The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

NADPH-flavin reductase in human erythrocytes and the reduction of methemoglobin through flavin by the enzyme

A NADPH-dehydrogenase of human erythrocytes was exhaustively purified to a homogeneous protein judging from the electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate. Studies on the specificity for the electron acceptor of this enzyme suggest that flavins serve as the natural and direct electron acceptor. The enzyme showed a broad specificty for flavins and the Michaelis constants for flavins were estimated to be 5 × 10^?5 M for both FMN and riboflavin. Rapid reduction of methemoglobin by the enzyme in the presence of flavin was demonstrated, and the reduction was explained by the reduction of flavin by the enzyme, and subsequent non-enzymatic reduction of methemoglobin by the reduced flavin. The therapeutic significance of flavins was discussed with reference to the flavin reductase activity in hereditary methemoglobinemia.     

Reduction of extracellular methemoglobin by erythrocytes

Erythrocytes, suspended in a glucose-containing buffer, catalyzed the partial reduction of extracellular methemoglobin. Physiological concentrations of ascorbic acid or dehydroascorbic acid greatly enhanced the rate of reaction and the ultimate extent of reduction. The relationship between erythrocyte concentration and initial reaction rate was nonlinear, which suggested that the rate limiting factor was not an erythrocyte membrane enzyme. Also, significant dehydroascorbate-stimulated reduction occurred even when the erythrocytes and methemoglobin were separated by a dialysis membrane. The above observations indicate that the transfer of reducing equivalents across the erythrocyte membrane and reduction of extracellular methemoglobin can be accomplished by release and recycling of ascorbic acid.     

Comparative aspects of methemoglobin formation and reduction in opossum (Didelphis virginiana) and human erythrocytes

Glucose-depleted, nitrite-treated opossum erythrocytes effectively reduce methemoglobin in an environment of physiological saline and added glucose does not accelerate the rate of reduction. In autologous plasma or 25 mM phosphate-buffered saline pH 7.4, added glucose significantly accelerates methemoglobin reduction in glucose-depleted, nitrite-treated opossum erythrocytes. Human red cells require added glucose to carry out reduction of methemoglobin and increased phosphate concentration or autologous plasma does not alter the rate of this process. Within the opossum red cell in vitro, autooxidation of hemoglobin proceeds at a much slower rate than that observed in human erythrocytes.     

Studies on the energy metabolism of opossum Didelphis virginiana erythrocytes--III. Metabolic depletion with 2-deoxyglucose markedly accelerates methemoglobin reduction in opossum but not in human erythrocytes

1. Glucose-depleted, nitrite-treated erythrocytes reduce ferriheme in vitro in an environment 100 mM to 2-deoxy-D-glucose at a rate of 2.4 microM/ml cells/hr (opossum) and 0.37 microM/ml cells/hr (human). 2. During the process of methemoglobin reduction the breakdown of adenine ribonucleotides is more rapid in opossum (0.9 microM/g hg/hr) than in human (0.36 microM/g hg/hr) erythrocytes. 3. Radiolabelled ribose from [U-14C] ATP is catabolized exclusively to [14C] lactate in opossum, and to [14C] pyruvate and [14C] lactate in human red cells.     

