Methemoglobin Reduction In Erythrocytes Research Articles

Type I Methemoglobinemia Caused by the Cytochrome b5 Reductase 806C>T Mutation Is Present in the Indigenous Evenk People of Yakutia.

Abstract 2588Poster Board II-564 Background:Congenital methemoglobinemia is an autosomal recessive metabolic disorder due to NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) deficiency. This enzyme exists in soluble and membrane-bound forms. The soluble erythrocytic cytb5r isoenzyme is involved in cytochrome b5 reduction and in erythrocyte methemoglobin reduction; the membrane-bound microsomal enzyme participates in a fatty acid desaturation complex and in drug metabolism. The cytb5r isoforms are a product of a single gene locus, DIA1 (or CYB5R3), on chromosome 22. Type I methemoglobinemia, a benign form in which cyanosis is the only phenotype, is characterized by cytb5r deficiency restricted to red blood cells. In less common type II methemoglobinemia, chronic cyanosis is associated with severe neurological and developmental deficits, including mental retardation, microcephaly, generalized dystonia and movement disorders. More then 40 mutations have been reported to date in the DIA1 gene, which either cause type I or type II methemoglobinemia; the majority are missense mutations and are associated with type I disease. Both methemoglobinemia types are sporadic worldwide but are claimed to be endemic among the Yakut people in Siberia, the Aleutians in Alaska and the Navajo tribe in the continental US. In 2006, a new mutation in exon 9 of the CYB5R3 gene (806C>T, Pro269Leu) was identified in 38 patients from the indigenous population of Yakutia in northeastern Siberia, a part of the Russian Federation (1). The frequency of homozygotes was reported to be 1 in 5677. The Sakha region of the Yakutia Republic has an area of 1,200,000 sq miles and a population <1 million composed of 45.5% Yakuts, 41.2% Russians, 3.7% Ukrainians and indigenous people including 1.9% Evenks, 1.2% Evens, 0.1% Dolgans, and 0.1% Yukagirs. Methods:We screened DNA of 162 subjects' samples taken from children of indigenous people from 4 different places in Sakha for the mutation 806C>T in the CYB5R3 gene using the the AluI and AciI restriction enzymes that recognize this mutation, and the results were confirmed by sequencing of the PCR product. Results:The study sample included 70 Evenks, 35 Evens, 26 Sakha (Yakuts), 23 Yukagirs, 4 Chukchas, 2 Dolgans, 1 Nenets and 1 Tatar. We found 2 806C>T heterozygous samples detected by the AciI restriction enzyme and confirmed by sequencing; both subjects were Evenks. The enzyme AluI produced a partial cut in another 22 samples that could not by confirmed as a true mutation by sequencing. Thus, the analysis with the AluI restriction enzyme frequently falsely identified heterozygotes among these 162 participants. Conclusion:In screening for the CYB5R3 806C>T mutation, the AciI restriction enzyme should be used instead of the AluI enzyme. Based on these results and according to Hardy-Weinberg equilibrium, the predicted frequency of homozygotes for the CYB5R3 806C>T gene mutation is 1 in 4,444 among the Evenks. These data suggest that the CYB5R3 806C>T mutation may be endemic among the Evenks indigenous people. Further, it remains to be determined whether the CYB5R3 806C>T mutation is also causative of type I methemoglobinemia in the Aleutian and Navajo peoples whose ancestors migrated to North America from Siberia. These data are preliminary and larger population-based studies using the AciI restriction enzyme are planned. Acknowledgments:This work was supported by the ASH Visitor Training Program Award and by NHLBI Grant 2 UH1-HL03679. References1) Гaлeeвa H.M., Haзapeнкo Л.П., Haзapeнкo C.A., Tвepcкaя C.M., Пoлякoв A.B. Moлeкyляpнo-гeнeтичecкaя пpичинa нacлeдcтвeн|Ryoй мeтгeмoглoбинeми|Rb лepвoгo типa в Якyти|Rb. Meдицинcкaя Гeнeтикa (2006). Disclosures:No relevant conflicts of interest to declare.

