Proc. NatI. Acad. Sci. USAVol. 88, pp. 10976-10980, December 1991Physiology

In vivo biodistribution of a radiolabeled blood substitute:99mTc-labeled liposome-encapsulated hemoglobin in ananesthetized rabbitALAN S. RUDOLPH*, ROBERT W. KLIPPERt, BETH GoINS*, AND WILLIAM T. PHILLIPSt*Center for Biomolecular Science and Engineering, Code 6090, Naval Research Laboratory, Washington, DC 20375-5000; and tDepartment of Radiology,University of Texas Health Science Center, San Antonio, TX 78284

Communicated by John D. Baldeschwieler, July 24, 1991

ABSTRACT Liposome-encapsulated hemoglobin (LEH) isan erythrocyte substitute that is a potential resuscitative fluidfor the in vivo delivery of oxygen. We have noninvasivelyimaged radiolabeled LEH in vivo with technetium-99m (""Tc)to study the biodistribution in an anesthetized rabbit. Rabbits(2.5 kg, n = 8) were infused with 30 ml of LEH (200 mg ofphospholipid, 2.5 g of hemoglobin per kg of body weight) andimaged with a y camera continuously for 2 hr. At 20 hrpostinfusion, the animals were inaged again and sacrificed; theorgans were weighed and their radioactivity was determinedfor autopsy organ distribution. Organ uptake from the imageswas corrected for organ-associated blood pool, which wasdetermined by infusion of "9mTc-labeled rabbit erythrocytes.Blood pool and decay-corrected biodistribuion data reveal thekinetics ofLEH distribution, with an initial rapid uptake by theliver, 8% at 30 min and 15% at 2 hr. The spleen accumulatesless LEH initially, 3% at 30 min and 7% at 2 hr, with anapparent linear uptake of LEH over this time period. Imagebiodistribution data was also validated at 20 hr by tissuesampling. At 20 hr postinfusion, autopsy biodistribution datareveals approximately 42.6% of the total counts remaining inthe blood, 15.4% in the liver, 18.1% in spleen, 3.2% in thelungs, 2.4% in muscle, 1.6% in urine, and trace levels in thekidney, brain, and heart (<1%). There is no evidence ofhemoglobin release from LEH or kidney dysfunction (normalcreatinine and blood urea nitrogen) at any time over the courseof the study.

The need for blood in trauma care, its uncertain supply, andthe risk of transmittable antigens associated with using thecurrent blood reserves has fueled the search for a substituteoxygen-carrying resuscitative fluid. One effort in the searchfor an adequate substitute has been to use modified stroma-free hemoglobin because infusion ofthe hemoglobin tetramerhas been demonstrated to cause a variety of toxic responsesin vivo (1). Modifications such as chemical cross-linking andpolymerization of the tetramer have improved some of thesedeleterious properties (2). Although modified hemoglobinsolutions 'may show promise, the remaining problems asso-ciated with stroma-free hemoglobin preparations are poten-tial nephrotoxicity and antigenicity (3). The development ofrecombinant technology to produce hemoglobin may allevi-ate the problems of finding adequate sources and potentiallytainted blood supplies, and recent advances in this directionhave been reported (4).One solution to the problems associated with hemoglobin-

based blood substitutes regardless of the source is the en-capsulation of hemoglobin within a liposome. This has thepotential to reduce the toxicity of hemoglobin whether in thetetrameric, cross-linked, polymerized, or recombinant forms

as liposomes have been proven to reduce the toxicity of anumber of bioactive agents (5-7). In addition, the encapsu-lation of active agents into liposomes increases the circula-tion persistence of pharmacological materials (8). Sufficientcirculation persistence is essential for an oxygen-carryingmaterial in applications where oxygen delivery may be re-quired for prolonged periods.Some of the desired properties of an encapsulated oxygen-

carrying material have been realized in the development ofliposome-encapsulated hemoglobin (LEH) (for a recent re-view, see ref. 9). Efficacy of this material (as determined byin vivo oxygen-carrying capacity) has been demonstrated intotal isovolemic exchange transfusions in anesthetized rats(10-12). Hemodynamic consequences of small doses ofLEH('10 mg/kg of body weight) have been studied in a normo-volemic conscious rat model (13-15). These studies revealeda variety of transient effects (thrombocytopenia, leukocyto-sis, rise in blood pressure, and drop in cardiac output), whichwere alleviated by the substitution of a synthetic hydroge-nated phosphatidylcholine for natural soy lecithin (whichcontained lysolecithin contamination) and by a syntheticplatelet-activating factor antagonist (13, 14). Circulation per-sistence of LEH has been measured in rodents and is '15 hrat moderate doses (10, 15, 16). Other important milestones inthe development of LEH have been the sterile large-scaleproduction and long-term shelf-life of LEH in the freeze-dried state (17-19). Although recent studies have focused onin vivo effects of LEH, little is known regarding the biodis-tribution of LEH and the interaction with'the reticuloendo-thelial system (RES).The large body of evidence regarding liposome removal

