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PubMed1989

Methemoglobin reduction under near physiological conditions.

Pure methemoglobin was prepared from fresh red cells and was used as substrate for methemoglobin reduction reaction. Two sources of methemoglobin reductase were used: (a) red cell hemolysate which was prepared by freezing and thawing of unwashed red cells; (b) purified methemoglobin reductase from bank blood. Methemoglobin reduction rate was measured in a mixture of pure methemoglobin (substrate) and hemolysate (enzyme). In other experiments the rate of methemoglobin reduction was measured in the above mixture with the addition of various other compounds such as NADH, cytochrome b5, and pure methemoglobin reductase. Only the addition of pure enzyme accelerated the rate of methemoglobin reduction. In other experiments, the rate of methemoglobin reduction was measured when the reduction reaction was carried out in the presence of various amounts of deoxyhemoglobin, globin, or albumin. It was shown that all proteins tested here decreased the reduction rate. It is concluded that (a) in the red cell, under normal conditions, only the activity of the methemoglobin reductase controls the speed of methemoglobin reduction, and (b) the inhibition of methemoglobin reduction by reduced hemoglobin is mostly nonspecific suggesting a noncompetitive reaction.

Body Temperature↗
PubMed1971

Electrophoretic and functional variants of NADH-methemoglobin reductase in hereditary methemoglobinemia.

The electrophoretic mobility and activity of NADH-methemoglobin reductase in erythrocytes of patients with hereditary methemoglobinemia, obligatory heterozygotes, and normal subjects were examined. Six distinct electrophoretic variants were found in studies of erythrocytes from members of ten different families. Five variants (Boston Slow, Duarte, Princeton, Puerto Rico, and California) were associated with significant methemoglobinemia and moderate to marked decreases in enzymic activity. Precise correlations between levels of NADH-methemoglobin reductase activity, electrophoretic mobility, and clinical severity of methemoglobinemia, however, could not be drawn. One variant (Boston Fast) was associated with almost normal activity and very minimal methemoglobinemia. Nine members from three generations of two Italian families were found to have two bands with NADH-methemoglobin reductase activity in their erythrocytes, one with normal mobility and one with a mobility identical with that of Boston Fast. No functional or clinical impairment could be attributed to this abnormality. The observations made in this investigation were consistent with an autosomal recessive mode of inheritance of multiple alleles for NADH-methemoglobin reductase. As has been shown to be true for hemoglobin and glucose-6-phosphate dehydrogenase, multiple aberrations in the NADH-methemoglobin reductase of human erythrocytes apparently exist, some with and some without functional consequences. Two bands with NADPH-methemoglobin reductase activity with electrophoretic mobilities distinct from those of the NADH-methemoglobin reductase were found in human erythrocytes. These bands were normal in hemolysates of erythrocytes from patients with hereditary methemoglobinemia, but were absent from the hemolysate of erythrocytes deficient in NADPH-methemoglobin reductase activity. These latter erythrocytes, however, contained normal concentrations of methemoglobin and had a normal ability to reduce methemoglobin in vitro. These observations were most consistent with the thesis that the NADH-methemoglobin reductase, distinct from any NADPH-methemoglobin reductase, was the major system responsible for the reduction of methemoglobin to hemoglobin in human erythrocytes.

Alleles↗
PubMed2003

Methemoglobin formation in children with congenital heart disease treated with inhaled nitric oxide after cardiac surgery.

