Anti-CYB5R3 (C-terminal) polyclonal antibody

Genetic modifiers of anemia inPlasmodium falciparuminfection and sickle cell disease (SCD) are not fully known. Both conditions are associated with oxidative stress, hemolysis and anemia. TheCYB5R3gene encodes cytochrome b5 reductase 3, which converts methemoglobin to hemoglobin through oxidation of NADH.CYB5R3(c.350C > G)encoding CYB5R3(T117S), the most frequent recognized African-specific polymorphism, does not have known functional significance, but its high allele frequency (23% in African Americans) suggests a selection advantage. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection from oxidants; its African-polymorphic X-linked A+ and A- alleles, and other variants with reduced activity, coincide with endemic malaria distribution, suggesting protection from lethal infection. We examined the association ofCYB5R3(c.350C > G)with severe anemia (hemoglobin <5 g/dL) in the context ofG6PDA+ and A- status among 165 Zambian children with malaria.CYB5R3(c.350C > G)offered protection against severe malarial anemia in children without G6PD deficiency (G6PDwild type or A+/A- heterozygotes) (odds ratio 0.29,P= .022) but not inG6PDA+ or A- hemizygotes/homozygotes. We also examined the relationship ofCYB5R3(c.350C > G)with hemoglobin concentration among 267 children and 321 adults and adolescents with SCD in the US and UK and found higher hemoglobin in SCD patients without G6PD deficiency (beta = 0.29,P= .022 children; beta = 0.33,P= .004 adults). Functional studies in SCD erythrocytes revealed mildly lower activity of native CYB5R3(T117S)compared to wildtype CYB5R3 and higher NADH/NAD+ ratios. In conclusion,CYB5R3(c.350C > G)appears to ameliorate anemia severity in malaria and SCD patients without G6PD deficiency, possibly accounting forCYB5R3(c.350C > G)selection and its high prevalence.

Objectives: Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiencies is an autosomal recessive disorder that occurs sporadically worldwide, A sensitive, accurate, and rapid analysis of NADH-CYB5R enzyme concentrations is necessary for the diagnosis of RCM. Here we present an alternative microplate method that is based on a standard 96-well microplate format and microplate reader that simplify the quantification of NADH-CYB5R activity. Methods: TECAN (Infinite 200 PRO series) microplate reader with Tecan's proven Magellan software measured the NADH-CYB5R enzyme activity in 250 normal controls and previously diagnosed 25 cases of RCM due to NADH-CYB5R deficiency in the Indian population using 96-well microplates using 200 mu l of total reaction mixture and also compared with standard spectrophotometric assay. We have also studied stability of the hemolysate stored at 4 and -20 degrees C temperature. Results and discussion: Enzyme activity in all 25 samples ranged from 6.09 to 10.07IU/g Hb (meanSD: 8.08 +/- 1.99IU/g Hb) where as normal control ranged (n=250) between 13.42 and 21.58IU/g Hb) (mean +/- SD: 17.5 +/- 4.08IU/g of Hb). Data obtained from the microplate reader were compared with standard spectrophotometer method and found 100% concordance using both methods. Microplate method allows differentiating between normal, deficient and intermediate enzyme activity. It was observed that samples had significant loss of activity when stored at 4 degrees C and retained stable activity at -20 degrees C for 1 week time. Conclusion: Our new method, incorporating a whole process of enzyme assay into a microplate format is readily applicable and allows rapid monitoring of enzyme assay. It is readily applicable to quantitative assay on pediatric sample as well as large number of samples for population screening.