from circulation indicates that the RES is largely responsiblefor liposome removal (20-26). Modifications to liposomessuch as lipid composition (e.g., cholesterol, degree of fattyacid saturation, and chain length), surface charge, and size allinfluence the physiological traffic pattern of liposomes (22).The use ofradiolabels has proven quite useful in following thefate of liposomes in vivo and as diagnostic tools in nuclearmedicine (24-28). The methods employed to label liposomesinclude entrapment of the radiolabel in the aqueous compart-ment, attachment of the label to lipid components prior toliposome formation, and the addition of label after theirmanufacture.We have previously reported the use of hexamethylpro-

pylene amine oxime (HMPAO), a lipophilic transporterof 9'Tc, to radiolabel LEH with 199Tc (29). This method isrelatively simple, can be performed on preformed unilamellarvesicles, and has a high labeling efficiency. In the presentreport, we investigate the biodistribution of 99mTc-radiolabeled LEH (99Tc-LEH) in an anesthetized rabbit as

Abbreviations: LEH, liposome-encapsulated hemoglobin; RES, re-ticuloendothelial system; HMPAO, hexamethylpropylene amine ox-ime; SGOT, serum glutamic-oxaloacetic transaminase; SGPT, serumglutamic-pyruvic transaminase.

10976

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an important step in understanding the effects of LEHadministration as a resuscitative fluid.

MATERIALS AND METHODS

LEH Preparation. LEH was prepared with bovine hemo-globin (Biopure, Newton, MA) as described (15, 16). Theantioxidant glutathione (30 mM) was added during the prep-aration ofLEH to prevent the oxidation ofoxyhemoglobin tothe methemoglobin form.LEH Labeling. HMPAO (Ceretec; Amersham) was hy-

drated from a lyophilized kit (which contains 0.5 mg ofHMPAO and 4 Ag of SnC12) with 5 ml of saline containing5-10 mCi (1 Ci = 37 GBq) of sodium [99mTc]pertechnetate.The solution was allowed to hydrate for 5 min and then wasimmediately added to the LEH pellet. The LEH pellet wasresuspended in an equivalent volume of phosphate-bufferedsaline (-5 ml) before adding 9'9Tc-HMPAO (5 ml). The991Tc-HMPAO complex was allowed to incubate with LEHfor 15 min before centrifugation to remove extravesicularlabel. The pellet was collected and resuspended to bring thefinal concentration of LEH to 25 mM phospholipid (30) witha total activity of 3-5 mCi. This procedure results in a highlabeling efficiency (>90%) with little loss of label in vitro (29).Animal Protocol. Male rabbits (2-2.5 kg, n = 8) were

anesthetized intramuscularly using a 5:1 (vol/vol) mixture ofketamine (50 mg/kg) and xylazine (10 mg/kg) 30 min prior toinfusion with LEH. One ear of the rabbit was catheterizedwith a 20-gauge venous line, and the other ear was catheter-ized with a 20-gauge arterial line. Blood samples were drawnfrom the arterial line and LEH was infused in the venous line.Rabbits were then placed in the prone position under the ycamera (Searle Radiographics; model 6413), and image ac-

quisition began with a low-energy all-purpose collimator asthe LEH solution was introduced. The total dose given inthese experiments was =200 mg of phospholipid per kg and2.5 g of hemoglobin per kg of body weight in a 30-ml volume.Image acquisition was accomplished using a Medasys A2computer with a 64 x 64 matrix acquisition (Medasys, AnnArbor, MI). One-minute dynamic images were acquired overa continuous 2-hr period. A small sample of 9'9Tc-LEH (0.5ml, -1/60th of the total dose) in a plastic cuvette was usedas a standard reference for intensity and placed next to theanimal for dose calibration of image biodistribution analysis.At 15, 30, 45, 60, 90, and 120 min from the start of theinfusion, blood was drawn into capillary tubes from thearterial line. Capillary tubes were spun in a hematocritcentrifuge for 10 min. The LEH is clearly visualized as adistinct pellet on top of the erythrocyte fraction. All of thecapillary tubes drawn were imaged at the end of the 2 hr todetermine if the label remained associated with the LEHpellet after infusion and to determine LEH activity remainingin the blood pool. The amount of radioactivity in thesecapillary tubes was also determined in a well counter after theactivity had decayed to a detectable range to determine thecirculation persistence of LEH over the course of the exper-iment.