Cytochrome b5 Reductase T116S Mutation and Hemolysis in Sickle Cell Disease.

Abstract 903Abstract Background:Deficiency of NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) is responsible for congenital methemoglobinemia. This enzyme exists in soluble and membrane-bound forms. The soluble erythrocytic cytb5r isoenzyme is involved in cytochrome b5 reduction and in erythrocyte methemoglobin reduction; the membrane-bound microsomal enzyme participates in a fatty acid desaturation complex and in drug metabolism. The cytb5r isoforms are the product of a single gene locus, DIA1 (or CYB5R3), on chromosome 22. More then 40 mutations which cause methemoglobinemia have been reported to date; the majority are missense mutations and are associated with mild type I methemoglobinemia. The CYBR5 T116S mutation is the most common genetic polymorphism among African Americans known (gene frequency as high as 20%) and it has not yet been detected in other ethnic and racial groups. This polymorphism is not associated with methemoglobinemia and its functional significance is not yet known. We studied the relationship of CYBR5 T116S with the degree of hemolysis and the tricuspid regurgitation velocity (which correlates with systolic pulmonary artery pressure) in patients with sickle cell disease. Methods:Two hundred sixty one children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Patients with other sickle genotypes were excluded from this analysis of CYBR5 T116S. Principal component analysis was used to develop a hemolytic component from reticulocyte count and concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of the CYBR5 T116S mutation. Multivariate models were employed to determine the independent effects of this genotype on degree of hemolysis and tricuspid regurgitation velocity. Results:Ninety-eight of the patients (38%) were CYBR5 T116S heterozygotes and 26 (10%) were homozygotes, consistent with Hardy-Weinberg equilibrium. Both heterozygosity (beta = -0.4) and homozygosity (beta = -0.5) were associated with reduction in the hemolytic component (N = 261; P for trend = 0.002) (Figure 1). This relationship persisted after adjusting for α-thalassemia, hemoglobin F percent and hydroxyurea treatment in a subset of 113 patients with all of this information available (P for trend = 0.037) and it also persisted in a subset of 87 patients with no α-globin gene deletion who were not being treated with hydroxyurea (P for trend = 0.029). In none of these analyses did G6PD-202/-376 have an effect on hemolysis. Both heterozygosity (beta = -0.04) and homozygosity (beta = -0.14) for the CYBR5 T116S mutation were also associated with lower tricuspid regurgitation velocity (P for trend = 0.024). Conclusions:CYBR5 T116S is a common polymorphism among patients with sickle cell disease that appears to be associated with less hemolysis and lower tricuspid regurgitation velocity. We speculate that this polymorphism may be related to a previously reported subpopulation of African Americans with increased cytochrome b5 reductase activity, and that increased anti-oxidant activity may explain the polymorphism's hemolysis-reducing effect. Functional studies to investigate this possibility are planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Simulation study of methemoglobin reduction in erythrocytes

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.

Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.

An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes with Kd values ranging from 1 to 5 microM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 microM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 microM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.

Impaired Erythrocyte Methemoglobin Reduction in Sickle Cell Disease: Dependence of Methemoglobin Reduction on Reduced Nicotinamide Adenine Dinucleotide Content

Abstract We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Impaired erythrocyte methemoglobin reduction in sickle cell disease: dependence of methemoglobin reduction on reduced nicotinamide adenine dinucleotide content

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Magnetic circular dichroism studies of hemoglobin. The reduction of ferrihemoglobin by ferrocytochrome b5 and characterization of the high-spin hydroxy species of mixed-valence hemoglobin.

The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b5 to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8, 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b5. This analysis generated a KD that depends on ionic strength and hemoglobin tetramer conformation, a Vmax that was independent of these factors, and an inhibition constant that was equal to KD. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b5 and hemoglobin and the electron transfer.

