Protocol for measuring erythrocyte glutathione reductase activity coefficient to assess riboflavin status

Riboflavin (vitamin B2) is a component of the co-enzyme flavin adenine dinucleotide (FAD). The activity coefficient of erythrocyte glutathione reductase (EGRAC), a FAD-dependent enzyme, is a biomarker of riboflavin status. Here, we describe a protocol for measuring unstimulated (basal) and FAD-stimulated (activated) erythrocyte glutathione reductase activity to calculate EGRAC. We describe the steps for preparing washed red blood cells and hemolysates; preparing reagents; loading, incubating, and reading the 96-well plate; and calculating the results. For complete details on the use and execution of this protocol, please refer to Hess et al.¹.