After 2 hr of continuous imaging, the catheters were

removed, and the rabbits were allowed to recover fromanesthesia and were housed in cages with food and water adlibitum. Urine from housed animals was collected for deter-mination of the amount of radioactivity and biodistribution.At 20 hr postinfusion, rabbits were anesthetized, and a staticimage and blood samples of the rabbit was taken. Rabbitswere then sacrificed, and the major organs and knownvolumes of blood and urine were removed for weighing anddetermining the amount of radioactivity. Approximately 2- to3-g samples of each organ were weighed and assayed on amultichannel analyzer (Canberra, Meriden, CT) to determinecounts per g of tissue and total organ activity. Organ and fluid

counts too high for detection at the time of sacrifice werestored until decay allowed for accurate determination.Blood Pool Correction. Blood pool correction was per-

formed on imaged biodistribution data by first calculatingblood pool contribution in various organs by imaging 'mTc-labeled rabbit erythrocytes. Heparinized blood (6.0 ml) wasdrawn from 2.5-kg New Zealand White rabbits (n = 3) andadded to a Cadema RBC kit (Middleton, NY). The blood andfreeze-dried material were reconstituted by gentle agitationfor 5 min, after which time 1.0 ml of 4.4% EDTA was added.The tube was inverted and centrifuged at 1300 x g for 5 min.While the tube was inverted, 2.0 ml of packed erythrocyteswere removed and transferred to a sterile vial containing 2.0ml of saline and 4 mCi of sodium [9'Tc]pertechnetate. Thesample was mixed gently and incubated for 10 min at roomtemperature before injection into the ear vein. A 0.1-mlaliquot (0.2 mCi) was included in the field of view as astandard reference dose. After adequate equilibration of theinjected erythrocytes (usually within S min postinfusion), a1-min image was acquired and analyzed. Regions of interestwere drawn around selected organs. Blood pool contributionof each organ was measured as follows: (counts in organ xattenuation correction factor)/(counts in 0.1-ml referencedose x 20) (the total dose injected was 2.0 ml). Labelingefficiencies using this procedure were >95%, and there wasminimal loss of free label in the early images. Table 1 showsthe blood pool correction for the heart, lung, liver, spleen,and left kidney. The percentage of blood pool counts sub-tracted from each organ is based on the percentage of countsin a particular organ as determined from Table 1 multiplied bythe estimated percentage of total LEH dose remaining in theentire blood pool. The estimated percentage of total LEHdose given that remains in the blood pool at any one time isbased on counts taken from the extracted capillary bloodsamples taken over the 2 hr.Image Analysis. Camera sensitivity was determined over

the millicurie range of the injected dose (3-7 mCi) and wasfound to be linear. An attenuation correction factor was alsoempirically derived and used for final calculations of imagedbiodistributions. Image analysis for LEH organ biodistribu-tion was performed after the images were transferred to anuclear medicine analysis workstation (Pinnacle computer,Medasys). Whole images were corrected for decay and theattenuation factor using software written for the image work-station. Regions of interest were drawn around the heart,lungs, liver, spleen, kidney, bladder, and the LEH standardfor each rabbit.Blood clearance and biodistribution studies were also

performed with free 99"Tc-HMPAO for comparison with99ITc-LEH. The HMPAO kit was hydrated with 5 ml ofsaline containing 5 mCi of [pTc]pertechnetate. After S minat room temperature, the 5-ml sample was injected over 30sec into the ear vein. At 2, 5, 10, 30, 60, and 120 minpostinjection and at 20 hr just prior to sacrifice, a 0.1-mlsample of blood was withdrawn. A 0.1-ml aliquot of 99mTc-HMPAO was used as reference for imaging and tissuestudies. The percentage of the total injected dose remaining

Table 1. Blood pool image contribution% of injected9'9Tc-labeled

Organ erythrocytes

Heart 18.1 ± 1.15Lung 4.0 ± 1.19Liver 25.4 + 1.17Kidney 1.9 ± 0.04Spleen 3.8 ± 0.16

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in the blood pool was calculated based on the total estimatedblood volume of the rabbit.

