Transfusion and Apheresis Science 34 (2006) 25–32

intl.elsevierhealth.com/journals/tras

Oxygen carriers: A selected review

Mohammed S. Inayat a, Andrew C. Bernard b, Vincent S. Gallicchio c,Beth A. Garvy d, Howard L. Elford e, Oliver R. Oakley a,¤

a Department of Clinical Sciences, University of Kentucky, Room 209b, Charles T. Wethington Building, 900 South Limestone Street, Lexington, KY 40536-0200, USA

b Department of Surgery, University of Kentucky, Lexington, KY, USAc Department of Biological Sciences, Clemson University, Clemson, SC, USA

d Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, KY, USAe Molecules for Health Inc., Richmond, VA, USA

Received 22 June 2005; received in revised form 8 September 2005; accepted 8 September 2005

Abstract

The most common and widely transplanted tissue world wide is blood, which in 2000 resulted in the transfusion of12.5 million units of blood in the US alone [Goodnough LT, Shander A, Brecher ME. Transfusion medicine: looking tothe future. Lancet 2003;361:161–9]. The current use of donated blood products is relatively safe; however, there areinherent problems with allogeneic blood transfusions. The wide spread use of blood in procedures results in problemsinvolving inadequate supply exacerbated in times of war and disasters and by the limited storage life of blood donations(30–42 days). Blood contamination due to patient pre-disposition, poor collection, sterilization, or storage is the secondmost common cause of death from transfusion in the US [Hillyer CD, Josephson CD, Blajchman MA, Vostal JG,Epstein JS, Goodman JL. Bacterial contamination of blood components: risks, strategies, and regulation: joint ASHand AABB educational session in transfusion medicine. Hematology (Am Soc Hematol Educ Program) 2003:575–89].Blood is a complex tissue involved in a plethora of homeostatic roles, including immunity, wound healing and the trans-port of nourishment, electrolytes, hormones, vitamins, heat, oxygen and the removal of metabolic waste products. How-ever, by far the principle role of blood transfusions is the replacement of red cell volume and the maintenance of oxygenlevels within the circulation. Creation of investigational new drugs (INDs) which would function as oxygen carriers andprolong shelf life is now a very active arena of scientiWc research. Several such IND products are now in clinical trials.This article gives an easy to follow concise evaluation of major areas of focus and current testing for each type of bloodsubstitution molecule. 2005 Elsevier Ltd. All rights reserved.

1473-0502/$ - see front matter 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.transci.2005.09.005

* Corresponding author. Tel.: +1 859 323 1100x80849; fax: +1 859 257 2454.E-mail address: oroakl1@uky.edu (O.R. Oakley).

26 M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32

Keywords: Hemoglobin; Oxygen carriers; PerXuorocarbon; Hemoglobin-based oxygen carriers

1. Hemoglobin

The three-dimensional structure of hemoglobin(Hb) was Wrst fully elucidated in 1969 and consistsof a tetrameric protolytic molecule consisting oftwo pairs of non-covalently bonded globular pep-tides (�, � subunits) [3]. Each subunit has as its corea heterocyclic ring (porphyrin) which holds an iron(Fe2+) atom and is the site of oxygen binding(Fig. 1A). Each Hb molecule has a molecularweight of approximately 64.4 kDa and has a bind-ing capacity of four oxygen molecules.

The oxygen aYnity of Hb is principallycontrolled by 2,3-diphosphoglycerate (2,3-DPG)

which binds to the Hb molecule resulting in a con-formational change that increases its oxygen ten-sion (P50: 50% Hb saturation) by the formation ofsalt bridges between the � chains (T-state). Whenoxygen is bound it results in the release of 2,3-DPGwhich induces the breaking of the salt bridgesculminating in the relaxation of the Hb moleculeand an increase in oxygen aYnity (R-state). Thedissociation of oxygen presents with a sigmoidrelationship which allows a great deal of oxygen tobe released even with a small drop in oxygen ten-sion of its surroundings. A decrease in pH (Bohrshift) or an increase in temperature or 2,3-DPGconcentration will result in a shift to the right of

Fig. 1. (A) Heme, a porphyrin ring structure containing an iron core, the critical component of oxygen transport via red blood cells. (B)Hemoglobin disassociation curve. Percent saturation of hemoglobin represents the number of bound oxygen molecules. The sigmoidcurve represents the physical relationship between 2,3-DPG, a by-product of glycolysis. In the diagram, represents an oxygen mole-cule and represents a heme molecule.

