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 Rats were sacrificed after 24 h, blood was withdrawn and plasma and erythrocytes were isolated.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.

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.

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.

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).

Neurometabolic disorder with microcephaly, dystonia, and central cyanosis masquerading as cerebral palsy.

Many neurodegenerative diseases can be misdiagnosed as cerebral palsy. The correct diagnosis is reached when the condition recurs in families or when there are specific clinical signs. The clinical and imaging features of 3 children, from 2 unrelated families, presenting with global developmental delay and dystonia are described, in whom the presence of cyanosis and methemoglobinemia confirmed the diagnosis of recessive hereditary methemoglobinemia type 2. Magnetic resonance imaging showed significant cerebellar atrophy in 2 of the 3 babies. In dark-skinned children, this condition is underdiagnosed, as mild cyanosis is difficult to detect. Screening for methemoglobinemia in children with dystonia, microcephaly, and progressive cerebellar atrophy can be helpful in identifying more cases. As there is no curative treatment for this autosomal recessive condition, the exact diagnosis offers the best chance for prenatal screening, by detecting deficient NADH--cytochrome b5 reductase enzyme activity or by identifying the specific mutation in cultured amniotic fluid cells.

[Effect of L-arginine on antioxidant system and NADH2-depended methemoglobinereductase activity in rat blood].

We investigated the influence of L-arginine, a nitric oxide precursor, on the state of hemoglobin and basic parts of the antioxidant system. We revealed the negative influence of NO hyperproduction on the oxygen transport by haemoglobin, which is manifested by the increase of MetHb concentration and decrease of the MetHb-reductase activity. Also increase of glutathioneperoxidase, glutathionereductase and glutathion-etransferase activities were documentated; at the same time catalase activity in the erythrocyte hemolisate was decreased. This can point on the dominating role of glutathione in the protection of erythrocyte membrane and hemoglobin under the condition of oxidative stress, which was induced by NO hyperproduction.

Red cell enzymes.

Mutations leading to red cell enzyme deficiencies can be associated with diverse phenotypes that range from hemolytic anemia, methemoglobinemia, polycythemia, and neurological and developmental abnormalities. While most of these mutations occur sporadically, some such as common glucose-6-phosphate dehydrogenase (G6PD) mutants are endemic and rarely cause disease. Common G6PD mutants likely reached their prevalence because they provide some protection against severe malarial complications. In this review G6PD, pyruvate kinase, 5' nucleotidase, and cytochrome b5 reductase deficiencies will be discussed in greater detail. Limitations of commonly used screening tests for detection of these disorders will also be emphasized, as well as emerging knowledge about non-enzymatic function of the glycolytic enzymes.

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.

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.

The value of methemoglobin reduction test as a screening test for neonatal glucose 6-phosphate dehydrogenase deficiency.

UNLABELLED: Glucose 6-phosphate dehydrogenase (G-6-PD) deficiency is common in the Thai population and is the cause of neonatal hyperbilirubinemia and hemolytic anemia. This X-linked disorder is much more common in males than females. The objectives of this study were to compare the result of the screening methemoglobin reduction test (MRT) with the gold standard G-6-PD activity, and also to determine the prevalence of G-6-PD deficiency in the cord blood and blood of neonates with hyperbilirubinemia. Five hunderd and twenty two randomly selected cord blood (350 males, 172 females) and 229 peripheral blood from neonates with hyperbilirubinemia were assayed for G-6-PD enzyme activity using a WHO-recommended standard test as well as methemoglobin reduction (MR) test. The results showed that prevalence of G-6-PD deficiency from the cord blood was 11.1 per cent in males, and 5.59 per cent in females. Among newborns with neonatal jaundice, the prevalence of G-6-PD deficiency was 22.1 per cent in males and 10.1 per cent in females. MRT in cord blood G-6-PD deficiency screening had acceptable sensitivity (85.7%) and high specificity (98.1%). The sensitivity of MRT in jaundiced infants was low (60.0%) whereas the specificity was acceptable (92.1%). The negative predictive values were more than 90 per cent while the positive predictive values were low (61-65%) from both specimens. CONCLUSIONS: G-6-PD deficiency is common in the Thai population, both in males and females and can be screened from cord blood by using low cost MRT. G-6-PD deficiency contributes to 20 per cent of neonatal jaundice, and screening with MRT yields low sensitivity.