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.

Acceleration of methemoglobin reduction in erythrocytes by selenium.

Selenium accelerated the reduction of methemoglobin in erythrocytes. Its mode of action is suggested as a catalysis of the methemoglobin reduction by glutathione.

Neural, hematologic and bacteriologic effects of nitrofurantoin in renal insufficiency

Eleven subjects with normal kidney function and six with renal insufficiency were given nitrofurantoin, 100 mg orally; urine concentrations of the drug sufficient to inhibit growth of sensitive microorganisms were obtained during the next six hours in normal subjects but not in those with azotemia. Despite decreased excretion, the serum drug concentration did not increase in patients with renal failure. In nine uremic patients with urinary tract infection treated with nitrofurantoin, control peripheral nerve and/or sensory nerve conduction velocities were at the lower limits of normal in all but one. After nitrofurantoin at a lower dose as recommended by the manufacturer had been given, three had a minimal further decrease in conduction velocities, three others did not. In the three affected patients, normal conduction velocities were regained within two weeks of discontinuation of therapy. Serum ascorbate and folate concentrations were not influenced by treatment nor did their control values discriminate patients who had altered nerve function. Blood counts and erythrocyte methemoglobin reduction did not vary significantly during this study. Our findings suggest that when renal function is seriously compromised, nitrofurantoin can be used in reduced dosage without affecting peripheral nerve function, but effective antimicrobial levels in the urine cannot be achieved. On the other hand, if dosage is raised to attain effective antimicrobial levels, neurologic side effects may be anticipated.

Reduction of Methemoglobin in Erythrocytes of a Patient with Congenital Methemoglobinemia, Subjects with Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency, and Normal Individuals

Reduction of Methemoglobin in Erythrocytes of a Patient with Congenital Methemoglobinemia, Subjects with Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency, and Normal Individuals

The Reduction of Methemoglobin in Erythrocytes of a Patient with Congenital Methemoglobinemia, Subjects with Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency, and Normal Individuals

AbstractThe pathways for the reduction of methemoglobin to hemoglobin that are dependent upon the generation of reduced pyridine nucleotides were studied in normal human erythrocytes, in erythrocytes deficient in G-6-PD activity and in erythrocytes of a subject with congenital methemoglobinemia. Reduction of methemoglobin, produced by treatment with nitrite, occurred at equivalent rates in normal and G-6-PD deficient erythrocytes, but failed to occur in the erythrocytes of the patient with congenital methemoglobinemia upon incubation with glucose or inosine. The DPNH-utilizing diaphorase-like system was normal in hemolysates of G-6-PD deficient erythrocytes, but was markedly deficient in hemolysates of erythrocytes of the subject with congenital methemoglobinemia. The marked acceleration of methemoglobin reduction that occurred upon the addition of methylene blue to normal erythrocytes and to the erythrocytes of the woman with congenital methemoglobinemia did not occur with G-6-PD deficient erythrocytes. The TPNH-utilizing methemoglobin reductase system was normal in hemolysates of erythrocytes of the patient with congenital methemoglobinemia, but was reduced to about 50 per cent of normal activity in hemolysates of G-6-PD deficient erythrocytes.The reduction of methemoglobin to hemoglobin in intact normal and G-6-PD deficient human erythrocytes probably proceeds by way of a DPNH-utilizing, diaphorase-like system that is deficient in the erythrocytes of one type of congenital methemoglobinemia. The TPNH-utilizing methemoglobin reductase appears to be a reserve system that requires an artificial electron carrier, such as methylene blue, to become fully effective in reducing methemoglobin to hemoglobin. The TPNH-methemoglobin reductase system is impaired in G-6-PD deficient erythrocytes not only because of a deficient source of TPNH, but, perhaps, also because of a defect in this methemoglobin reductase system itself.