M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32 27

the oxygen–hemoglobin dissociation curve whichculminates in the release of oxygen to tissues withhigher than normal oxygen partial pressures(Fig. 1B). Conversely, the increase in pH or thedecrease in temperature or 2,3-DPG concentrationwill increase the aYnity of hemoglobin for oxygen.

The current goal for researchers is to produceoxygen carriers that can successfully be used inlarge volumes without the occurrence of signiWcantassociated kidney toxicities. This carrier systemwould ideally; (1) have a long storage capacity, (2)be universally compatible i.e. would not requirecross-matching, (3) be eVective in transport anddelivery/release of oxygen, and (4) be pathogen free.

Current research has focused on bio-artiWcialoxygen carriers which are modiWed Hb solutions(hemoglobin-based oxygen carriers: HBOC) andsynthetic oxygen carriers which include syntheticmetal chelates, lipid vesicles, and perXuorocarbonoxygen carriers (PFOC) emulsions, which are cur-rently in clinical trials.

2. Hemoglobin-based oxygen carriers

The Wrst clinical trial using unmodiWed Hbadministered in saline was undertaken by Amber-son and his colleagues in 1949 which indicatedcomplications associated with severe kidney toxic-ity [4]. This problem was overcome by the modiW-cation of the Hb extract to remove any stromallipid (SFHb: Stromal Free Hb); however, due tothe puriWcation methods to remove endotoxins, the2,3-DPG was also removed, which resulted in theSFHb having a high oxygen aYnity and a shortcirculatory life. This problem has been addressedby a variety of methods:

1. polymerization,2. stabilization,3. conjugation,4. hybridization,5. encapsulation.

2.1. Polymerization

Currently two hemoglobin-based oxygen carri-ers have been submitted for US FDA biologic

license applications. Both are glutaraldehyde cross-linked polymers. Poly-SFH-P Injection, (Poly-Heme, NorthWeld Laboratories, Evanston, Illinois)is a polymerized human hemoglobin while HBOC-201 (Hemopure, Biopure Corporation, Cambridge,MA) is a bovine Hb based product. PolyHeme,intended for use in circumstances of blood unavail-ability, has been shown to support human lifewhen native hemoglobin levels fall to below 3 g/dl[5]. PolyHeme is currently under testing in a land-mark Phase III “ambulance” trial. In this trial,patients with evidence of traumatic bleedingreceive PolyHeme in the pre-hospital setting whereblood is not available. Hemopure a bovine Hbpolymer developed by Biopure Corporation hasshown signiWcant promise in both preclinical andrandomized singled blinded clinical trials [6]. It hascurrently completed phase III trials in the US andis pending regulatory Wling but has gainedapproval for use in Europe and South Africa in1998 for non-cardiac surgery (www.biopure.com).Biopure is closely associated with the US NavalMedical Research Center (NMRC) in the develop-ment of Hemopure, highlighting the potential clin-ical utility of HBOC in a military scenario.

Other polymerization methods include the reac-tion of human Hb with O-raYnose (a polyalde-hyde obtained through oxidation of the trisaccharideraYnose) to give O-R-polyHb. During the cross-linking process, the aldehyde of O-raYnose com-bines with the amino groups of Hb to form reversibleSchiV base linkages. These are then reduced tostable amines. O-raYnose is implicated in stabiliz-ing the tetrameric structure of the Hb (T-state) bycovalently cross-linking the two �� dimers at theamino groups within the 2,3-diphosphoglycerate(2,3-DPG) binding site, and also to oligomerizethe tetramer. The dissociated products of O-R-poly-Hb have been shown to consist of HbA oligomers:32 kDa dimer (65%); 64 kDa tetramer (33 § 10%);128–600 kDa 2–10 tetramers (63 § 12%); 7600kDa larger oligomers (63%) [7]. This hexa func-tional cross-linker O-raYnose Hb has been shownto deliver comparative amounts of oxygen undernormal physiological circulatory conditions as redblood cells. It has been shown to reduce systemicand renal vasoconstrictor eVects compared tounmodiWed Hb [8] with recent studies indicating

