Cytochrome B5 Reductase Activity Research Articles

Activity of NAD(P)H-Oxidoreductases in Ovarian Cancer.

Reactive oxygen species (ROS) play an important and controversial role in carcinogenesis. Microsomal redox chains containing NADH- and NADPH-dependent oxidoreductases are among the main sites of intracellular ROS synthesis, but their role in the oxidative balance has not been fully studied. Here, we studied the activity of cytochrome b5 reductase (CYB5R) and cytochrome P450 reductase (CYPOR) in ovarian cancer tissues and cells isolated from peritoneal fluid, along with the antioxidant capacity of peritoneal fluid. We used the developed a chemiluminescence assay based on stimulation with NADH and NADPH, which reflects the activity of CYB5R and CYPOR, respectively. The activity of CYB5R and CYPOR was significantly higher in moderately and poorly differentiated ovarian adenocarcinomas compared with well-differentiated adenocarcinomas and cystadenomas. For the chemotherapy-resistant tumors, the activity of tissue CYB5R and CYPOR was lower compared to the non-resistant tumors. In the peritoneal fluid, the antioxidant capacity significantly increased in this series, benign tumors < well-differentiated < moderately and poorly differentiated adenocarcinomas, so the antioxidant excess was observed for moderately and poorly differentiated adenocarcinomas. The antioxidant capacity of peritoneal fluid and the activity of CYB5R and CYPOR of cells isolated from peritoneal fluid were characterized by a direct moderate correlation for moderately and poorly differentiated adenocarcinomas. These results indicate the significant role of NAD(P)H oxidoreductases and the antioxidant potential of peritoneal fluid in cancer biochemistry. The parameters studied are useful for diagnostics and prognostics. The developed assay can be used to analyze CYB5R and CYPOR activity in other tissues and cells.

Hypoxia Dysregulates the Transcription of Myoendothelial Junction Proteins Involved with Nitric Oxide Production in Brain Endothelial Cells.

Myoendothelial junctions (MEJs) are structures that allow chemical signals to be transmitted between endothelial cells (ECs) and vascular smooth muscle cells, which control vascular tone. MEJs contain hemoglobin alpha (Hbα) and endothelial nitric oxide synthase (eNOS) complexes that appear to control the production and scavenging of nitric oxide (NO) along with the activity of cytochrome b5 reductase 3 (CYB5R3). The aim of this study was to examine how hypoxia affected the regulation of proteins involved in the production of NO in brain ECs. In brief, human brain microvascular endothelial cells (HBMEC) were exposed to cobalt chloride (CoCl2), a hypoxia mimetic, and a transcriptional analysis was performed using primers for eNOS, CYB5R3, and Hbα2 with ΔΔCt relative gene expression normalized to GAPDH. NO production was also measured after treatment using 4,5-diaminofluorescein diacetate (DAF-DA), a fluorescent NO indicator. When HBMEC were exposed to CoCl2 for 48 h, eNOS and CYB5R3 messenger RNA significantly decreased (up to -17.8 ± 4.30-fold and -10.4 ± 2.8, respectively) while Hbα2 increased to detectable levels. Furthermore, CoCl2 treatment caused a redistribution of peripheral membrane-generated NO production to a perinuclear region. To the best of our knowledge, this is the first time this axis has been studied in brain ECs and these findings imply that hypoxia may cause dysregulation of proteins that regulate NO production in brain MEJs.

