Carnosine and N-acetyl cysteine protect against sodium nitrite-induced oxidative stress in rat blood.

Sodium nitrite (NaNO2 ) is widely used in the food industry as a preservative and colorant in meat and fish products. Industrialization and improper agricultural practices have greatly increased human exposure to high nitrite levels, mainly through contaminated drinking water, causing various health disorders. We have investigated the protective effect of carnosine (CAR) and N-acetyl cysteine (NAC) on NaNO2 -induced toxicity in rat blood. CAR is a bioactive dipeptide found in mammalian muscle while NAC is a synthetic sulfhydryl amino acid and an important precursor of glutathione. Animals were given a single acute oral dose of NaNO2 at 60 mg/kg body weight with or without prior administration of either CAR or NAC. Rats were sacrificed after 24 h, blood was withdrawn and plasma and erythrocytes were isolated. Administration of NaNO2 alone increased methemoglobin levels and methemoglobin reductase activity, decreased the activities of antioxidant defense and metabolic enzymes and significantly weakened the total antioxidant capacity of rat erythrocytes. Similar effects were seen in plasma of NaNO2 -treated rats. In contrast, administration of CAR or NAC, prior to NaNO2 treatment, markedly attenuated the NaNO2 -elicited deleterious effects. Thus, CAR and NAC can mitigate nitrite-induced metabolic alterations and oxidative damage probably due to their intrinsic biochemical antioxidant properties. This study suggests that CAR and NAC can be potentially used as therapeutic/protective agents against NaNO2 toxicity.

Methemoglobin reductase activity in intact fish red blood cells.

Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence of counteracting oxidation. This assay was used to compare metHb reduction in rainbow trout and carp RBCs under both oxygenated and deoxygenated conditions. Washing resulted in effective wash-out of nitrite to low and safe values (~2µM). The subsequent decline in [metHb] with time followed first-order kinetics, allowing characterization of metHb reductase activity through the first order rate constant k. In oxygenated RBCs at 25°C, the k values for rainbow trout and carp were slightly below or above 0.01min-1, respectively; which is double the value reported for mammals at 37°C. We conclude the higher metHb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs was high (Q10 ~2.8) and instrumental in handling increased Hb autoxidation with temperature.

Enzymopenic Congenital Methemoglobinemia in Children of the Republic of Sakha (Yakutia).

Type I congenital methemoglobinemia is an autosomal recessive disorder. A high frequency of congenital methemoglobinemia has been reported among Native Americans inhabiting the Yukon-Kuskokwim Delta. Other rare cases of congenital methemoglobinemia of types I and II have been reported in Japan and other countries. In Russia-namely, in Yakutia-a high frequency of type I congenital methemoglobinemia has been reported. In 2009, the Consultation Polyclinic of the Pediatric Center in Yakutsk city established a registry of children with congenital methemoglobinemia. In total, 43 patients were registered between 2005 and 2009. The median methemoglobin level was 13.5% (ranging between 4.2% and 33.9%) and physical examination revealed cyanosis of the skin and mucus membranes. There were significant positive relationships between percentage of methemoglobin and erythrocyte count, hemoglobin concentration, and hematocrit among male patients, consistent with an upregulation of the hypoxic response. The prevalence per 100,000 children ranged from 12.7 to 47.0 in 3 geographic regions of Yakutia. Further research is needed to clarify the clinical consequences of congenital methemoglobinemia in the children of Yakutia and the reasons for the high variability in the prevalence of the condition.

Membrane-bound cytochrome b5 reductase (methemoglobin reductase) in human erythrocytes. Study in normal and methemoglobinemic subjects.