Green hemoprotein of erythrocytes: methemoglobin superoxide transferase

Influences of base (pH 10), heat (50 degrees C), microwave radiation (2450 MHz, 103 +/- 4 W/kg), and hydrogen peroxide (5.6 mM) generated by glucose oxidase on oxidation of human oxyhemoglobin to methemoglobin were examined. Conversion of oxyhemoglobin to methemoglobin was followed by the difference in absorbancy of 540 or 542 nm and 576 nm wavelength light versus time. Fresh basic hemolysates auto-oxidized on heating with a zero order rate constant, implying that hemoglobin or another protein saturated with oxyhemoglobin catalyzed the oxidation. Simultaneous microwave irradiation inhibited thermally induced auto-oxidation on the average by 28.6%. However, there was great variability among samples and a decrease in auto-oxidation with aging of individual samples. The auto-oxidation rate was independent of initial oxyhemoglobin concentration. Oxidation of partially purified oxyhemoglobin by hydrogen peroxide was not influenced by microwave irradiation. Adding green hemoprotein isolated from human erythrocytes to the oxyhemoglobin/glucose oxidase reaction mixture yielded absorption spectra (500-600 nm) that were a combination of oxyhemoglobin, deoxyhemoglobin, and methemoglobin spectra. Green hemoprotein was labile in hemolysates but stable in a partially purified ferric form. These results imply that thermally unstable reduced green hemoprotein can reverse oxidation of oxyhemoglobin by hydrogen peroxide and could mediate the thermally induced and microwave inhibited auto-oxidation of oxyhemoglobin.     

Simulation study of methemoglobin reduction in erythrocytes. Differential contributions of two pathways to tolerance to oxidative stress

Methemoglobin (metHb), an oxidized form of hemoglobin, is unable to bind and carry oxygen. Erythrocytes are continuously subjected to oxidative stress and nitrite exposure, which results in the spontaneous formation of metHb. To avoid the accumulation of metHb, reductive pathways mediated by cytochrome b5 or flavin, coupled with NADH-dependent or NADPH-dependent metHb reductases, respectively, keep the level of metHb in erythrocytes at less than 1% of the total hemoglobin under normal conditions. In this work, a mathematical model has been developed to quantitatively assess the relative contributions of the two major metHb-reducing pathways, taking into consideration the supply of NADH and NADPH from central energy metabolism. The results of the simulation experiments suggest that these pathways have different roles in the reduction of metHb; one has a high response rate to hemoglobin oxidation with a limited reducing flux, and the other has a low response rate with a high capacity flux. On the basis of the results of our model, under normal oxidative conditions, the NADPH-dependent system, the physiological role of which to date has been unclear, is predicted to be responsible for most of the reduction of metHb. In contrast, the cytochrome b5-NADH pathway becomes dominant under conditions of excess metHb accumulation, only after the capacity of the flavin-NADPH pathway has reached its limit. We discuss the potential implications of a system designed with two metHb-reducing pathways in human erythrocytes.     

Effects of ascorbate on methemoglobin reduction in intact red cells

An endpoint of 75% HbO₂/25% methemoglobin (MetHb) was approached in red cells incubated with a greater than physiologic concentration of ascorbate (10 mm). The presence of glucose (5 mm) with ascorbate shifted the endpoint to 90% HbO₂/10% MetHb while lactate (2 mm) plus pyruvate (0.1 mm) had no effect. These endpoints were approached regardless of the $\text{HbO}{}_2\text{MetHb}$ ratio at zero time. No hemoglobin degradation was observed. When red cells containing 100% MetHb at zero time were used, analysis of the initial rate of HbO₂ formation in the presence of various substrates showed synergistic interaction between ascorbate (10 mm) and glucose, additive activity with ascorbate and lactate, and less than additive activity with glucose and lactate. Incubation of red cells with a phsyiologic concentration of ascorbate (0.1 mm) resulted in no significant HbO₂ formation in the absence of other additions. When red cells were incubated with glucose and/or lactate plus pyruvate, an endpoint of about 99% HbO₂/1% MetHb was approached regardless of the HbO₂/MetHb ratio at zero time or the presence or absence of physiologic ascorbate. Physiologic ascorbate slightly but consistently increased the rate of HbO₂ formation in red cells incubated with glucose but not with lactate. HbO₂ formation was not increased by ascorbate in red cells which contained more than about 90% HbO₂ at zero time. The results indicate that excess ascorbate functions stoichiometrically driving cellular chemistry to a steady state between HbO₂ and MetHb formation whereas physiologic ascorbate functions catalytically allowing electron transport from glucose to MetHb via the hexose monophosphate shunt.