RESULTSThe location of 99"Tc in labeled LEH following the labelingprocedure is an important determinant of the observed bio-distribution. An organic extraction procedure used to sepa-rate LEH components revealed that 9.5% ± 1.0% of theactivity remains in the organic phase, 22.1% ± 0.7% is foundin the aqueous phase, and 67.5% ± 2.5% is observed asso-ciated with the hemoglobin fraction. This may indicate thatthe label may have a higher affinity for the hemoglobin thanfor HMPAO once it is carried across the liposome bilayer.Previous reports indicate that the (8 chain of hemoglobin maybe a potential 9'Tc binding site (31, 32). The conversion oflipophilic HMPAO to a intravesicular hydrophilic form mayalso be aided by the presence of encapsulated glutathione asthis reductant is thought to play a role in trapping 99,Tc-HMPAO once it has crossed the blood-brain barrier for usein brain imaging (33). Alternatively, the 991Tc-HMPAOcomplex may precipitate with the hemoglobin (or glu-tathione) in the extraction protocol, and the identification of9'Tc with the hemoglobin fraction may not indicate a directprotein association.A typical example of the image acquired at the end of the

infusion (after =30 min) is shown in Fig. 1. The majority ofthe intensity visualized at this time is due to the presence ofLEH in blood vessels and organ-associated blood pools. It isimportant to note that little intensity is visualized in thebladder, stomach, or thyroid at this time point, indicating thatthe 9I1Tc does not readily dissociate from LEH. Furtherevidence of the stability of 99mTc in vivo is seen in Fig. 2A,which shows the y emission profile from a single blood-drawncapillary tube that was centrifuged. The imaged intensity hasa narrow peak where the LEH pellet is found. This is alsoobserved in the capillary blood samples drawn at 15-minintervals following infusion of LEH (Fig. 2B). These imagesshow activity only in the LEH fraction above the erythrocytepellet. No activity was observed from images of capillarytubes in the plasma or erythrocyte fraction at any time pointexamined.The disappearance of activity from the LEH fraction in the

capillary blood samples has been used to generate a clearanceprofile based on the percent of injected dose in the total

A*- HEAD

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estimated blood level (Fig. 3). The clearance profile suggeststhat LEH is removed rapidly over the first 2 hr (20% isremoved at 30 min). A slower secondary removal phase isseen between 4 and 20 hr, with -45% remaining in the bloodpool at 20 hr. In contrast, the clearance of99mTc-HMPAO isquite rapid with only 10%o remaining in the blood pool 2 minafter the infusion. This is in agreement with previous studiesof 991Tc-HMPAO clearance, which show little circulationpersistence and rapid metabolic conversion ofHMPAO (34).The images collected over the 2 hr following LEH infusion

show the slow disappearance of LEH from the blood poolwith increasing organ uptake. Fig. 4A shows the percent ofinjected dose in each organ region from the collected images2 hr postinfusion (not corrected for organ blood pool). Thesedata indicate that the liver accumulates the greatest quantityof LEH, with intensity that increased rapidly (almost 20o at15 min) and that remained at high levels ('30% of totalcounts) over the 2-hr dynamic image acquisition. The countsin the spleen increase linearly over 2 hr to almost 7%, withsimilar counts in the heart. The remaining imaged organregions (lung, kidney, and bladder) show little activity (<2%)even at 2 hr postinfusion. Fig. 4B shows the blood poolcorrected biodistribution over the 2-hr period and reveals thatthe liver and spleen are the only organs that show measurable

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FIG. 3. Blood clearance of99mTc-LEH and 99"Tc-HMPAO. Thelevel of circulating label was measured by imaging blood-drawncapillary tubes.

10978 Physiology: Rudolph et al.

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FIG. 4. (A) Organ biodistribution of 99mTc-LEH generated fromthe dynamic images collected during the first 2 hr postinfusion. Thedistribution was generated by drawing regions of interest aroundimaged organs. (B) Blood pool corrected image counts over the 2-hrcontinuous imaging period.

