A microplate reader-based method to quantify NADH-cytochrome b5 reductase activity for diagnosis of recessive congenital methaemoglobinemia

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?μl of total reaction mixture and also compared with standard spectrophotometric assay. We have also studied stability of the hemolysate stored at 4 and ?20°C temperature.

Results and discussion: Enzyme activity in all 25 samples ranged from 6.09 to 10.07?IU/g Hb (mean?±?SD: 8.08?±?1.99?IU/g Hb) where as normal control ranged (n?=?250) between 13.42 and 21.58?IU/g Hb) (mean?±?SD: 17.5?±?4.08?IU/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°C and retained stable activity at ?20°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.