Oxidation of hemoglobin: mechanisms of control in vitro and in vivo

Abstract

SUMMARY Hemoglobin (Hb) within red blood cells (RBC) is protected from oxidative processes by enzymatic and small molecule antioxidants as well as the RBC membrane that provides a physical barrier against oxidation. When Hb is introduced into the circulation as a result of hemolysis or following infusion of Hb‐based oxygen carriers (HBOCs; ‘blood substitutes’), the control of oxidative processes becomes dependent on plasma oxidative status. In vitro studies clearly demonstrate that Hb and HBOCs undergo oxidative modification at the site of heme iron and at multiple amino acid sites as a result of autoxidation, nitrosylation and peroxidation. Recent findings from in vitro studies provide a strong basis for understanding of potential toxicity in vivo. The antioxidative status of both plasma and tissue becomes an important factor in the control of Hb and HBOC oxidation and nitrosylation in the circulation. Several studies that performed ex vivo and in vivo have demonstrated a critical role for plasma antioxidants such as ascorbic acid and uric acid in maintaining Hb and HBOCs in functional non‐oxidized states. In the present review we discuss mechanisms of Hb and HBOC autoxidation, nitrosylation and peroxidation in vitro and in vivo. Additionally, we explore the role(s) of plasma antioxidants in maintaining functional HBOC status that may potentially be exploited as part of protective strategies against Hb and HBOC oxidative toxicity.

Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin

“…Hemolysis and the transfusion of banked blood or Hb-based therapeutics can result in varying quantities of circulating acellular Hb which can induce life threatening radical generating reactions in patients with a compromised vascular system [60]. Small to moderate levels of free Hb (<10 μM) have been reported to arise from transfused red blood cells and in sickle cell anemia [61].…”

“…Biological systems express several globin and heme scavengers that can, under normal physiological states, deal effectively with acellular Hb in circulation [60]. Hp is the first-line scavenger that binds and accelerates the clearance of Hb in the circulation, although the macrophage CD163 receptor has also been the focus of several recent investigations [66–68].…”

Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide

“…The pseudo-peroxidase activities of Hb have been under intense investigation in vitro, and more recent experimental evidence from animal studies supports the notion that these reactions occur in vivo with some potentially serious consequences (35,36 Low temperature EPR was used to monitor the amino acid ferryl radicals following H 2 O 2 addition to the ferric form of the mutants (data not shown). A signal was observed at g ϭ 6 and represented high spin ferric heme, and a second signal was observed at g ϭ 2 and represented the tyrosine free radical as described in previous publications (22,37).…”

Blood substitutes: why haven’t we been more successful?

“…Indirect EPR measurements of ferryl radicals in rabbits infused with HBOCs were also reported recently [39]. Subtle oxidative changes at the amino acid levels in proteins after infusion of HBOCs or stroma-free Hb were recently identified by more sensitive mass spectrometric methods [40]. Intramolecular Hb cross-links, porphyrin-globin covalent adducts modifications have been found in Hb recovered from the spinal fluid after subarachnoid hemorrhage [41] and from the urine [42], suggesting that peroxidative reactions may contribute to Hb toxicity in vivo .…”