28 M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32

that high molecular weight lower aYnity O-raY-

nose Hb can result in lesser hemodynamic altera-tions [9] (Hemolink, Hemosol, Canada). In orderto enhance oV-loading, cross-linkers that stabilizehemoglobin in the T-state have been sought fortheir potential eYcacy as oxygen carriers [10]. How-ever, the physiological ramiWcations of locking Hbin the T-state conformation with the O-raYnoseare still unknown [11]. Hemosol have suspendedphase III clinical trials with the O-raYnose Hb andare instead focusing on a second generation hydro-ethanol starch conjugated Hb molecule which hasa higher molecular weight and has volume expand-ing capabilities.

2.2. Stabilization

Pyridoxal-5�-phosphate (PLP) is an analog of2,3-DPG which binds to speciWc alpha and betaNH2-terminal regions of the DPG binding site onthe Hb [12]. PLP-Hb has low circulating oxygenaYnity; however, requires dimeric cross-linkagesto prevent dissociation by the kidneys. Thishas been achieved predominately in two ways: (1)bi-functional aldehydes [13] and (2) �-speciWccross-linker [14] such as diaspirin which has beenshown to eVectively restore pancreatic microcircu-latory failure in hemorrhagic shock [15]. Morerecently in a prospective trial, diaspirin cross-linked Hb solution given to patients after repair ofabdominal aortic aneurysm was well toleratedwithout severe organ dysfunction or toxicity [16].Diaspirin cross-linked hemoglobin (DCL-Hb,HemAssist) was evaluated as a resuscitationadjunct in a phase III clinical trial of hemorrhagicshock but the trial was halted prior to targetenrollment because of increased mortality in theHemAssist group [17]. The vasoactive nature ofthe compound, though beneWcial in raising bloodpressure, may have contributed to the disparitybetween the groups but the precise etiology isunclear.

2.3. Conjugation

Initial work focused around increasing SFHbvascular retention due to its small hydrodynamicvolume by the covalent coupling of Hb with dex-

tran derivatives which artiWcially increases its size[18]. Moreover, the use of sulfated dextran alsoreinforced the lowering in oxygen aYnity [19].Conjugation with polyethylene glycol/polyoxyeth-ylene (PEG/POE) can increase the intravascularcirculation times and delay dissociation and excre-tion of Hb by the kidneys [20]. Recently, modiWca-tions of PEG-Hb have led to the development ofMP4 which has been designed with a larger molec-ular radius that results in an increased oncoticpressure and viscosity accompanied by increasedoxygen aYnity [21]. Observations in the hamsterskin fold model demonstrate that the MP4 is eVec-tive in conserving oxygen in pre-capillary vessels,releasing oxygen in capillaries, and inhibitingvasoconstriction that together culminate in theselective delivery of oxygen to hypoxic tissues [22].A human hemoglobin tetramer modiWed bysurface polyoxyethylene (pyridoxalated hemoglo-bin polyoxyethylene conjugate, PHP, Curacyte,Munich Germany and Durham, NC) has beendeveloped which intentionally exploits nitric oxidescavenging as its mechanism of action. PHP hasbeen used in a recent phase II trial of septic shockand was found to reduce the requirement for vaso-active drugs (unpublished abstract, www.cura-cyte.de). A phase III trial is planned but is not yetactive.