Oxidative Metabolism Genes in Ovarian Neoplasms

BACKGROUND: Reactive oxygen species play important and ambiguous role in carcinogenesis, and local oxidative metabolism may differ significantly from systemic metabolism and determine the processes occurring in tumor tissues.&#x0D; AIM: This study aimed to examine the expressions of key oxidative metabolism genes, particularly CYB5R, POR, NOX4, SOD1, NF-B, and NRF2, in ovarian neoplasm tissues, and determine cytochrome b5 reductase and cytochrome P450 reductase activity, blood neutrophil activity, and antioxidant indices in the blood plasma and peritoneal fluid.&#x0D; MATERIALS AND METHODS: The study included two groups of patients: a study group (n = 10) with ovarian adenocarcinoma and a comparison group (n = 6) with benign ovarian neoplasms. The expressions of CYB5R1, CYB5R2/R4, CYB5R3, POR, BIRC3, NOX4, NRF2, NF-B, SOD1, HMOX1, and BCL2 genes, cytochrome b5 reductase, and cytochrome P450 reductase activity, oxidative activity of blood neutrophils, and antioxidant potential of plasma and peritoneal fluid were evaluated in these two groups of women.&#x0D; RESULTS: The expression levels of CYB5R3 and BCL2 were significantly lower in adenocarcinoma tissues. The activities of cytochrome b5 reductase and cytochrome P450 reductase increased in the group with adenocarcinoma. On average, the activity of blood neutrophils corresponded to the reference values. For blood plasma, the antioxidant capacity were not different, whereas the antioxidant capacity in the peritoneal fluid increased approximately twofold in ovarian cancer.&#x0D; CONCLUSIONS: Significantly increased cytochrome b5 reductase activity in adenocarcinoma tissues may be a response to intracellular oxidative stress, whereas CYB5R3 gene expression may be reduced by a negative feedback mechanism. The activities of cytochrome P450 reductase and its gene change to a lesser extent in the presence of ovarian adenocarcinoma. The oxidative balance in the blood and peritoneal fluid correlated with the tissue expressions of NF-B and NRF2.

АНАЛИЗ АКТИВНОСТИ МИКРОСОМАЛЬНЫХ РЕДУКТАЗ ТКАНИ ЯИЧНИКОВ ПОСЛЕ КРИОКОНСЕРВАЦИИ МЕТОДОМ АКТИВИРОВАННОЙ ХЕМИЛЮМИНЕСЦЕНЦИИ

The aim of the study was to investigate the activity of NADH-dependent cytochrome b5 reductase (CYB5R) and NADPH-dependent cytochrome P450 reductase (CYPOR) in ovarian tissues after cryopreservation by lucigenin-enhanced chemiluminescence with NADH and NADPH stimulation, respectively. The results indicate that both mitochondrial and microsomal reductase activities are preserved in cryopreserved ovarian tissues. After cryopreservation, the level of production of superoxide anion radical by mitochondria drops by 3–10 times, while the presence or absence of chemotherapy has no effect, and this parameter is also not affected by the severity of the disease. Compared to the control group (patients with benign tumors), the activity of CYB5R and CYPOR of ovarian tissue in a malignant cancer process decreases 1.5–10 times depending on the disease severity, and the presence of chemotherapy dramatically affects these parameters — the activity of microsomal reductases decreases by 50–100 times in chemotherapy compared to the control group. Thus, microsomal reductase activity is a more informative parameter for assessing the functionality of cryopreserved ovarian tissue than superoxide-producing capacity of mitochondria, because, firstly, it depends on the stage of disease and prior chemotherapy, and secondly, the analytical signal of NADH/NADPH stimulated chemiluminescence is characterized by approximately 30 times higher intensity than mitochondrial chemiluminescence, which leads to higher analytical sensitivity of the technique.