In this study we present evidence that in human erythrocytes NADH-cytochrome b5 reductase (methemoglobin reductase) is not only soluble but also tightly bound to the membrane. The membrane methemoglobin reductase-like activity is unmasked by Triton X-100 treatment, and represents about half of the total activity in the erythrocytes. Like the amphiphilic microsomal-bound cytochrome b5 reductase, the erythrocyte membrane-bound enzyme is solubilized by cathepsin D. Because this treatment is effective on unsealed ghosts but not on resealed (inside-in) ghosts, it is concluded that the enzyme is strongly bound to the inner face of the membrane. The erythrocyte membrane enzyme is antigenically similar to the soluble enzyme. The two forms of enzyme are specified by the same gene, in that both were found defective in six patients with recessive congenital methemoglobinemia. We suggest that the cytochrome b5 reductase of the erythrocyte membrane is the primary gene product. A posttranslational partial proteolysis probably gives rise to the soluble form of the enzyme, which serves as a methemoglobin reductase.

Congenital methemoglobinaemia due to Hb F-M-Fort Ripley in a preterm newborn.

Methemoglobinaemia is a rare cause of cyanosis in newborns. Congenital methemoglobinaemias due to M haemoglobin or deficiency of cytochrome b5 reductase are even rarer. We present a case of congenital methemoglobinaemia presenting at birth in a preterm infant. A baby boy born at 29 weeks and 3 days of gestation had persistent central cyanosis immediately after delivery, not attributable to a respiratory or cardiac pathology. Laboratory methemoglobin levels were not diagnostic. Cytochrome b5 reductase levels were normal and a newborn screen was unable to pick up any abnormal variants of fetal haemoglobin. Genetic testing showed a γ globin gene mutation resulting in the M haemoglobin, called Hb F-M-Fort Ripley. The baby had no apparent cyanosis at a corrected gestational age of 42 weeks. Although rare, congenital methaemoglobin aemia should be considered in the differential in a preterm with central cyanosis and investigated with genetic testing for γ globin chain mutations if other laboratory tests are non-conclusive.

Immunogenicity of trimethoprim/sulfamethoxazole in a macaque model of HIV infection.

BACKGROUND: Sulfonamide hypersensitivity has a high incidence in HIV infection and correlates with low CD4+ counts, but the mechanisms are not understood. The aims of this study were to determine whether trimethoprim/sulfamethoxazole (TMP/SMX) led to SMX adduct formation, immunogenicity, or signs of drug hypersensitivity in SIV-infected rhesus macaques, and whether differences in antioxidants, pro-inflammatory mediators, or SMX disposition were predictive of drug immunogenicity. METHODS: Nine macaques chronically infected with SIVmac239 and 7 non-infected controls were studied. Baseline blood ascorbate, glutathione, IFN-γ, LPS, sCD14, and cytochrome b5 reductase measurements were obtained, macaques were dosed with TMP/SMX (120mg/kg/day p.o. for 14days), and SMX metabolites, lymph node drug adducts, drug-responsive T cells, and anti-SMX antibodies were measured. RESULTS: Four of 9 of SIV-positive (44%), and 3 of 7 SIV negative (43%) macaques had drug-responsive T cells or antibodies to SMX. Two macaques developed facial or truncal rash; these animals had the highest levels of lymph node drug adducts. Antioxidants, pro-inflammatory mediators, and SMX metabolites were not predictive of drug immunogenicity; however, the Mamu DRB1*0401/0406/0411 genotype was significantly over-represented in immune responders. CONCLUSIONS: Unlike other animal models, macaques develop an immune response, and possible rash, in response to therapeutic dosages of TMP/SMX. Studying more animals with CD4+ counts <200cells/µl, along with moderately restricted ascorbate intake to match deficiencies seen in humans, may better model the risk of SMX hypersensitivity in HIV-infection. In addition, the role of Mamu-DRB1 genotype in modeling drug hypersensitivity in retroviral infection deserves further study.

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes.

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65-70% of the rate with NADH. Added cytochrome b5 stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b5 and cytochrome b5 reductase determine the rate of methemoglobin reduction in hemolysates.

HbM methaemoglobinaemia as a rare case of early neonatal benign cyanosis.