activity. The lung shows only trace activity over this timeperiod.At 20 hr, the validity of the imaged biodistribution was

examined by tissue sampling. Table 2 compares the imaged(both corrected and background subtracted) and autopsybiodistribution at 20 hr for both 9'9Tc-LEH and 'Tc-HMPAO. The autopsy biodistribution at 20 hr reveals thatthe blood pool still has the greatest 99"Tc-LEH-associatedactivity with 42.6% ± 6.0% remaining in circulation. Theorgan with the most accumulation of LEH is the spleen with18.1% ± 3.3%, followed by the liver with 15.4% ± 2.1%. Itis important to note the validity of the blood pool correctedimage data by comparing the autopsy biodistribution valuesfor these organs (in which the organ-associated blood pool is

washed out) to the blood pool corrected image data. Theblood pool corrected percent of injected dose/imaged organregion is 14.6% ± 2.8% for the liver, compared to the autopsyvalue of 15.4% ± 2.1%. For the spleen, the blood poolcorrected percent of injected dose/imaged organ region is18.0%o ± 4.5%, whereas the autopsy biodistribution is 18.1%+ 3.3%. It may be that the blood pool correction for thoseorgans that have very little associated accumulation of LEHsuch as the heart, lungs, and kidneys is more sensitive tocorrection and thus accounts for the reported negative valuesfor the blood pool corrected image data for the heart andkidney. This is supported by the autopsy biodistribution at 20hr, which shows very little accumulation in the heart andkidney with only minimal accumulation (3% or less) in thelungs, muscle, and urine. The total activity accounted for inthe autopsy was 84.2%. The remaining activity could be insites of LEH accumulation not measured such as the bonemarrow, which is an active site of RES activity.

Select blood chemistries were taken from the animals at thebeginning of the experiment (before infusion of LEH) and at2 and 20 hr postinfusion. Standard blood chemistry analysisof samples taken at the same time points reveals no signifi-cant change in indicators of kidney dysfunction such ascreatinine, blood urea nitrogen, and bilirubin. The serumenzymes glutamic-oxaloacetic transaminase (SGOT) and glu-tamic-pyruvic transaminase (SGPT) were unchanged at 2 hrbut slightly elevated at 20 hr (SGOT baseline = 46.7 ± 16, at20 hours = 134.8 ± 54; SGPT baseline = 40.2 + 9.3, at 20hours = 85.8 ± 18). Alkaline phosphatase and lactate dehy-drogenase were normal at 2 and 20 hr compared to baselinevalues. In addition, there was no significant change in albu-min, bilirubin, glucose, triglycerides, cholesterol, glucose, orelectrolytes (Na, K, and Cl) at 2 and 20 hr postinfusion.

DISCUSSIONOne of the essential requirements for an effective oxygen-carrying resuscitative fluid is the ability to carry oxygen forprolonged periods in scenarios where an oxygen deficit maybe encountered (e.g., bleeding injury or organ perfusion). Thebiodistribution data presented here indicate that there issignificant retention of LEH (43%) in circulation at 20 hr,which is significantly longer than most modified hemoglobinpreparations (2, 3). The image biodistribution of LEH 2 hrafter infusion reveals that LEH accumulates in the liver andspleen. The kinetics over this period of LEH uptake showthat the liver accumulates LEH rapidly, with a slower uptakeby the spleen. Additional LEH is removed by the spleen over20 hr to result in almost equivalent removal of LEH in liverand spleen at 20 hr. These results indicate that LEH accu-mulates in RES organs, and at the given dose, the RES maybecome saturated over the course ofthe rapid removal phase.

Table 2. Biodistribution of ImTc-LEH and 19"Tc-HMPAO at 20 hr postinfusion as a percentage of the injected dose (±SEM)Autopsy Image

% 99mTc-LEH (n = 8) % 99'Tc-HMPAO (n = 3) % 99'Tc-LEH per organ (n = 8) t 99mTc-HMPAOOrgan Per organ Per g of tissue Per organ Per g of tissue Uncorrected* Correctedt per organtBlood 42.6 ± 6.0 0.3 ± 0.015 1.8 ± 0.2 0.01 ± 0.001 -Heart 0.1 ± 0.01 0.02 ± 0.002 0.3 ± 0.05 0.05 ± 0.01 5.0 ± 0.7 -2.5 ± 0.7 0.8 ± 0.1Lung 3.2 ± 0.6 0.28 ± 0.04 1.8 ± 0.4 0.18 ± 0.04 3.3 ± 0.7 1.61 ± 0.7 1.3 ± 0.2Liver 15.4 ± 2.1 0.21 + 0.03 3.9 ± 0.8 0.05 ± 0.01 25.5 ± 2.8 14.6 ± 2.8 4.3 ± 0.6Spleen 18.1 ± 3.3 13.0 ± 1.7 0.1 ± 0.02 0.09 ± 0.02 18.9 ± 4.5 18.0 ± 4.5Kidney 0.8 ± 0.15 0.05 ± 0.01 5.1 ± 1.2 0.27 ± 0.02 1.0 ± 0.15 -0.7 ± 0.1 3.9 ± 0.3Muscle 2.4 ± 0.6 0.002 ± 0.001 7.5 ± 1.1 0.01 ± 0.001Urine 1.6 ± 0.6 0.04 ± 0.01 30.4 ± 5.5 0.17 ± 0.01*Without blood pool correction.tWith blood pool correction.tWith background subtraction only.