Hemospan (Sangart, San Diego, CA) is pro-duced from human hemoglobin bound with poly-ethylene glycol (PEG) to reduce toxicity andprolong the half-life. Johns Hopkins Universityhas recently undertaken a 40-patient single-centerphase II trial of Hemospan (MP4) designed toassess its safety and eYcacy. (www.clinicaltri-als.gov). Hemotech is a bovine HBOC (Hemobio-tech, Dallas, TX) cross-linked with o-adenosinetriphosphate and o-adenosine, however no clinicaltrials are currently being conducted using thisproduct. Another promising HBOC exploited theuse of recombinant human hemoglobin (BaxterHealthcare, Boulder, CO) to avoid the less desir-able nitric oxide scavenging characteristics ofmodiWed animal or human hemoglobin. Theperformance of rHb2.0 the second generationrecombinant appeared otherwise similar to heter-ologous blood [23], however Baxter discontinuedresearch and development of this product in 2003.

M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32 29

2.4. Hybridization

Early hybrid models included iron–cobalt hybridHb with oxygen equilibrium studies [24]; however,the clinical usefulness and possible applicationsstill remain to be elucidated. The use of metal Hbhybrids are showing promise especially with thenickel–iron hybrid which does not bind to oxygenor carbon dioxide mimics, and it was also notedthat the aYnities of this asymmetric hybrid forthe Wrst oxygen molecule were as low as those ofnative Hb [25]. More recent studies have alsoindicated that iron–zinc hybrids of Hb which haveshown a T-state structure that shows plasticitywhich can adopt a range of tertiary conWgurations[26].

2.5. Encapsulation

Initial research into encapsulation has focusedon the use of phospholipids (PL) vesicles. TheseHb containing vesicles are formed by a relativelynon-disruptive technique of extrusion throughpolycarbonate membranes [27]. Recent data sug-gest the choice of extrusion buVer has the greatesteVect on the PL-Hb size distribution, compared toeither Hb concentration or extruder membranepore size [28]. The nature of the membrane alsoplays a critical role in determining the stability ofthe encapsulated Hb. Hb encapsulated in lipo-somes made from negatively charged phospholip-ids were less stable than Hb encapsulated inisoelectric phosphatidylcholine and equimolaramounts of cholesterol in the phospholipid bilayercan aVord a stabilizing eVect on Hb [29]. Whencompared to red blood cells, the increased surfaceto volume ratio of synthetic red cells, coupled withtheir lower concentration of hemoglobin, resultsin more rapid rate of oxygen uptake and release.The eYcacy of encapsulated Hb was furtherdemonstrated by Farmer and his colleagueswho showed that following a 90% transfusionexchange, rats were able to maintain normal car-diac and respiratory function [30]. Data obtainedfrom studies in Male Wistar rats given intrave-nous Hb vesicle suspensions (10 ml/kg/day for 14days), showed good tolerance and that detoxiciW-

cation was preferentially via the reticulo-endothe-

lial system, which is the normal physiologicalcompartment for degradation of senescent redblood cells [31]. Nanocapsule technology usingpolymers such as polylactic acid and polyisobutyl-cyanoacrylate have also shown promise. Theyhave slightly lower viscosity than human blood,and the in vitro P50 (containing bovine Hb) isidentical to free bovine Hb solution. ModiWcationof the surface with polyethylene glycol (PEG) canincrease its survival in the circulation. Earlywork on non-phospholipid liposomes show lowencapsulation eYciency, the greatest being 30%[32].

3. PerXuorocarbon oxygen carriers

This approach relies on the ability of certaininert organic chemicals to dissolve large amountsof gases [33]. PerXuorocarbons are the mostcommonly used chemicals which include perXu-orodecalin (Green Cross Corp., Osaka, Japan),perXuorooctyl bromide (Alliance Corp., San Diego,CA), and dodecaXuoropentane (Sonus Corp., Seat-tle, WA), which are aliphatic molecules thatpossess strong intramolecular bonding [34] whichhelp prevent degradation in the blood stream.These compounds are not miscible in water andhence cannot transport water soluble metabolitesor waste products in the circulation, which maylimit their ability to sustain life over long periods.The reticulo-endothelial system is responsible forthe systemic removal of perXuorcarbons that areWnally exhaled via the alveolar surfaces in the lungs[35] which result in a short dose dependent circula-tory half-life [36,37]. This combined with the linearoxygen binding relationship results in a narrowphysiological pO2 [36] which in its self raises possi-ble toxicities related to prolonged exposure to highconcentrations of oxygen [38]. Research under-taken in hemodiluted dogs has revealed an increasein mixed venous pressure resulting in increasedsurvival following cardiopulmonary bypass. Thesestudies indicate that perXuorochemical emulsionscan be used as a temporary erythrocyte substituteto reduce the need for allogeneic transfusions dur-ing cardiac operations [39,40]. To aid dispersionand bioavailability of PFOC in the circulation,

