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Ultraviolet light oxidation of fresh hemoglobin eliminates aggregate formation
seen in commercially sourced hemoglobin.
Afzal A, Beavers WN, Skaar EP, Calhoun MC, Richardson KA, Landstreet SR, Cliffel
DE, Wright D, Bastarache JA, Ware LB
Submitted Externally on 3/10/2023
Blood cells, molecules & diseases
Volume : Pages
98 : 102699
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) hemoglobin
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 hemoglobin did not lead to CAgg formation. These
studies suggest that commercially sourced CFH may contain stabilizers and
additives which contribute to CAgg formation.
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Financial support for this work was provided by the NIDDK Mouse Metabolic Phenotyping Centers (National MMPC, RRID:SCR_008997,
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