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.

Erythrocyte enzyme activities in cord blood of extremely low-birth-weight infants.

To investigate the features of erythrocyte metabolism in extremely immature infants, we assayed 21 enzyme activities and glutathione level in cord erythrocytes from 28 extremely low-birth-weight infants (ELBWI; defined as birth weight <1,000 g). The results were compared with those from normal adults and non-neonatal reticulocyte-rich controls. Statistical analysis revealed that activities of six enzymes (glucosephosphate isomerase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, glucose-6-phosphate dehydrogenase (G6PD), and glutathione reductase) were significantly higher, and those of eight other enzymes (phosphofructokinase, 6-phosphogluconate dehydrogenase (6PGD), glutathione peroxidase, adenylate kinase, adenosine deaminase, acetylcholinesterase, NADH methemoglobin reductase, and catalase) were lower in ELBWI taking their marked reticulocytosis into consideration. The 6PGD/G6PD ratio, which is consistently unchanged under various physiological and pathological conditions, was markedly reduced in ELBWI. Our results support the previous reports that neonatal erythrocytes have a unique metabolic pattern which is different from that of adult erythrocytes, and also suggest that the 6PGD/G6PD ratio might be an index for the developmental immaturity of fetal erythrocytes. This is the first report describing the pattern of erythrocyte enzyme activities in ELBWI.

Human erythrocytes are protected against chromate-induced peroxidation.

In previous studies performed in this laboratory it was realized that in a broad concentration range (0.5-8 mM) dichromate does not induced red blood cell (RBC) peroxidation. To investigate the reasons behind RBC protection against chromate-induced peroxidation, the effects of 8 mM dichromate on white ghost and RBC peroxidation, RBC antioxidant system and hemoglobin status, as well as RBC osmotic fragility and morphology, were studied in more detail. It was observed that the peroxidation level induced by dichromate on RBCs is practically negligible when compared with the peroxidation induced in white ghosts. Furthermore, the osmotic fragility of RBCs exposed to dichromate is not altered, but the cells undergo echinocytic transformation, probably due to chromate-induced structural RBC membrane modifications. The activities of catalase, gluthatione peroxidase, and superoxide dismutase of RBCs exposed to dichromate were similar to those observed in controls, but the gluthatione reductase and GSH levels were significantly reduced (P<0. 05). Concomitantly, GSSG and methemoglobin levels increased and NADH-methemoglobin reductase activity decreased. These results indicate that chromate does not induce RBC peroxidation, but does promote echinocytic shape transformation, oxidation of hemoglobin and GSH, and inhibition of gluthatione reductase and methemoglobin reductase. The enzymatic antioxidant defense system and hemoglobin oxidation are probably involved in the mechanism of RBC proctection against chromate-induced peroxidation, as is discussed.

Cyanosis and congenital methemoglobinemia in a puppy.

A six-month-old Pomeranian was referred for evaluation of cyanosis. The puppy had been cyanotic since it was acquired at six weeks of age, but otherwise appeared normal. Diagnostics were aimed at the most common causes (i.e., congenital defects in the cardiovascular and respiratory systems) of cyanosis in a juvenile animal. No clinically significant abnormalities were detected on evaluation of thoracic radiographs, echocardiogram, cardiac color-flow Doppler examination, or blood gases. At this point, a dysfunction in hemoglobin was considered as a possibility. Methemoglobinemia due to deficiency of methemoglobin reductase enzyme was diagnosed based on a specific assay.

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.