Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation seen in commercially sourced hemoglobin

Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe3+, ferric) hemo-globin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemo-globin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain sta-bilizers and additives which contribute to CAgg formation.

Automated summary

Elevated levels of circulating cell-free hemoglobin (CFH) play a significant role in several clinical conditions, and it is important to study the pathophysiology of these diseases using commercially sourced CFH. This study found that treating endothelial cells with commercial ferric hemoglobin led to the formation of dense cytoplasmic aggregates (CAgg), while fresh ferric hemoglobin did not. Chemical analysis revealed that these CAgg contain inorganic components and are not pure hemoglobin, suggesting that stabilizers and additives in commercially sourced CFH contribute to CAgg formation.