second EditionThis chapter provides a basis for how clinical pathology parameters are used to correctly diagnose toxicity and naturally occurring disease in the rat. Hematology data are generally collected by highly automated instrumentation that differentiates and quantifies the formed elements in blood based on their size, internal structures, staining, and/or biochemical characteristics. Such analyses then use species-specific algorithms to classify cells according to type. An evaluation of bone marrow cells is generally not necessary for the interpretation of peripheral blood changes. However, it is important to prepare bone marrow smears at the time of sacrifice for potential future examination. An examination of bone marrow alterations may provide additional information to the interpretation made from peripheral blood analyses, particularly in cases of unexplained cytopenias or malignancies. Routine clinical chemistry tests are run on automated clinical chemistry analyzers. These instruments use a variety of reagents to determine concentrations or activities of analytes in serum or plasma.
Acute hematopoietic toxicity of aniline in rats
1997, Toxicology LettersIn the present study, acute hematopoietic toxicity of aniline as a function of time was investigated in rats. The animals were given a single oral dose of aniline hydrochloride (2 mmol/kg) and euthanized at zero (control), 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h following the treatment. The blood methemoglobin level increased dramatically and attained a peak level of 37% (31 fold greater than the controls) at 0.5 h. Thereafter, the increases were less pronounced and the level declined with time. Spleen weight to body weight ratio remained unchanged up to 24 h, but increased ∼25% at 48 h. Lipid peroxidation (MDA content) in the spleen increased by 39% at 24 h and remained steady even at 48 h. MDA-protein adducts, as quantitated by a competitive ELISA, showed 94, 126 and 265% increases in the spleen homogenates at 12, 24 and 48 h, respectively, following the treatment. However, no changes were observed in the splenic protein oxidation. Morphological examination showed congestion of splenic blood vessels and marked expansion of red pulp at 24 and 48 h. These studies suggest that aniline related changes in the blood are reflected very early as evident from increases in the methemoglobin content, whereas changes in the spleen appear later and are characterized by splenic weight changes, increased lipid peroxidation, MDA-protein adduct formation and morphological changes after a single high dose exposure. The increased lipid peroxidation in the spleen also suggests that free radical-mediated reactions could be the potential mechanism of splenic toxicity of aniline and lipid peroxidation precedes protein oxidation.
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Presented in part at the August 1965 meeting of the American Society for Pharmacology and Experimental Therapeutics. This investigation was supported by Grant AP-00260, Division of Air Pollution, and by Training Grant 1T1 GM 1370-1, National Institutes of General Medical Sciences, United States Public Health Service