Effects of Endogenous Ascorbate on Oxidation, Oxygenation, and Toxicokinetics of Cell-Free Modified Hemoglobin after Exchange Transfusion in Rat and Guinea Pig

Abstract

Chemically modified hemoglobin (Hb) solutions are promising oxygen therapeutics; however, these agents are prone to intravascular oxidation. Using a 50% exchange transfusion (ET) model with bovine polymerized hemoglobin (PolyHbBv), we examined heme oxidation, oxygenation markers, and toxicokinetics in rats, an ascorbic acid (AA)-producing species, and in guinea pigs, a non-AA-producing species. Plasma AA decreased by 50% in guinea pigs after ET, but it was unchanged in rats for the first 20 h post-ET. Both species cleared PolyHbBv from the circulation at similar rates. However, exposure to ferric PolyHbBv over time was 5-fold greater in the guinea pig. Mass spectrometry analysis of plasma revealed oxidative modifications within the tetrameric fraction of PolyHbBv in guinea pig. Oxygen equilibrium curves of PolyHbBv measured in plasma after ET were more left-shifted in guinea pigs compared with rats, consistent with increased ferric PolyHbBv formation. Renal hypoxia-inducible factor (HIF)-1␣, whose activity strictly depends on the partial pressure of oxygen increased over time, and it correlated inversely with circulating ferrous PolyHbBv in both species. Interestingly, HIF-1␣ activity was greater in guinea pigs compared with rats at 72 h post-ET. Mean arterial pressure increases were also greater in guinea pigs; however, minimal differences in cardiac and renal pathology were observed in either species. The present findings suggest the importance of plasma AA in maintaining the stability of acellular Hb susceptible to oxidation, and they may be relevant to humans, which display a similar plasma/tissue antioxidant status to guinea pig.Hemoglobin (Hb)-based oxygen carriers (HBOCs) represent a class of complex biological entities being developed as oxygen-bridging agents with volume-expanding properties. Despite their therapeutic promise, HBOCs demonstrate a significant potential for toxicity based on administration of large quantities of Hb into the plasma compartment. Normally Hb remains protected in the red blood cell, where processes exist to reduce oxidized Hb and to modulate nitric oxide (NO) binding. It has become increasingly evident that Hb oxidative toxicity can limit the safety and efficacy of current generation HBOCs (Alayash, 2004). This prompted the design of new strategies aimed at reducing or controlling Hb-oxidative side reactions. In vivo oxidation of cell-free Hb is driven spontaneously and/or chemically by variety of oxidants, including hydrogen peroxide (H 2 O 2 ) and NO. NOinduced oxidation of heme iron has the added complication of producing an immediate elevation in blood pressure as a result of removal of NO (a vasodilator) by Hb. Thus, two primary safety concerns with HBOCs in the extracellular space include hypertension and oxidative stress (Riess, 2001;Alayash, 2004). The latter effect depends on plasma and tissue reductive capacity to maintain the HBOC in a reduced and functional state.HBOCs have generally demonstrated promising safety and efficacy in animals, and, in many cases...

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase‐1

“…Several animal studies have reported the accumulation of large quantities of oxidized Hb (ferric) (30–40%) after infusion of ferrous Hb or chemically modified Hb‐based oxygen carries (HBOCs) 30, 31. Recently it was shown that intravascular hemolysis in guinea pigs and beagle dogs (with acute kidney injury) led to intrarenal conversion of ferrous to ferric Hb, accumulation of free heme and Hb‐cross‐linked products, enhanced 4‐hydroxynonenal reactivity in renal tissue, and acute tubule injury.…”

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs

“…While the redox reactions initiated at heme iron are well characterized from a chemical perspective, there is limited evidence that these reactions occur in vivo during hemolysis. Evaluating animal models of hemoglobinuria and exchange transfusion with a long circulating HBOC, we recently obtained evidence of globin and heme oxidative modifications, including oxidation of key β-chain amino acids and protein cross-linking, in animal blood and urine (9,30,57). Here, we found no evidence of heme oxidation within the circulation, as less than 2% of free intravascular Hb was observed in the oxidized Fe 3+ state and no Hb was observed in the higher oxidation ferryl (Fe 4+ ) state throughout the infusion time of up to 8 hours.…”

“…The dog is therefore a unique model, in which the effects of intravascular Hb can be analyzed at a range of targeted endogenous Hp plasma concentrations. The guinea pig, on the other hand, allowed us to analyze both vascular and extravascular effects of extracellular Hb in the presence or absence of exogenously administered, purified human Hp in a nonascorbate-producing species, with blood and tissue antioxidant status comparable to that of humans (30).…”

“…All animals were fed normal diets (Teklad guinea pig chow; HarlanTeklad) throughout the acclimatization period and weighed 350-450 g at the time of study. Catheterization of the right common carotid artery and left external jugular vein was performed as described previously (30).…”

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…The disparity between in vitro biochemical observations and in vivo findings may be because of the shifted balance between oxidation and reduction reactions in in vivo conditions. 12 The availability of large quantities of small-molecule and enzymatic reductants may reduce the stability of higher-oxidation-state Hb species and Hb-derived radicals to undetectable levels. Intramolecular Hb cross-links, porphyrin-globin covalent adducts, and globin chain amino acid oxidations have been defined as surrogate markers for Hb-Fe 4ϩ formation.…”