OBJECTIVE: Inhaled nitric oxide (NO) is used as a therapy of pulmonary hypertension in children after cardiac surgery. Hemoglobin binds to NO with great affinity and forms methemoglobin by oxidation in the erythrocyte. Once produced, methemoglobin is unable to transport and unload oxygen in the tissues. The amount of available hemoglobin in the body for oxygen transport is thereby reduced. Anemia, acidosis, respiratory compromise and cardiac disease may render patients more susceptible than expected for a given methemoglobin level. The goal of the present study was to review the cumulative effect of inhaled NO on methemoglobin formation in critically ill children. We therefore looked for methemoglobin levels in children with congenital heart disease after cardiac surgery who were treated with inhaled NO in a range of 5-40 ppm. METHODS: We retrospectively reviewed the medical charts of 38 children with congenital heart disease after cardiac surgery. We extracted demographic data and physiological measurements at the following time points: (1) T0 = before starting inhaled NO therapy, (2) T1 = 24 h after the beginning of inhaled NO therapy, (3) T2 = half-time therapy, (4) T3 = end of therapy, (5) T4 = 24 h after finishing inhaled NO therapy. RESULTS: The median duration of inhaled NO therapy was 5.5 days (interquartile range 6, range 2-29), NO concentrations at T1 and T2 were 16 ppm (10, 5-40) and 12.5 ppm (12.3, 2-40), respectively. The median cumulative dose of inhaled NO was 1699 ppm (2313, 193-7018). Methemoglobin levels increased moderately, but significantly, during therapy ( T0 vs T1 p<0.05 and T0 vs T2 p<0.001). The highest methemoglobin level measured was 3.9%. Methemoglobin levels correlated positively with the inhaled NO doses applied at T1 ( r(2)=0.8376; p<0.01) and at T2 ( r(2)=0.8945; p<0.01). At T1 the methemoglobin level correlated negatively with the T1 blood pH value. The overall mortality rate was 13.2% (5 of 38 study patients died). There was no significant difference in methemoglobin levels between survivors and non-survivors. CONCLUSION: We conclude from our data that the use of inhaled NO therapy for children with congenital heart disease after cardiac surgery in the described range of 5-40 ppm, resulting in a maximum of 4% methemoglobin blood level, is feasible and safe. However, we recommend the use of the minimal effective dose of inhaled NO and continuous monitoring of methemoglobin levels, especially in cases of anemia or sepsis in critically ill children.

Administration, Inhalation↗
PubMed1993

Reversal of copper(II)-induced methemoglobin formation by thiols.

Oxyhemoglobin is oxidized to methemoglobin by copper(II) in a two-stage reaction that results in conversion of Fe(II) to Fe(III) in heme of the beta subunits, but not the alpha subunits. Glutathione and other thiols, which are slowly oxidized by Cu(II), protect oxyhemoglobin from Cu(II)-induced oxidation. In the present studies, when oxyhemoglobin was first oxidized to methemoglobin by Cu(II) and thiols such as glutathione added to the sample, methemoglobin was reduced to oxyhemoglobin. Once reduction of methemoglobin stopped, as the thiol was oxidized, the oxyhemoglobin formed was reoxidized by Cu(II). The addition of the same thiols to methemoglobin formed by autoxidation did not reduce it to oxyhemoglobin. The addition of thiols such as cysteine, which are rapidly oxidized by Cu(II), to methemoglobin formed by incubation with Cu(II) also resulted in reduction of methemoglobin, but the period of reversal was much shorter than that seen with glutathione and other less reactive thiols. When cysteine and glutathione were added together to Cu(II)-induced methemoglobin, the rate of reduction and reoxidation was intermediate to that seen when either was added separately. When EDTA was added to a system in which oxyhemoglobin was undergoing Cu(II)-induced oxidation, oxidation of oxyhemoglobin ceased and there was no reduction of the methemoglobin to oxyhemoglobin. When both glutathione and EDTA were added to this system, the response was the same as with EDTA alone, suggesting that Cu(I) or (II) may be required for the reduction of copper-induced methemoglobin by thiols. These studies show that thiols that are slowly oxidized by Cu(II) both protect oxyhemoglobin from Cu(II)-induced oxidation, and reduce the methemoglobin formed to oxyhemoglobin.

Copper↗
PubMed1999

Accuracy of methemoglobin measurements: comparison of six different commercial devices and one manual method.