Early neonatal central cyanosis that is unrelated to cardiopulmonary causes, alerts clinicians to possibility of methaemoglobinaemia. Congenital methaemoglobinaemia due to haemoglobin M is an autosomal dominant disorder characterised by lifelong cyanosis. We report a case presentation and review of diagnostic pitfalls of a newborn who presented with central cyanosis; investigations revealed a low methaemoglobin reductase (2.2 IU/g Hb), with normal maternal levels (9.1 IU/g Hb). Therefore, haemoglobinopathy investigations were completed on the mother and her baby, which showed an α-globin variant in both. The maternal α2 globin gene sequencing showed heterozygosity for haemoglobin M Boston (α58 His → Tyr).

Influence of dietary iron deficiency on hemoglobin, myoglobin, their respective reductases, and skeletal muscle mitochondrial respiration.

Male weanling rats were fed a control diet (46 ppm iron) or an iron-deficient diet (11 ppm iron) for 7 wk to determine the influence of iron deficiency on heme proteins and skeletal muscle mitochondrial respiration. At the end of 7 wk, the hemoglobin in the blood of the iron deficient rats was 35% less and skeletal muscle myoglobin was 20 to 37% less than in the control animals. The concentration of myoglobin in the heart was not appreciably diminished by iron deficiency. Cytochrome c concentration was 20% less in the heart and 35% less in the mixed-fiber gastrocnemius in the iron-deficient animals. Iron deficiency did not influence the activity of metmyoglobin reductase in either heart or skeletal muscle. There was about 30% more methemoglobin reductase activity in the red blood cells of the iron-deficient animals, which resulted in methemoglobin levels that were so low as to be virtually unmeasurable. In the iron-deficient rats, skeletal muscle mitochondrial respiration with either pyruvate-malate or palmitylcarnitine as substrate was 17 to 20% less than in the control animals. This study demonstrates that dietary iron deficiency of sufficient severity to reduce blood Hb and skeletal muscle myoglobin or cytochrome c also results in an impaired skeletal muscle oxidative capacity. The study also illustrates the preferential utilization of iron, not only between tissues, but within tissues, and tissue specific adaptive responses to iron deficiency.

Reduction of methemoglobin through flavin at the physiological concentration by NADPH-flavin reductase of human erythrocytes.

The reduction of methemoglobin by NADPH-flavin reductase of human erythrocytes through flavin was studied under various conditions using a reconstituted methemoglobin reductase system. The reduction of methemoglobin by the reconstituted enzyme system could be easily detected with flavin at the physiological concentration (e.g., 0.1-1.0 microM), and the rates obtained with 0.1 and 1.0 microM FMN were 0.19 and 2.2 nmol heme reduced per min per ml, respectively, in the absence of oxygen. FMN was more effective than FAD in reduction by the reconstituted enzyme system, and oxygen decreased the rate of the reduction. The reduction of methemoglobin by the reconstituted enzyme system with flavin at a physiological concentration proceeded as a zero order reaction. These results apparently suggest that the NADPH-flavin reductase system is able to reduce methemoglobin in erythrocytes at a moderate speed with about 1 microM flavin, and the reduction was estimated to vary from less than 1% to about 20% of that by the NADH-cytochrome b5 reductase system with 1 microM cytochrome b5, depending on the uptake of flavin by human erythrocytes.

Purification and characterization of NADPH-dependent methemoglobin reductase from the nucleated erythrocytes of bullfrog, Rana catesbeiana.

