Fluorescent Heme Degradation Products Are Biomarkers of Oxidative Stress and Linked to Impaired Membrane Integrity in Avian Red Blood Cells.

Abstract

Oxidative stress is generally understood to be an important mediator of life-history traits, yet the specific relationships between oxidative stress and life-history traits have been difficult to describe because there is often a lack of covariation among biomarkers of oxidative stress. For instance, although oxidative damage to red blood cell (RBC) membranes can lead to pathological conditions (i.e., anemia), in some cases there is not a clear relationship between lipid oxidation and RBC membrane resistance to pro-oxidants. Alternatively, oxidative damage to hemoglobin may be an indirect mechanism contributing to RBC membrane damage. To better understand the mechanisms contributing to oxidative damage and probe new approaches to measuring oxidative stress, we used a series of in vitro and in vivo procedures in zebra finches (Taeniopygia guttata) to explore (1) whether avian RBCs exposed to a pro-oxidant generate fluorescent heme degradation products (HDPs), (2) whether HDPs interact with RBC membranes, and (3) whether HDPs are linked to impaired RBC integrity. We found that finch RBCs exposed in vitro to hydrogen peroxide produced fluorescent HDPs and HDPs associated with RBC membranes. Exposure to hydrogen peroxide also caused a reduction in hemoglobin and an increase in percent methemoglobin (a hemoglobin oxidation product), further indicating hemoglobin degradation. Moreover, HDP fluorescence correlated with impaired membrane integrity and erythrocyte osmotic fragility in vivo. This study suggests that reactive oxygen species may indirectly impair RBC membrane integrity via hemoglobin degradation products that associate with RBC membranes and that HDPs may be an inexpensive and logistically simple tool for measuring oxidative stress.