30 M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32

emulsiWcation of PFOC was developed which wasstill suitable for administration with an injection. Itwas observed that at 60% w/v the viscosity ofPFOC was less than that of human blood, has ashelf life of over a year, and exhibited only mildside eVects in Phase I clinical trials [41,42]. PFOCwhen administered in Phase I randomized, double-blinded, placebo-controlled studies (48 healthyvolunteers) showed no adverse alterations in coag-ulation [43]. A recent single-blind randomizedstudy in general surgery evaluated the eYcacy ofPFOC as an artiWcial oxygen carrier and foundthat it augmented acute normovolemic hemodilu-tion and reduced the need for transfusion inpatients undergoing non-cardiac surgical proce-dures with blood loss greater than 20 ml/kg [44]. Ina separate study, swine were given a highly concen-trated Xuorocarbon emulsion which resulted in thedevelopment of Xu-like symptoms [45]. Paxian andhis colleagues subjected rats to hemorrhagic hypo-tension (mean arterial pressure D 35–40 mmHg for120 min) and then resuscitated with perXubronemulsion. They reported that perXubron emulsionwas superior to stored blood when comparing therestoration of hepatocellular energy metabolism,but the improved oxygen availability failed tonegate early hepatocellular injury [46]. To datePFOC have not been approved by the FDA in theUS; however, Perfuoromethyl-cyclohexylpiperidin

(Perftoran) has been approved for use by the Rus-sian Ministry of Health [47].

4. Conclusion

The demand for transfusions continues to growand supply seems to remain alarmingly constant.This coupled with blood safety concerns [2] espe-cially in developing countries [1], has necessitated aneed for an alternate to red blood cell transfusions.Incredible progress has been made with bothimproving oxygen carrying capacity and in thedelivery eYciency of oxygen carriers. CurrentlyHemospan (Sangart) is in phase II clinical trialsand human POE-Hb (Curacyte) and pyridoxalpolyHb (NorthWelds Laboratories) are in phase IIIclinical trials. In contrast to HBOC, currently theonly approved perXuorocarbon is Perftoran, whichwas approved by the Russian ministry of Healthfor clinical use in 1999 [47]. However, the diVerencein binding capacity of perXuorocarbons whichhave linear oxygen dissociation characteristicscompared to cell free hemoglobin results in twoinherent problems: (1) oxygen supply to peripheralextremities is limited and (2) the patient has to beconstantly exposed to high concentrations of oxy-gen. Despite minor side eVects (Xu-like symptoms),PFOC have and do show promise as potential

Table 1Developmental stage of hemoglobin-based oxygen carriers

HBOC Manufacturer Location Construct P50(mmHg)

Half-life(h)

Shelf life(yr)

Status inUS trials

Poly-SFH-P(PolyHeme)

NorthWeldLaboratories

Evanston, IL Polymerization of humanhemoglobin with glutaraldehyde

26–32 24 1 (2–4 °C) Phase IIIongoing

HBOC-201(Hemopure)

BiopureCorporation

Ontario,Canada

Polymerization of bovinehemoglobin with glutaraldehyde

40 18–24 3 Phase IIIcomplete

O-R-polyHb(Hemolink)