LATE DIAGNOSIS OF CONGENITAL METHEMOGLOBINEMIA IN A 33-YEAR-OLD CYANOTIC PATIENT, CASE REPORT AND REVIEW OF LITERATURE

Due to mild symptoms, congenital methemoglobinemia is rarely diagnosed and reported as a cause of the cyanosis, especially in adults. Despite the benign nature of congenital methemoglobinemia, it is crucial to keep it in the differential diagnosis list when assessing cyanotic patients, mainly if he has a normal PaO2. Patients are usually asymptomatic and are treated for cosmetic purposes, but they might suffer from severe complications if exposed to oxidative agents. A 33-year-old lady presented to ED with difficulty breathing and bluish discoloration gradually increased over days, without fever or cough. she mentioned having recurrent similar episodes since childhood. family history positive for similar episodes in the sister. physical examination positive for central and peripheral cyanosis, with o2sat of 88% and RR of 20, the rest of examination within normal limits. laboratory tests normal except for ht 48.9%, PO2 160 on ABGs. The patient's clinical picture of cyanosis with no evidence of cardiovascular or pulmonary diseases and the discrepancy between PaO2 and O2 saturation on oximeter required thinking of methemoglobinemia as a possible diagnosis despite the patient's age and the absence of any exposures. Methemoglobin level 20.9% (0-1.5%). Hemoglobin electrophoresis did not detect any abnormal hemoglobin. The activity of NADH cytochrome b5 reductase or the level were not done. We searched PubMed and Google Scholar, we found 22 articles with a total of 30 patients with congenital methemoglobinemia. The mean age of the patients was 25 years (range 2 days-61 years); most of them were previously healthy. Out of 30 patients, 16 were treated with ascorbic acid or methylene blue or both with improvement, 14 either were not treated or treatment not mentioned in the report. Our patient received ascorbic acid 500 mg BID orally and improved clinically and laboratory. Due to mild symptoms, congenital methemoglobinemia is rarely diagnosed and reported as a cause of the cyanosis, especially in adults. Despite the benign nature of congenital methemoglobinemia, it is crucial to keep it in the differential diagnosis list when assessing cyanotic patients, mainly if he has a normal PaO2. Patients are usually asymptomatic and are treated for cosmetic purposes, but they might suffer from severe complications if exposed to oxidative agents.

Microsomal reductase activity in patients with thyroid neoplasms.

Cytochrome b5-reductase (CYB5R) and cytochrome P450 reductase (CYPOR) are important for cell metabolism; however, their role in thyroid hormonogenesis and carcinogenesis has not been elucidated yet. The activity of CYB5R correlates with the metastasis in breast cancer, but there are no similar studies for CYB5R and CYPOR for thyroid tumors. The aim of this study was to elucidate the activity of CYB5R and CYPOR changes in benign euthyroid and hyperthyroid neoplasms and in papillary thyroid cancer for their potential application as biomarkers for diagnosis and prognosis prediction of thyroid cancer. Thirty-six patients with thyroid diseases participated in the study. The control euthyroid nodular goiter group included ten patients; the thyrotoxic nodular or diffuse goiter group included 14 patients; the papillary thyroid cancer T1-2N0-1M0 (PTC) group included 12 patients. The activity of CYB5R and CYPOR was assessed with lucigenin-enhanced chemiluminescence stimulated by NADH and NADPH, respectively. Compared to the control euthyroid nodular goiter group, activity of CYB5R and CYPOR increased ~5 and 10 times, respectively, in toxic goiter, and 15 and 30 times, respectively, in half of cases of PTC. The change in activity of CYPOR was more pronounced compared to CYB5R. Within the PTC group, the subgroups with low and high activities of microsomal reductases were identified. Microsomal reductases in follicular adenoma was 2-4-fold less active compared to the euthyroid nodular goiter and the low-activity PTC group. Activity of tissue microsomal reductases varies in thyroid pathology and can be considered as a promising biomarker for differential diagnostics of benign and malignant thyroid tumors.

Purified NADH-cytochrome b5 reductase is a novel superoxide anion source inhibited by apocynin: sensitivity to nitric oxide and peroxynitrite

Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb5R acting in concert with other cellular components. In this work, we have purified Cb5R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb5R at 37°C are Vmax = 3.0±0.5μmol/min/mg of purified Cb5R and KM(NADH) = 2.8±0.3μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb5R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the KM(NADH) value of Cb5R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb5R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb5R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb5R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb5R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb5R for NADH.