Two protein components having a NADPH-dependent methemoglobin reductase activity were purified to electrophoretic homogeneity from the erythrocytes of the bullfrog, Rana catesbeiana. Their molecular properties were investigated. The components were separated by isoelectric focusing, having discrete bands of pI 5.0 and 7.5, respectively. The pI 5.0 component, designated F-5.0, was faint yellow, with a broad absorption in the range of 400-450 nm, while the pI 7.5 component, designated F-7.5, was colorless and did not absorb in that range. The molecular weight was estimated to be 22,000 for both components by gel filtration and SDS-PAGE. When F-5.0 was subjected to isoelectric focusing repeatedly, the protein part of that component gradually moved to and refocused at pH 7.5, leaving a yellow color at acidic pH. Both F-5.0 and F-7.5 were highly specific for NADPH and had the same kinetic properties in catalyzing the reduction of MB, DCPIP, FMN, or FAD, and that of methemoglobin or cytochrome c in the presence of a certain dye. They were also indistinguishable from one another in their amino acid compositions and were completely identical in the N-terminal sequence of 24 amino acid residues. These findings strongly suggest that the two components can be attributed to the same enzyme molecule, carrying an identical protein moiety but interacting differently with some unidentified biological pigments, and that they are equivalent in their molecular and kinetic properties to the NADPH-dependent enzyme(s) occurring in human erythrocytes.

Automated determination of red cell methaemoglobin reductase activity by a continuous-flow system for screening hereditary methaemoglobinaemia.

A flow diagram for the automated determination of ferricyanide reductase activity in red blood cells was prepared in the modules from AutoAnalyzer AA I (Technicon Instruments Inc). Ferricyanide reductase assay can be substituted for assay of cytochrome b5 reductase (EC 1.6.2.2), which plays a major role in reducing methaemoglobin in erythrocytes, and is defective specifically in the erythrocytes of patients with hereditary methaemoglobinaemia. The effective sampling rate of the analysis is 30/h, and less than 0.05 ml of whole blood is required. Interference of haemoglobin with absorption by potassium ferricyanide at 420 nm is effectively exculded by dialysis. This automated method was compared with the accepted diaphorase method, and it distinguished clearly the ferricyanide reductase activity of cord bloods from that of adult bloods. The activity of the blood from a patient with hereditary methaemoglobinaemia was only residual. It is suggested that the method is useful as a mass screening test for hereditary methaemoglobinaemia.

Methemoglobin reductase: inactivation and protection against inactivation during disc gel electrophoresis.

Methemoglobin reductase was found to be inactivated during electrophoresis on ammonium persulfate polymerized polyacrylamide gels. Hemoglobin in various states of ligation offers protection from inactivation. Thiols such as dithiothreitol also offer protection but the effect due to hemoglobin is not provided by its sulfhydryl groups.

Methemoglobin formation and glutathione disappearance in cord blood red cells exposed to acetylphenylhydrazine.

Methemoglobin formation in the presence of acetylphenyl hydrazine occurs faster in red cells from cord blood than in normal adult erythrocytes. Under the same conditions, the rate of disappearance of glutathione is slower in cord blood erythrocytes. The mean NADH-methemoglobin reductase activity of cord blood red cells is less than half of the adult value. The results point to a potential danger in the administration of oxidant drugs to expectant mothers or new-born infants.

Cadmium and vanadate oligomers effects on methaemoglobin reductase activity from Lusitanian toadfish: in vivo and in vitro studies.

Cadmium and two vanadate solutions as 'metavanadate' (containing ortho and metavanadate species) and 'decavanadate' (containing decameric species) (5 mM) were injected intraperitoneously in Halobatrachus didactylus (Lusitanian toadfish), in order to evaluate the effects of cadmium and oligomeric vanadate species on methaemoglobin reductase activity from fish red blood cells. Following short-term exposure (1 and 7 days), different changes were observed on enzyme activity. After 7 days of exposure, 'metavanadate' increased methaemoglobin reductase activity by 67% (P < 0.05), whereas, minor effects were observed on enzymatic activity upon cadmium and 'decavanadate' administration. However, in vitro studies indicate that decameric vanadate, in concentrations as low as 50 microM, besides strongly inhibiting methaemoglobin reductase activity, promotes haemoglobin oxidation to methaemoglobin. Although decameric vanadate species showed to be unstable in the different media used in this work, the rate of decameric vanadate deoligomerization is in general slow enough, making it possible to study its effects. It is concluded that the increase in H. didactylus methaemoglobin reductase activity is more pronounced upon exposition to 'metavanadate' than to cadmium and decameric species. Moreover, only decameric vanadate species promoted haemoglobin oxidation, suggesting that vanadate speciation is important to evaluate in vivo and in vitro effects on methaemoglobin reductase activity.