Hemosol Toronto,Canada

Polymerization of humanhemoglobin with O-raYnose

27–51 12–24 >2 Phase IIIsuspended

PHP Curacyte Munich,GermanyDurham, NC

ModiWed conjugated humanhemoglobin

20 8–12 Notpublished

Phase IIcomplete

Hemospan MP4 Sangart Inc. San Diego,CA

Polyethylene glycol modiWedhuman hemoglobin

5–6 24 (rat) >2 (¡20 °C) Phase IIinitiated

Hemotech Hemobiotech Dallas, TX Cross-linked bovinehemoglobin

21–23 24 0.5–1 (4 °C) Pre-clinical

rHb2.0 Baxter Healthcare

Boulder, CO Second generation recombinant 34 >24 Notpublished

Pre-clinicaldiscontinued

M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32 31

RBC substitutes with respect to oxygen carryingand delivery eYcacy.

Acknowledgements

The authors would like to thank NorthWeldLaboratories, Biopure Corporation, Hemosol,Curacyte, Sangart Inc., Hemobiotech and BaxterHealthcare for kindly providing information abouttheir HBOC which was presented in Table 1.

References

[1] Goodnough LT, Shander A, Brecher ME. Transfusionmedicine: looking to the future. Lancet 2003;361:161–9.

[2] Hillyer CD, Josephson CD, Blajchman MA, Vostal JG,Epstein JS, Goodman JL. Bacterial contamination ofblood components: risks, strategies, and regulation: jointASH and AABB educational session in transfusion medi-cine. Hematology (Am Soc Hematol Educ Program)2003:575–89.

[3] Perutz MF. Structure and function of hemoglobin. HarveyLect 1969;63:213–61.

[4] Amberson W, Jennings J, Martin Rhode C. Clinical experi-ence with hemoglobin-saline solutions. J Appl Physiol1949;1:469–89.

[5] Gould SA, Moore EE, Hoyt DB, Ness PM, Norris EJ, Car-son JL, et al. The life-sustaining capacity of human poly-merized hemoglobin when red cells might be unavailable.J Am Coll Surg 2002;195:445–52 [discussion 452–445].

[6] Sprung J, Kindscher JD, Wahr JA, Levy JH, Monk TG,Moritz MW, et al. The use of bovine hemoglobin glutamer-250 (Hemopure) in surgical patients: results of a multicen-ter, randomized, single-blinded trial. Anesth Analg2002;94:799–808 [table of contents].

[8] Lieberthal W, Fuhro R, Freedman JE, Toolan G, LoscalzoJ, Valeri CR. O-raYnose cross-linking markedly reducessystemic and renal vasoconstrictor eVects of unmodiWedhuman hemoglobin. J Pharmacol Exp Ther 1999;288:1278–87.

[9] Scatena R, Giardina B. O-raYnose-polymerised haemoglo-bin. A biochemical and pharmacological proWle of an oxy-gen carrier. Expert Opin Biol Ther 2001;1:121–7.

[10] Riess JG. Oxygen carriers (“blood substitutes”)—raisond’etre, chemistry, and some physiology. Chem Rev2001;101:2797–920.

[11] Jia Y, Ramasamy S, Wood F, Alayash AI, Rifkind JM.Cross-linking with O-raYnose lowers oxygen aYnity andstabilizes haemoglobin in a non-cooperative T-state con-formation. Biochem J 2004;384:367–75.

[12] Schnackerz KD, Benesch RE, Kwong S, Benesch R,Helmreich EJ. SpeciWc receptor sites for pyridoxal 5�-phos-

phate and pyridoxal 5�-deoxymethylenephosphonate at thealpha and beta NH2-terminal regions of hemoglobin. J BiolChem 1983;258:872–5.

[13] Bleeker WK, van der Plas J, Agterberg J, Rigter G, BakkerJC. Prolonged vascular retention of a hemoglobin solutionmodiWed by cross-linking with 2-nor-2-formylpyridoxal 5�-phosphate. J Lab Clin Med 1986;108:448–55.

[14] Nelson D, Azari M, Brown R, Burhop K, Bush S, CatarelloJ, et al. Preparation and characterization of diaspirin cross-linked hemoglobin solutions for preclinical studies. Bioma-ter Artif Cells Immobil Biotechnol 1992;20:423–7.