Methemoglobinemia In Rhesus Monkeys Due To M-Hemoglobinopathy

M-hemoglobinopathies in humans are usually due to amino acid changes, which tend to stabilize the heme iron in ferric state and inaccessible to methemoglobin reductase. Consequently, blood methemoglobin concentrations are elevated despite normal methemoglobin reductase activity. No naturally-occurring animal models for M-hemoglobinopathies have been described. In contrast to humans where thousands of hereditary disorders and genetic predispositions to disease have been described, inborn errors of metabolism in non-human primates have been very rarely documented (e.g. globoid cell leukodystrophy). As part of an NIH-supported National Referral Center for Animal Models of Human Genetic Disease, which focuses on homologous hereditary diseases in dogs and cats, we present here the discovery and characterization of an M-hemogobinopathy in rhesus monkeys (Macaca mulatta). Adult rhesus monkeys of Chinese origin with persistent cyanotic mucous membranes and healthy controls were studied from two institutions. Despite the apparent cyanosis, these primates did clinically not show any other overt signs. Routine complete blood cell counts, chemistry screens and urinalyses as well as cardiopulmonary examinations did not reveal any abnormalities. Fresh EDTA blood samples were analyzed to determine the degree and cause of methemoglobinemia and results were compared to healthy rhesus monkeys from the same institutions. Blood methemoglobin concentrations were persistently increased to 15-30% in affected monkeys, while control animals had &lt;1% methemoglobin levels. Methemoglobin (cytochrome b5) reductase activities in erythrocytes were repeatedly measured in vitro and found to be normal compared to controls. Based upon the hemoglobin-oxygen dissociation curves and p50 values (14.8 mmHg; compared to 14.5 and 14.6 mmHg for controls) the oxygen transport function was normal. Both cation-exchange as well as reversed-phase and high performance liquid chromatography analyses revealed normal elution patterns of hemoglobin isotype and globin chains. DNA studies identified a disease-associated polymorphism in the alpha globin gene (CAC → CAA) in the affected animals. This change in the alpha globin protein was confirmed by mass spectrometry (α78 His → Gln). Our findings indicate an electrophoretically silent α-globin polymorphism is associated with methemoglobinemia in these rhesus monkeys. This is the first description of methemoglobinemia in a non-human primate and represents the first animal model of an M-hemoglobinopathy. While clinical signs were relatively mild in these rhesus monkeys, our findings represent one of the first inborn errors of metabolism described in non-human primates, thus emphasizing how unusual disorders caused by “experiments of nature” provide rare opportunities to gain a better understanding of both medicine and biology. Rhesus monkeys represent unique, valuable animal models for developing and testing new treatment options for hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

NADH-dependent cytochrome b5 reductase and NADPH methemoglobin reductase activity in the erythrocytes of Oncorhynchus mykiss

Methemoglobin is oxidized hemoglobin that cannot bind to or dissociate from oxygen. In fish, it is most commonly caused by exposure to excess nitrites and can lead to abnormal swimming, buoyancy, or death. The methemoglobin concentration in mammals is determined by the balance of oxidizing agents versus reducing enzymes in erythrocytes. The objective of our studies was to characterize the enzymes that reduce methemoglobin in fish erythrocytes. Whole blood was collected from healthy rainbow trout. Methemoglobin was induced in vitro by NaNO(2) exposure. Methemoglobin reduction in controls was compared to reduction in samples with added NADH, NADPH, or NADPH and methylene blue. Rainbow trout whole blood was also fractionated into cytosol, microsomal, and mitochondria/plasma membranes/nuclei fractions. The fractions were compared for NADH-dependent cytochrome b5 reductase (CB5R) activity and for nitrite induction of methemoglobin. The CB5R activity in rainbow trout erythrocytes was compared to the CB5R activity in equine, feline, and canine erythrocytes. Rainbow trout erythrocytes had significant NADPH methemoglobin reductase activity in the presence of methylene blue (P < 0.001). The CB5R activity was greatest (P < 0.001) in the plasma membrane/mitochondria/nuclei fraction. The CB5R activity in rainbow trout erythrocytes was not significantly different than canine or equine activity but was significantly lower than feline CB5R activity (P < 0.0001). Methemoglobin in rainbow trout erythrocytes can be reduced by CB5R or NADPH-dependent methemoglobin reductase. Unlike mammalian anuclear erythrocytes, which are dependent on soluble CB5R, the nucleated RBCs of rainbow trout use membrane-bound CB5R to reduce methemoglobin.

