Protocol for measuring erythrocyte glutathione reductase activity coefficient to assess riboflavin status.
STAR protocols 2023 ; 4: 102726.
PubMed ID : 37988268
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..
Vitamin B2 (riboflavin) is an essential nutrient required for many processes in the body related to growth, development and metabolism. It is found in a broad range of foods including meat, dairy products and fortified foods, such as breakfast cereals and bread flour. Riboflavin levels are not frequently measured in the clinic or in studies because the measurement is difficult and treatment of severe deficiency is fast and cheap. Therefore, little is known about the frequency of sub-clinical deficiency of riboflavin in different populations.
An important measure of riboflavin is the “erythrocyte glutathione activity coefficient” (EGRAC). Glutathione is an enzyme in erythrocytes (red blood cells) that need riboflavin to function properly, so measurement of activity of this enzyme tells us how much riboflavin is available in the cells. Although this method has been available for many years, it is poorly standardised and different laboratories may get different results.
We have written a step-by-step protocol to help other researchers to measure EGRAC. The protocol provides a foundation for the introduction of the method in other laboratories around the world and will help researchers produce robust and comparable results. Ultimately, it can help researchers better understand the frequency of riboflavin deficiency, its causes, and its consequences, with the aim to better inform public health policy.