OBJECTIVE: During nitric oxide inhalation, methemoglobinemia needs to be monitored. We compared six commercially available instruments and one manual method for methemoglobin measurements. In addition, we studied whether and to what degree methylene blue interferes with methemoglobin measurements. DESIGN: In vitro methodologic study. SETTING: Research laboratory in a university hospital. PATIENTS: Five healthy volunteers from whom red blood cells were obtained. INTERVENTIONS: Methemoglobinemia was generated in a red blood cell suspension by nitric oxide; methemoglobin was measured with six commercial instruments and one manual photometric method to calculate variation coefficients and to determine the differences between the devices. Methemoglobin was measured with and without the addition of methylene blue with two instruments. Measurements were performed immediately after the addition of methylene blue. MEASUREMENTS AND MAIN RESULTS: All six commercially available instruments had variation coefficients of <0.1 at methemoglobin concentrations of 5%, whereas the manual photometric method did not reach a variation coefficient of <0.1 at 8% of methemoglobin. Apart from two devices that measured slightly but significantly higher methemoglobin levels, all instruments measured similar values of methemoglobin when the same samples were determined simultaneously. Higher concentrations of methylene blue (10, 40, 100 microM) reduced substantially the apparent concentrations of methemoglobin. Interference by methylene blue was most pronounced at low methemoglobin levels. CONCLUSIONS: With some limitations, all commercial instruments that were tested performed adequately for the monitoring of methemoglobinemia. Methylene blue interferes with the methemoglobin measurements in a dose-dependent manner.

Adult↗
PubMed1978

Nuclear magnetic resonance studies of hemoprotein. Proton hyperfine shifts and structural characterization of the different heme environments in methemoglobin and metmyoglobin.

The different features of the nature of heme iron-proximal binding in methemoglobin and metmyoglobin were investigated by high resolution proton nuclear magnetic resonance spectroscopy at 220 MHz. From the comparison of the hyperfine-shifted heme methyl resonances of various methemoglobin and metmyoglobin derivatives it was revealed that the metHb derivatives exhibit smaller hyperfine shifts than the corresponding metMb derivatives. This observation was interpreted in terms of the stronger interaction between proximal histidine and ferric heme iron in methemoglobin than in metmyoglobin. The spectral contributions from the alpha and beta subunits in methemoglobin in high spin state were successfully separated by the analysis of the temperature dependent heme methyl shifts of the methemoglobin azide complex which is in the high and low thermal spin equilibrium. It was shown that the beta heme methyl resonances have smaller hyperfine shifts than the alpha heme ones in ferric high spin state. This tendency in methemoglobin derivatives was also interpreted in terms of the stronger iron-histidine binding in the beta subunits than in the alpha subunits within the tetramer. From the comparisons between methemoglobin and metmyoglobin, and between the alpha and beta subunits in methemoglobin, the order of the strength of the iron-histidine bond interaction was deduced as follows: metmyoglobin less than alpha subunits in methemoglobin less than or equal to beta units in methemoglobin.

Hemeproteins↗
PubMed1979

Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A.

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the alpha-hemes (g = 6.0) and the beta-hemes (g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A (g = 6.0). The beta-hemes are found to be comparatively insensitive to both effectors while the alpha-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the alpha-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the beta-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The beta-heme spectral changes appear to be pH independent while those arising from the alpha-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the alpha-hemes which undergoes spectral change upon inositol hexaphosphate binding to the beta-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the alpha-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the beta-hemes are in the high spin form.

Chemical Phenomena↗
PubMed1995

Measurement of methemoglobin formation from oxyhemoglobin. A real-time, continuous assay of nitric oxide release by human polymorphonuclear leukocytes.

We have evaluated the spectrophotometric measurement (at 401 vs. 411 nm) of nitric oxide (NO)-dependent methemoglobin formation from oxyhemoglobin in order to assess NO release from human polymorphonuclear neutrophil leukocytes (PMN). S-nitroso-D,L-acetyl-penicillamine (SNAP, 25-200 microM), a donor of NO, induced a dose-dependent methemoglobin formation. Furthermore, when PMN were activated with N-formyl-methionylleucyl-phenylalanine or phorbol myristate acetate in the presence of superoxide dismutase (SOD) and catalase, methemoglobin formation ensued. The amount of methemoglobin formed was dependent on the amounts of oxyhemoglobin and stimulus used, and the number of PMN in the assay. The NO synthase (NOS) inhibitors NG-monomethyl-L-arginine or nitro-L-arginine methyl ester did not affect methemoglobin generation from oxyhemoglobin induced by SNAP but inhibited that mediated by activated PMN with IC50 values of 250 microM and 340 microM, respectively. The substrate for NO formation from NOS, L-arginine in concentrations up to 1 mM did not significantly influence the methemoglobin formation either induced by SNAP or activated PMN. Exclusion of SOD did not affect SNAP-dependent oxidation of oxyhemoglobin. Exclusion of SOD from the cell-containing system attenuated methemoglobin formation, and if catalase was also excluded the response was further reduced. Finally, PMN from a patient with X-linked chronic granulomatous disease, unable to produce superoxide anions, showed a similar production of methemoglobin from HbO2 as did healthy PMN, activated with the respective agonists. We conclude that spectrophotometric measurement of methemoglobin formation from oxyhemoglobin in the presence of SOD and catalase is a suitable method for the measurement of NO release from PMN, with the benefits of a real-time, continuous assay.