Comparison of NADH-dependent cytochrome b5 reductase activity and in vitro methemoglobin induction by sodium nitrite in Oncorhynchus mykiss, Salmo salar, and Salvelinus fontinalis

Methemoglobin is hemoglobin containing ferric iron. Methemoglobin cannot bind to oxygen and at high concentrations causes tissue hypoxia. Brook trout (Salvelinus fontinalis) develop significantly greater methemoglobinemia than Atlantic salmon (Salmo salar) or rainbow trout (Oncorhynchus mykiss) following general anesthesia with benzocaine or tricaine methanesulfonate. The objective of this study was to compare the activity of the major methemoglobin reducing enzyme, NADH-dependent cytochrome b5 reductase (CB5R), in brook trout erythrocytes to the activity of CB5R in Atlantic salmon and rainbow trout erythrocytes. Methemoglobin levels were compared using co-oximetry following in vitro incubation of erythrocytes with sodium nitrite (NaNO(2)). The CB5R activity was measured using a ferricyanide assay. There was significantly greater methemoglobin at time 0 in brook trout erythrocytes than in rainbow trout or Atlantic salmon erythrocytes (2.79 ± 0.29 %, 2.19 ± 0.23 %, 2.08 ± 0.14 %), (P < 0.001). There was significantly greater methemoglobin induction by NaNO(2) in brook trout erythrocytes (33.14 ± 3.32 %) than in rainbow trout or Atlantic salmon erythrocytes (28.73 ± 2.92 % and 24.85 ± 1.40 %, respectively), (P < 0.001). The CB5R activity was significantly less in brook trout erythrocytes (median of 3.05 μmol/min/μl) than in rainbow trout erythrocytes (median of 6.73 μmol/min/μl). The CB5R activity in Atlantic salmon erythrocytes (median 4.09 μmol/min/μl) was not significantly different than in brook or rainbow trout erythrocytes. Total methemoglobin at any one time is a balance between induction by oxidants and reduction by antioxidants. Lower CB5R activity in brook trout erythrocytes may contribute to a species-specific sensitivity to methemoglobin induction; however, there are likely additional factors.

Methemoglobinemia in children

All pigments of hemoglobin (oxyhemoglobin, methemoglobin, sulphemoglobin and carboxyhemoglobin) are of clinical importance and each has a laboratorial characteristic in the absorption spectrum. Methemoglobin, in which the ferrous iron atom of the heme group has been oxidized to a ferric iron atom, is unable to carry molecular oxygen. Various red cell enzymes convert methemoglobin back to oxyhemoglobin and keep the level of this pigment between 1% and 3%. High levels of methemoglobin (= 5%) are associated with chocolate-colored blood, cyanosis, headaches, tachycardia, dyspnea and tachypnea and in severe methemoglobinemia coma and death can occur.(1) Newborns are more susceptible to methemoglobinemia because fetal hemoglobin (Hb F) is easily oxidized.(2) Methemoglobin may arise from three different causes: 1. An excessive formation of methemoglobin due to contamination by chemical products, such as phenacetin, nitro or amino derivatives of benzene, derivatives of sulfur, etc. These products cause toxic methemoglobinemia.(3) 2. Inherited enzyme deficiencies including superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase and NADH-linked methemoglobin reductase or diaphorase. These enzyme deficiencies are inherited as Mendelian recessive conditions and are responsible for dramatic cases of cyanosis and hemolytic anemias.(4) 3. Methemoglobinemia due to abnormalities of the beta and alpha globins, especially those near to the heme group region. This abnormal form of oxidized hemoglobin is referred as hemoglobin M (Hb M) and there are at least six different types: Hb M Boston, a variant hemoglobin caused by a mutation in alpha globin (alpha 58 His → Tyr), Hb M Iwate (alpha 87 His → Tyr), Hb M Saskatoon (Beta 63 His → Tyr), Hb M Zurich (Beta 63 His → Arg), Hb M Hyde Park (Beta 92 His → Tyr) and Hb M Milwaukee (Beta 67 Val → Glu).(1,5) The article Reference values for methemoglobin concentrations in children(6) published in this issue of the Revista Brasileira de Hematologia e Hemoterapia showed that the methemoglobin levels in 6- to 10-year-old children are higher than those of healthy adults. The authors suppose that the difference in levels is due by a smaller amount of soluble cofactor cytochrome b5 and less activity of cytochrome b5 reductase in the red blood cells of children. However these enzymatic activities were not evaluated by the authors. So, should not factors (school, food, water, etc) in the environmental where the children of this investigation live be considered as a possible cause of the increased levels of methemoglobin?(3,4,7)

