title: Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates.
abstract: Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.
authors.affiliation: Sangart, Inc., 6175 Lusk Blvd, San Diego, California 92121 USA. kvandegriff@sangart.com
authors.forename: Kim D
authors.initials: KD
authors.lastname: Vandegriff
authors.forename: Ashok
authors.initials: A
authors.lastname: Malavalli
authors.forename: Charles
authors.initials: C
authors.lastname: Minn
authors.forename: Eva
authors.initials: E
authors.lastname: Jiang
authors.forename: Jeff
authors.initials: J
authors.lastname: Lohman
authors.forename: Mark A
authors.initials: MA
authors.lastname: Young
authors.forename: Michele
authors.initials: M
authors.lastname: Samaja
authors.forename: Robert M
authors.initials: RM
authors.lastname: Winslow
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001802:Blood Substitutes / Q000737:chemistry* / Q000493:pharmacokinetics; D002374:Catalase / Q000037:antagonists & inhibitors / Q000097:blood; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D008297:Male; D008301:Maleimides / Q000737:chemistry* / Q000493:pharmacokinetics; D008706:Methemoglobin / Q000378:metabolism; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000493:pharmacokinetics; D011190:Potassium Cyanide / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D008301:Maleimides; D017382:Reactive Oxygen Species; C493836:maleimide-polyethylene glycol-modified hemoglobin, MP4; D019820:Xanthine; D011092:Polyethylene Glycols; D006418:Heme; D008706:Methemoglobin; D002374:Catalase; D014969:Xanthine Oxidase; D011190:Potassium Cyanide; D001205:Ascorbic Acid