Amino Acid Oxidoreductases↗
PubMed1982

[Methemoglobin level in the blood of agricultural animals and poultry].

Studies were carried out with all kinds of farm animals and birds with regard to the quantitative determination of methemoglobin in their blood in connection of seasonal changes. Blood was immediately treated upon sampling. Methemoglobin determination was carried out spectrophotometrically. Evaluated was also the percent of nitrates in the rations of the animals and birds. It was found that during the winter the amound of methemoglobin in the blood of both animals and birds varied from 0.25 to 4.4 of the total amount of methemoglobin. Its percent content during the summer varied from 0.25 to 4.17 with animals, and from 9.5 to 15.7 with birds of the total amount of methemoglobin. The amount of blood methemoglobin was found to correlate directly with the percent of nitrates in the ration. Except for birds, the seasons had no effect on the values of methemoglobin. The breed, weight, age, and physiologic status (pregnancy, barrenness) did not either affect the amount of methemoglobin. There were individual variations of the methemoglobin values with animals of one and the same species.

Animal Feed↗
PubMed1998

Hemoglobin based oxygen carriers: how much methemoglobin is too much?

The oxidized form of hemoglobin, methemoglobin, is unable to deliver oxygen to tissues. Hemoglobin based oxygen carriers generally lack the natural oxidative-reductive machinery present within the red blood cell that converts methemoglobin to hemoglobin. This study examines tolerable levels of methemoglobin that can be present in solutions of polyethylene glycol (PEG) conjugated bovine hemoglobin without compromising its ability to deliver oxygen. Rodents were exchange-transfused to 30% of their estimated blood volume with solutions of six grams percent PEG-hemoglobin containing varying concentrations of PEG-methemoglobin. Tissue oxygenation was measured by an oxygen dependant phosphorescence quenching method. This study also looked at the level of methemoglobin formation following a top loaded infusion of low methemoglobin containing PEG-hemoglobin. Results of the oxygenation study showed that PEG-methemoglobin levels at or below 10% did not significantly alter the ability of solutions to deliver oxygen to intestines, liver, spleen and kidney. However, PEG-methemoglobin levels greater than 10% resulted in a significant decrease in PEG-hemoglobin's ability to oxygenate tissues. In addition, methemoglobin levels remain low (< 10%) for a substantial period of time following PEG-hemoglobin administration.

Animals↗
PubMed1985

Interspecies variability in propylene glycol dinitrate-induced methemoglobin formation.

Interspecies variability of propylene glycol dinitrate (PGDN)-induced methemoglobin formation was studied in vitro employing erythrocytes from four separate species. The net rate of methemoglobin formation was significantly different among species with dog greater than guinea pig greater than rat greater than or equal to human. This order of susceptibility was maintained in stroma-free hemolysates, indicating that interspecies variability was not a reflection of differences in red cell membrane permeability or intracellular transport of PGDN. The erythrocytic enzymes, catalase, superoxide dismutase, glutathione peroxidase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, methemoglobin reductase, and glutathione-S-transferase, were assayed by adaptation of existing methods to a centrifugal analyzer. The above enzymes were removed from hemoglobin derived from each species and the order of susceptibility to PGDN-induced methemoglobin formation remained essentially the same with dog greater than guinea pig greater than human = rat. However, the net rate of PGDN-mediated oxidation of hemoglobin to methemoglobin increased in purified hemoglobin preparations from each species. These results demonstrate that there is species variability in the net rate of PGDN-mediated methemoglobin formation. Total enzyme activity in erythrocytes may contribute to reduction in the net rate of methemoglobin formation. However, the primary determinant of the net rate of methemoglobin formation induced by PGDN appears to be the structure of each hemoglobin molecule.

Animals↗