Alteration of drug metabolizing enzymes in sulphite oxidase deficiency

The aim of this study was to investigate the possible effects of sulphite oxidase (SOX, E.C. 1.8.3.1) deficiency on xenobiotic metabolism. For this purpose, SOX deficiency was produced in rats by the administration of a low molybdenum diet with concurrent addition of 200 ppm tungsten to their drinking water. First, hepatic SOX activity in deficient groups was measured to confirm SOX deficiency. Then, aminopyrine N-demethylase, aniline 4-hydroxylase, aromatase, caffeine N-demethylase, cytochrome b5 reductase, erythromycin N-demethylase, ethoxyresorufin O-deethylase, glutathione S-transferase, N-nitrosodimethylamine N-demethylase and penthoxyresorufin O-deethylase activities were determined to follow changes in the activity of drug metabolizing enzymes in SOX-deficient rats. Our results clearly demonstrated that SOX deficiency significantly elevated A4H, caffeine N-demethylase, erythromycin N-demethylase and N-nitrosodimethylamine N-demethylase activities while decreasing ethoxyresorufin O-deethylase and aromatase activities. These alterations in drug metabolizing enzymes can contribute to the varying susceptibility and response of sulphite-sensitive individuals to different drugs and/or therapeutics used for treatments.

Cytochrome B5 Reductase T116S Polymorphism Is Associated with Decreased Risk of Severe Anemia Among Zambian Children with Malaria

AbstractAbstract 4233 Background.NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) exists in soluble and membrane-bound forms. Both cytb5r isoforms are the product of a single gene locus, DIA1 (or CYB5R3). The known functions of cytb5r include reduction of methemoglobin and cytochrome b5 for the soluble isoenzyme, a fatty acid desaturation complex for the membrane-bound microsomal enzyme, and participation in drug metabolism. Deficiency of cytb5r leads to congenital methemoglobinemia with more than 40 mutations reported to date, the majority being missense mutations associated with mild type I methemoglobinemia. CYBR5T116S is a common genetic polymorphism among African Americans (allele frequency of 23%) that has not yet been detected in other ethnic and racial groups. This polymorphism does not cause methemoglobinemia, but it may be associated with reduced hemolysis in patients with sickle cell disease [Blood (ASH Annual Meeting Abstracts), Nov 2009; 114: 903]. We postulated that this polymorphism may protect from severe anemia in African children with malaria, providing these individuals with a genetic advantage that has likely been the target of positive selection, since severity of anemia is a major risk factor for malaria survival in infants and young children. Methods.Children <6 years of age from the Tonga ethnic group of southern Zambia admitted to hospital with clinical malaria were studied. Sixty-eight had severe malarial anemia (hematocrit<15% and peripheral smear positive for Plasmodium falciparum), 31 had cerebral malaria (Blantyre coma score 0–2, hematocrit >18%, smear positive), and 69 had uncomplicated malaria (hematocrit >18%, smear positive). Children with overlapping severe anemia and cerebral malaria were excluded. Risk factors for severe anemia were determined by pathway analysis and logistic regression. The CYBR5T116S polymorphism was detected by Allele Specific-PCR and its relationship to severe anemia was assessed in the context of the identified risk factors. Results.The allele frequency of CYBR5T116S was 32.8% and the distribution was consistent with Hardy-Weinberg equilibrium. After adjusting for weight-for-age Z score, spleen size, history of treatment with sulfadoxine-pyrimethamine for the current illness, and plasma interleukin-10 concentration, CYBR5T116S was associated with a 3.72-fold reduction in the odds of severe malarial anemia at presentation to hospital (95% CI of 1.32–10.47; P = 0.013). Similarly, in an ANOVA model, the adjusted mean (SE) screening hematocrit was 18.8 (1.0) % in 58 CYBR5 wildtype children compared to 21.9 (0.9) % in 77 children with CYBR5T116S (P = 0.024). Conclusions.CYBR5T116S is a common polymorphism among African children that may be associated with protection from severe malarial anemia, an important cause of death in this population. 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 protective effect. Functional studies to investigate this possibility are underway. We submit that a protective effect from severe malaria-induced anemia may be the principal reason for the high prevalence of this African-specific genetic polymorphism. 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.

