An E
x Vivo Model of Oxidatively Stressed Red Blood Cells Demonstrates a Role for Exogenous Amino Acids in Enhancing Red Blood Cell Function and Morphology
Authors: Colvin Heather N. , Alipour Elmira , Buzzett Jordan , Marrs Glen , Kim-Shapiro Daniel B. , Cordy Regina Joice 
Published: November 2021

Abstract
The capacity of mature red blood cells (RBCs) to respond to oxidative stress is limited due to lack of a full complement of organelles and as such, when faced with an oxidative environment, they rely on their endogenous antioxidant capacity (including superoxide dismutase, catalase, peroxiredoxin and glutathione) to protect against cellular damage. Low blood glutathione activity has been reported in several red cell disorders leading to increased oxidative stress. Targeting oxidative stress has thus been proposed as a secondary treatment in multiple anemia-causing diseases, such as sickle cell disease (SCD) and malaria, although its overall efficacy remains unclear. As glutathione itself is not permeable through the RBC membrane, treatment with cell-permeable amino acid precursors of glutathione (glutamine, cysteine and/or glycine) is a potential strategy to expand the RBC's antioxidant capacity and alleviate oxidative stress. Indeed, L-glutamine has recently been approved as a therapeutic for SCD, although the mechanistic basis for its effect is not clear. To fill this gap in our understanding, we performed detailed characterization of biophysical phenotype, morphology, and intracellular redox environment of oxidatively stressed RBCs in environments with varying amounts of available precursor amino acids.
To assess the impact of exogenous amino acid precursors on the RBC's glutathione antioxidant capacity, we exposed mature RBCs from healthy adults to hydrogen peroxide (H 2O 2) and co-incubated with media that included glutamine, cysteine, and/or glycine. As catalase has the ability to scavenge high levels of exogenously fluxed H 2O 2, we performed these experiments using sodium azide to block catalase activity, enabling us to model oxidatively stressed RBCs. We performed osmotic gradient ektacytometry to quantify RBC deformability and hydration status, and assessed RBC morphology using osmotic-adjusted fixation techniques and scanning electron microscopy. As previously documented, H 2O 2 exposure in sodium azide-treated healthy RBCs was associated with decreased deformability, decreased hydration and increased numbers of echinocytes in a dose-dependent manner. We monitored red cell phenotypic changes following co-incubation with glutamine, cysteine, and/or glycine individually and in combination to test whether these amino acids extended the RBC's antioxidant abilities and contributed to improved function and morphology. We found that supplementation with all three amino acids in combination significantly improved both deformability and hydration of H 2O 2-stressed RBCs, as opposed to treatment with glutamine alone. To directly assess whether the exogenous amino acids were in fact contributing to less intracellular oxidative stress in RBCs, we quantified intracellular reactive oxygen species (ROS) using 2', 7' -dichlorofluorescein diacetate (DCFDA), a cell permeable dye used to measure ROS production. As expected, H 2O 2 exposure alone was associated with elevated intracellular ROS inside RBCs in both a time- and dose-dependent manner. Supplementation with the three amino acid cocktail during H 2O 2 stress resulted in a reduction in the level of intracellular ROS activity.
In summary, we documented that exogenous added amino acids reduce oxidative damage in RBCs and we hypothesize that this protection occurs via the glutathione antioxidant pathways. In future studies, we plan to investigate the impact of exogenous amino acids on sickled and irreversibly sickled RBCs (ISCs) in the context of SCD, and on uninfected and infected RBCs in the context of malaria.

Disclosures
Kim-Shapiro: Beverage Operations LLC: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: co-inventor on a patent related to the use of nitrite under cardiovascular conditions, and a co-author on patents related to treatment of hemolysis.