[15] von Dobschuetz E, HoVmann T, Messmer K. Diaspirincross-linked hemoglobin eVectively restores pancreaticmicrocirculatory failure in hemorrhagic shock. Anesthesi-ology 1999;91:1754–62.

[16] BloomWeld EL, Rady MY, Esfandiari S. A prospective trialof diaspirin cross-linked hemoglobin solution in patientsafter elective repair of abdominal aortic aneurysm. MilMed 2004;169:546–50.

[17] Sloan EP, Koenigsberg M, Brunett PH, Bynoe RP, MorrisJA, TinkoV G, et al. Post hoc mortality analysis of theeYcacy trial of diaspirin cross-linked hemoglobin in thetreatment of severe traumatic hemorrhagic shock.J Trauma 2002;52:887–95.

[18] Bonneaux F, Labrude P, Dellacherie E. Preparation andoxygen binding properties of soluble covalent hemoglobin-dextran conjugates. Experientia 1981;37:884–6.

[19] Bonneaux F, Dellacherie E, Labrude P, Vigneron C.Hemoglobin-dialdehyde dextran conjugates: improvementof their oxygen-binding properties with anionic groups.J Protein Chem 1996;15:461–5.

[20] Iwashita Y. Relationship between chemical properties andbiological properties of pyridoxalated hemoglobin-poly-oxyethylene. Biomater Artif Cells Immobil Biotechnol1992;20:299–307.

[21] VandegriV KD, Malavalli A, Wooldridge J, Lohman J,Winslow RM. MP4, a new nonvasoactive PEG-Hb conju-gate. Transfusion 2003;43:509–16.

[22] Winslow RM. MP4, a new nonvasoactive polyethylene gly-col-hemoglobin conjugate. Artif Organs 2004;28:800–6.

[23] Malhotra AK, Kelly ME, Miller PR, Hartman JC,Fabian TC, Proctor KG. Resuscitation with a novelhemoglobin-based oxygen carrier in a Swine model ofuncontrolled perioperative hemorrhage. J Trauma 2003;54:915–24.

[24] Tsuneshige A, Zhou YX, Yonetani T. Oxygen equilibriumstudies of cross-linked iron–cobalt hybrid hemoglobins.Models for partially ligated intermediates of cobalt hemo-globin. J Biol Chem 1993;268:23031–40.

[25] Shibayama N, Imai K, Morimoto H, Saigo S. Oxygen equi-librium properties of asymmetric nickel(II)–iron(II) hybridhemoglobin. Biochemistry 1993;32:8792–8.

[26] Samuni U, Juszczak L, Dantsker D, Khan I, Friedman AJ,Perez-Gonzalez-de-Apodaca J, et al. Functional and spec-troscopic characterization of half-liganded iron–zinchybrid hemoglobin: evidence for conformational plasticitywithin the T state. Biochemistry 2003;42:8272–88.

32 M.S. Inayat et al. / Transfusion and Apheresis Science 34 (2006) 25–32

[27] Gaber BP, Yager P, Sheridan JP, Chang EL. Encapsulationof hemoglobin in phospholipid vesicles. FEBS Lett1983;153:285–8.

[28] AriWn DR, Palmer AF. Determination of size distributionand encapsulation eYciency of liposome-encapsulatedhemoglobin blood substitutes using asymmetric Xow Weld-Xow fractionation coupled with multi-angle static lightscattering. Biotechnol Prog 2003;19:1798–811.

[29] Szebeni J, Di Iorio EE, Hauser H, Winterhalter KH.Encapsulation of hemoglobin in phospholipid liposomes:characterization and stability. Biochemistry 1985;24:2827–32.

[30] Farmer MC, Rudolph AS, VandegriV KD, Hayre MD,Bayne SA, Johnson SA. Liposome-encapsulated hemo-globin: oxygen binding properties and respiratory function.Biomater Artif Cells Artif Organs 1988;16:289–99.