GPR56-Regulated Granule Cell Adhesion Is Essential for Rostral Cerebellar Development

Mutations in GPR56, an orphan G-protein-coupled receptor (GPCR), cause bilateral frontoparietal polymicrogyria (BFPP), a disorder characterized by mental retardation, seizures, motor developmental delay, and ataxia. BFPP patients have structural abnormalities of the cerebral cortex, cerebellum, and pons. To shed light on the function of GPR56 and the anatomical and behavioral defects underlying BFPP, we analyzed the cerebellum of mice lacking this GPCR. Gpr56(-/-) mice display a severe malformation of the rostral cerebellum that develops perinatally. Defects involve fusion of adjacent lobules, disrupted layering of neurons and glia, and fragmentation of the pial basement membrane. At the age of defect onset, GPR56 expression is restricted specifically to developing granule cells in the rostral cerebellum, suggesting that GPR56 regulates properties of these cells. Indeed, granule cells from the rostral region of perinatal Gpr56(-/-) cerebella show loss of adhesion to extracellular matrix molecules of the pial basement membrane. Interference RNA-mediated knockdown of GPR56 recapitulates the loss of adhesion seen in knock-outs, and reexpression of GPR56 rescues the adhesion defect in knock-out granule cells. Loss of GPR56 does not affect cell proliferation, migration, or neurite outgrowth. These studies establish a novel role for GPR56 in the adhesion of developing neurons to basal lamina molecules and suggest that this adhesion is critical for maintenance of the pia and proper cerebellar morphogenesis.

Recessive hereditary methemoglobinemia: Two novel mutations in the NADH-cytochrome b5 reductase gene

We report the clinical and molecular characteristics of 6 new patients with recessive hereditary methemoglobinemia due to cytochrome b5 reductase deficiency. One patient was affected by Type-II disease with cyanosis and severe progressive neurological dysfunction, whereas the others displayed the benign Type-I phenotype. Methemoglobin levels ranged from 12.1% to 26.2% and cytochrome b5 reductase activity from 0 to 10% of normal. Eight different mutations were detected among the twelve mutated alleles identified, one splicing mutation, two stop codon, and five missense. Two mutations c. 82 C>T(Gln27STOP) and c. 136 C>T(Arg45Trp) are new. Prenatal diagnosis was performed in the family with Type-II disease.

Enhanced cytotoxicity of bioreductive antitumor agents with dimethyl fumarate in human glioblastoma cells

We compared the cytotoxicity of the bioreductive antitumor agents mitomycin C (MMC) and streptonigrin (SN) with or without the DT-diaphorase (DTD) inducer dimethyl fumarate (DMF) in four human glioblastoma cell lines with the conventional chemotherapeutic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). We also examined four other types of cancer cells to compare with glioblastoma cells. Cytotoxicity was measured with the sulforhodamine B (SRB) assay and was represented by 50% inhibition concentration (IC50). Enzymatic activities of DTD, cytochrome b5 reductase and glutathione-S-transferase (GST) in cells were measured spectrophotometrically. IC50 for BCNU was in a range of 28-300 microM in the glioblastoma cell lines. Glioblastoma cells were more sensitive to MMC or SN than to BCNU. Pretreatment with DMF significantly increased cytotoxicity of MMC and SN in glioblastoma cell lines and the NCI-H1299 lung cancer cell line, but had no effect on BCNU cytotoxicity. DMF significantly increased DTD and cytochrome b5 reductase activity, and decreased GST in three of four glioblastoma cell lines. Addition of the DTD inhibitor, dicumarol, significantly inhibited cytotoxicity of MMC and SN, and reversed the increased cytotoxicity seen when DMF was combined with either MMC or SN in all glioblastoma cell lines. Combining inducers of DTD and cytochrome b5 reductase with bioreductive agents may be a potential therapeutic strategy for glioblastoma.