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The blood clearance profile of 99mTc-LEH also supports twophases of LEH removal, with a fast removal over the first 2hr and slower disappearance out to 20 hr. It is unclear,however, whether LEH localized in the organs as observedby image biodistribution is located intracellularly (by phago-cytosis) or is sequestered by the organ without active uptakeand metabolism by cells of the RES.The fate of the encapsulated hemoglobin over the course of

this experiment apparently tracks the distribution of theliposomes. There is no evidence of leakage of intravesicularfree hemoglobin from the aqueous compartment of LEH.There is no free hemoglobin or label (99'Tc) observed in theplasma fraction of blood-drawn capillary tubes over thecourse of the experiment. This would also be evidenced by99mTc activity in the bile (intestine), kidney, or bladder, whichwas not observed, in contrast to the considerable activity ofthese organs in animals given '9Tc-HMPAO. Urine re-moved from the bladder upon autopsy did show a smallamount of label (2%), which may be due to some dissociationof label from LEH or, alternatively, LEH metabolism. Therewas negligible 99'Tc-LEH imaged in the kidney, and nomeasurable hemoglobin was found in the urine. This is animportant finding for a hemoglobin-based oxygen-carryingfluid because of the long history of accumulation of hemo-globin in the kidney and associated nephrotoxicity. Themeasurement of LEH uptake into the RES by imagingintensity in organ regions ofinterest is somewhat complicatedby blood pool contributions. Overall, our methods of bloodpool correction have resulted in accurate estimations oforganuptake from acquired images, especially when compared totissue sampling at 20 hr.The in vivo stability of9Tc-HMPAO could also affect the

observed biodistribution. Previous reports of the stability of9Tc-HMPAO in saline show that there is rapid loss ofradiochemical purity (60%6) over the course of 2 hr (35). Thesestudies showed that the in vivo administration of 9'Tc-HMPAO resulted in rapid clearance of the label from theblood pool, with metabolism and transfer of the label to otherblood elements. Previous biodistribution studies show accu-mulation of free 1'Tc-HMPAO in the kidney, bile, andintestine (28, 35). Our results confirm the rapid clearance of9'9Tc-HMPAO from the blood pool with significant accu-mulation in the urine. In contrast, the in vivo stability of99mTc-LEH shows long retention in the blood pool (45% at 20hours) and no transfer of the label to other blood elements,as evidenced by sampling peripheral blood from animalsgiven 99mTc-LEH. This suggests that the encapsulation ofthelabel within the liposome retards the metabolism of 9'9Tc-HMPAO while LEH resides in the RES. The efficient en-capsulation of the label in LEH may involve the reduction ofthe radiolabel by the coencapsulated glutathione. This la-beled liposome system may prove to be a superior technetiumlabel for clinical imaging diagnostics.The present report offers promising results toward the

further development of this oxygen-carrying resuscitativefluid. The distribution and uptake of the dose of LEH used inthis study results in dissemination of LEH to the RES:principally the liver and spleen with minimal involvement ofthe kidney. Blood chemistries taken over the course of theexperiment indicate that there are transient increases in SGOTand SGPI and no increase in bilirubin or serum creatinine.This correlates with the observed uptake in the liver with littledistribution to the kidney. The observed half-life of '18 hrmay be related to the two rates ofLEH accumulation in organsof the RES and could indicate that the liver is saturated at thegiven dose. The significant involvement of the RES in removalof LEH also suggests that further experiments into the met-abolic fate of LEH after handling by the RES are warranted.It may also be important to consider the effect of secondaryimmune challenge after LEH infusion, repeated LEH dosing,

and the oxygen-carrying capacity ofLEH in scenarios appro-priate to its eventual application.We thank Mr. Richard Cliff for his technical assistance and Ms.

Shelley DeLozier for technical editing of this manuscript. Theauthors gratefully acknowledge financial support from the Office ofNaval Research and the Naval Medical Research and DevelopmentCommand to A.S.R. and W.T.P.

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