[31] Sakai H, Masada Y, Horinouchi H, Ikeda E, Sou K, Take-oka S, et al. Physiological capacity of the reticuloendothe-lial system for the degradation of hemoglobin vesicles(artiWcial oxygen carriers) after massive intravenous dosesby daily repeated infusions for 14 days. J Pharmacol ExpTher 2004;311:874–84.

[32] VandegriV KD, Wallach DF, Winslow RM. Encapsulationof hemoglobin in non-phospholipid vesicles. Artif CellsBlood Substit Immobil Biotechnol 1994;22:849–54.

[33] Spahn DR, Leone BJ, Reves JG, Pasch T. Cardiovascularand coronary physiology of acute isovolemic hemodilu-tion: a review of nonoxygen-carrying and oxygen-carryingsolutions. Anesth Analg 1994;78:1000–21.

[34] Riess JG. Fluorocarbon-based in vivo oxygen transportand delivery systems. Vox Sang 1991;61:225–39.

[35] Rosenblum WI, HadWeld MG, Martinez AJ, Schatzki P.Alterations of liver and spleen following intravenous infu-sion of Xuorocarbon emulsions. Arch Pathol Lab Med1976;100:213–7.

[36] Zuck TF, Riess JG. Current status of injectable oxygencarriers. Crit Rev Clin Lab Sci 1994;31:295–324.

[37] Spence RK. PerXuorocarbons in the twenty-Wrst century:clinical applications as transfusion alternatives. Artif CellsBlood Substit Immobil Biotechnol 1995;23:367–80.

[38] Hess JR, Reiss RF. Resuscitation and the limited utility ofthe present generation of blood substitutes. Transfus MedRev 1996;10:276–85.

[39] Keipert PE, Faithfull NS, Bradley JD, Hazard DY, HoganJ, Levisetti MS, et al. Oxygen delivery augmentation bylow-dose perXuorochemical emulsion during profoundnormovolemic hemodilution. Adv Exp Med Biol 1994;345:197–204.

[40] Spruell RD, Ferguson ER, Clymer JJ, Vicente WV, Mur-rah CP, Holman WL. PerXuorocarbons are eVective oxy-gen carriers in cardiopulmonary bypass. Asaio J1995;41:M636–41.

[41] Riess JG, Dalfors JL, Hanna GK, Klein DH, KraVt MP, Pel-ura TJ, et al. Development of highly Xuid, concentrated andstable Xuorocarbon emulsions for diagnosis and therapy. Bio-mater Artif Cells Immobil Biotechnol 1992;20:839–42.

[42] Riess JG. [Fluorocarbon emulsions as injectable oxygencarriers. Recent progress and perspectives. Rev Fr TransfusHemobiol 1992;35:391–406.

[43] Leese PT, Noveck RJ, Shorr JS, Woods CM, Flaim KE,Keipert PE. Randomized safety studies of intravenous per-Xubron emulsion. I. EVects on coagulation function inhealthy volunteers. Anesth Analg 2000;91:804–11.

[44] Spahn DR, Waschke KF, Standl T, Motsch J, Van Huyne-gem L, Welte M, et al. Use of perXubron emulsion todecrease allogeneic blood transfusion in high-blood-lossnon-cardiac surgery: results of a European phase 3 study.Anesthesiology 2002;97:1338–49.

[45] Flaim SF, Hazard DR, Hogan J, Peters RM. Characteriza-tion and mechanism of side-eVects of Oxygent HT (highlyconcentrated Xuorocarbon emulsion) in swine. Artif CellsBlood Substit Immobil Biotechnol 1994;22:1511–5.

[46] Paxian M, Rensing H, Geckeis K, Bauer I, Kubulus D,Spahn DR, et al. PerXubron emulsion in prolongedhemorrhagic shock: inXuence on hepatocellular energymetabolism and oxygen-dependent gene expression. Anes-thesiology 2003;98:1391–9.

[47] Kim HW, Greenburg AG. ArtiWcial oxygen carriers as redblood cell substitutes: a selected review and current status.Artif Organs 2004;28:813–28.