Recurrent diarrhea in children living in areas with high levels of nitrate in drinking water.

Given that there was documented evidence of an association between diarrhea and high nitrate ingestion, the authors examined drinking water nitrate concentration and its possible correlation(s) with methemoglobin levels, cytochrome b5 reductase activity, and recurrent diarrhea. In addition, the authors studied histopathological changes in the intestines of rabbits in an animal model. Five village areas were studied, and nitrate concentrations (expressed in mg of nitrate per liter of water) of 26, 45, 95, 220, and 459 existed in the respective villages. The study included 88 randomly selected children who were 8 yr of age or younger; they represented 10% of the total population of each of the areas. Detailed histories of recurrent diarrhea were noted, and medical examinations were conducted. Cytochrome b5 reductase activity and methemoglobin levels were estimated biochemically. Collected data were analyzed statistically with Microsoft Excel software. In addition, the authors exposed rabbits to various levels of nitrate, and histopathological changes of the stomach and intestine (small and large) were evaluated. There was a strong relationship between nitrate concentration and recurrent diarrhea; 80% of the recurrent diarrhea cases were explained by nitrate concentration alone. In the rabbit intestines, lymphocytic infiltration and hyperplasia characterized the submucosa as nitrate concentrations increased.

Epidemiological evaluation of recurrent stomatitis, nitrates in drinking water, and cytochrome b5 reductase activity.

Our aim was to examine a possible correlation between drinking water nitrate concentration, recurrent stomatitis, and cytochrome b5 reductase activity. Dietary nitrate can form nitrite in vivo. This can cause methemoglobinemia in the red blood cells. Cytochrome b5 reductase is an enzyme in the red blood cells that reduces methemoglobin back to hemoglobin. Five areas were selected in the State of Rajasthan, India, having drinking water nitrate concentration (as nitrate) of 26, 45, 95, 222, and 459 mg of NO3/L. House schedules were prepared in these areas in accordance with a statistically designed protocol. We selected 193 age- and weight-matched persons, representing 10% of the total population in each of these areas. Detailed history was taken for recurrent stomatitis, medical examination was conducted, and blood samples were taken to ascertain cytochrome b5 reductase activity in the selected population. Collected data were statistically analyzed to ascertain a relationship between nitrate concentration, cytochrome b5 reductase activity, and percent stomatitis, using Microsoft Excel software. This study suggests that there is a significant interdependence between drinking water nitrate concentration, cytochrome b5 reductase activity, and recurrent stomatitis. Increased cytochrome b5 reductase activity primarily induced by the presence of high nitrate concentration in drinking water could be the cause for recurrent stomatitis.

Antibody-based spot test for NADH–cytochrome b5 reductase activity for the laboratory diagnosis of congenital methemoglobinemia

NADH (reduced Coenzyme I)–cytochrome b5 reductase (b5R) is a multifunctional redox enzyme, whose deficiency causes recessive congenital methemoglobinemia. A novel procedure for the detection of b5R activity in human hemolysates was developed, in which b5R monoclonal antibodies dot-blotted on nitrocellulose membrane was used to capture and enrich b5R from hemolysates, and the captured b5R activity was subsequently visualized with the substrate 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Application of this simple method to the detection of b5R activity in the hemolysates from different subjects demonstrated that it was both sensitive and reliable. Our method would be useful for the laboratory diagnosis of congenital methemoglobinemia.