NADH-methemoglobin reductase (cytochrome b5 reductase) levels in two groups of American blacks and whites.

BACKGROUND: Sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency and alpha-thalassemia trait are common genetic abnormalities among the American Black population. Under oxidative stress, the presence of any of these conditions would predispose the hemoglobin (Hb) to oxidation resulting in accelerated methemoglobin (metHb) formation. It was hypothesized that red cells phenotypic for these genetic variants should have more or different levels of metHb reductase (cytochrome b5 reductase) activity. METHODS: To test this hypothesis, we measured the red cell metHb reductase activity in 558 male subjects (316 Blacks and 242 Whites), by the procedure described by Beutler. All Black patients also had G6PD spot test and Hb electrophoresis. In addition, all patients had a complete blood count (CBC). If the hematocrit was < 35% a reticulocyte count was also done. Patients with corrected reticulocyte (retic count X hematocrit/45) index over 2% were excluded regardless of other findings. RESULTS: The results showed that Blacks had different metHb reductase activity levels than Whites (mean = 3.19 vs 2.89 IU/gHb, respectively with p = 0.03). However, the differences in metHb reductase activities in patients with sickle cell trait, G6PD deficiency, and low MCV < 80 micron3 (presumptively having alpha-thalassemia) in small subgroups did not reach statistical significance (p = 0.2), although, all 3 groups were comprised of small numbers. CONCLUSIONS: It is concluded that American Blacks have significantly different metHb reductase activity. The different metHb reductase activity in Blacks seems to be unrelated to the presence of G6PD deficiency, sickle cell trait, or alpha-thalassemia and it may be the result of genetic polymorphism. However, our study samples do not exactly represent the cross-sections of the Black and White populations. In addition, all patients were male in this study. Therefore, this study should be confirmed using larger and more population-representative samples. The clinical significance of this problem is not clear at this time.

Studies on erythrocytic methemoglobin reductase systems in Plasmodium yoelii nigeriensis infected mice.

BACKGROUND: The methemoglobin reductase system plays a vital role in maintaining the equilibrium between hemoglobin and methemoglobin in blood. Exposure of red blood cells to oxidative stress (pathological/physiological) causes an impairment in this equilibrium. OBJECTIVE: To study the status of methemoglobin and the related reductase system during Plasmoidum yoelii nigeriensis (P. y. nigeriensis) infection in mice. METHOD: Mice were divided into two groups viz., normal mice and P. y. nigeriensis infected mice. Malaria infection was induced by intraperitoneal inoculation of 10(6) infected erythrocytes. RESULTS: The present investigation revealed significant decrease in the activity of methemoglobin reductase, with concomitant rise in methemoglobin content during P. y. nigeriensis infection in mice erythrocytes. This was accompanied with a significant increase in reduced glutathione and ascorbic acid levels. Also the activities of the associated enzymes viz., lactate dehydrogenase, glucose-6-Phosphate dehydrogenase and glutathione reductase were found to increase with progressive rise in parasitemia. CONCLUSION: P. y. nigeriensis infection in mice results in impairment of methemoglobin reductase in the host.

Comparative study of the antioxidant defence systems in the erythrocytes of Australian marsupials and monotremes.

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes "detoxify" H2O2, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC.

Comparative red cell metabolism in the domestic dog and the dingo.

A comparative study of the red cell characters, glycolytic enzymes and intermediates was made in the domestic dog and the dingo. No significant differences were found in any of the parameters studied except the enzyme level of NADH-MR which was significantly lower in dingoes (P less than 0.05).