Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement

J

THoRAc CARDIOVASC SURG

1990;100:379-88

Hemodynamic effects and oxygen transport properties of a new blood substitute in a model of massive blood replacement Recent concerns regarding the safety of the national blood supply have rekindled interest in the development of blood substitutes. Clinical studies have dampened the initial enthusiasm for fluorocarbon solutions as blood substitutes. The potential of hemoglobin solutions as blood substitutes has continued to stimulate investigations. However, the development of an idealhemoglobin-derived blood substitute has eluded investigators for the past century. A persistent problem has been the inability to develop hemoglobin solutions that provide adequate oxygen and carbon dioxide exchange, wblle avoiding toxicity that precludes clinical safety and long-term survivaL Traditionally, investigators have focused on human hemoglobin solutions. The use of outdated banked blood or pedigree human donor blood as a hemoglobin source poses continued disease transmission risks and a prohibitively limited supply. We evaluated the hemodynamic and gas transport effects of a new purified, polymerized bovine hemoglobin preparation. Bovine hemoglobin oxygen affinity is regulated by chloride ion. The concentration of chloride ions in human plasma results in excellent oxygen transport properties in a stroma-free environment. In addition, unlike human blood, bovine blood is a more disease-free hemoglobin source that is available in large supply. We exchange-transfused eight conscious sheep with this new polymerized bovine hemoglobin solution. AU animals tolerated 2::: 95 % exchange transfusion to reach a final ovine hematocrit of 2.4% ± 0.5% with stable hemodynamics and no clinical evidence of distress. The exchange transfusion with bovine hemoglobin polymer resulted in a final plasma hemoglobin concentration of 6.1 ± 1.6 gm/dJ, which supported oxygen consumption at baseline levels. AU animals that were exchange transfused with this preparation survived long term with rapid resynthesis of ovine erythrocytes.

Gus J. Vlahakes, MD, Raymond Lee, MD, Edward E. Jacobs, Jr., MD, Paul J. LaRaia, MD, and W. Gerald Austen, MD, Boston. Mass.

Laditionally, patients undergoing cardiac, thoracic, vascular, or other major surgical procedures have frequently required blood replacement during or after the operation. Despite recent advances in blood conservation and the use of autologous blood, the need for homologous blood replacement still remains, particularly in patients in whom the need for surgery is acute. From the Departments of Surgery and Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Mass. Funded in part by the Alfred Blalock Research Scholarship of The American Association for Thoracic Surgery. Received for publication June 14, 1989. Accepted for publication Nov. 30,1989. Address for reprints: Gus J. Vlahakes, MD, Department of Surgery, Massachusetts General Hospital, Boston, MA 02114.

12/1/18510

The recent concern over the acquired immunodeficiency syndrome (AIDS) has brought the safety of our national blood supply under close scrutiny. The Centers for Disease Control has estimated that approximately 12,000 persons now living in the United States harbor transfusion-acquired human immunodeficiency virus (HIV) infection. I, 2 In attempts to improve the safety of our blood supply, measures such as donor self-deferral and HIV antibody screening have been implemented. Although these measures should decrease the disease transmission risk, the recent report of 14 cases of transfusion-associated AIDS since the implementation of HIV antibody testing' provides additional evidence that a completely safe human-derived blood supply is presently an unattainable goal. Many attempts have been made during the past century to develop a stroma-free hemoglobin blood sub379

The Journal oi Thoracic and Cardiovascular Surgery

3 8 0 Vlahakes et al.

Table I. Characteristics ofpure. polymerized bovine hemoglobin solution* Hemoglobin concentrationt Methemoglobin content Polymerized fraction

P50+

Colloid oncotic pressure§ Osmolarity Sodiumcontent Potassium content Chloride content Phospholipid contentII Endotoxin content~

II ± I gm/dl <10% 50%-60%

Approximately 20-23 torr Approximately 20 torr 250 rrrOsrn/kg 120mEq/L 4.0mEq/L 115 mEq/L I nmol/ml <0.5 EU/ml

• Kindly supplied by the Biopure Corporation, Boston, Mass. tIncludes hemoglobin + methemoglobin. :j:Pso determined by Hemox analyzer, model B, TCS Medical Products Co., Huntingdon Valley, Pa. §Oncotic pressure determined by the Weil Oncometer System 186, Instrumentation Laboratories, Lexington, Mass.

II Phospholipid determined with use of two extractions according to the method of

Bligh and Dyer (Bligh EG, Dyer WJ. Can J Biochem PhysioI1959;37:911-7) and silica gel thin-layer chromatography. 'l[Endotoxin determined by limulus amebocyte lysate chromogenic assay, validated according to the "Guideline on Validation of the Limulus Amebocyte Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices," Food and Drug Administration, December 1987.

stitute.v" In the last published clinical trial to evaluate a polymerized, stroma-free modified human hemoglobin solution, Savitsky and co-workers" demonstrated acute cardiovascular, renal, and coagulation toxicities. The problem that plagued Savitsky's preparation and all other historical attempts has been the inability to purify hemoglobin solutions on a large-scale basis, free of stromal phospholipid, endotoxin, and other contaminants. The effect of such contaminants is to produce documented renal and coagulation toxicity."!' In this study we determined the efficacy of this new blood substitute in the maintenance of hemodynamic stability and adequate oxygen and carbon dioxide transport in a conscious ovine model of near complete blood replacement. The long-term survival of this model was demonstrated after extensive blood replacement. . Materials and methods This study was conducted on 14 sheep of mixed Western breeds, weighing 20 to 25 kg (mean 24.6 ± 4.9 kg). The experimental protocol described herein was reviewed and approved by the Subcommittee on Animal Care, Massachusetts General Hospital. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 8D-23, revised in 1978). Because sheep may sequester erythrocytes in the spleen, ani-

mals underwent surgical splenectomy at least two weeks before this efficacy study to achieve stability of hematocrit after blood exchange, Sheep were fasted 24 hours before splenectomy except for access to potable water ad libitum. Anesthesia was induced by breathing 4% halothane, and after intubation anesthesia was maintained with 1.5%to 2% halothane in oxygen. By means of aseptic technique, splenectomy was performed through a left subcostal incision. Cefazolin 1 gm intramuscularly was given once on the day of operation and daily for 2 postoperative days. Meperidine 1 to 2 mg/kg intramuscularly was given every 6 to 8 hours as needed to control postoperative discomfort until animals were freely ambulatory. At least 2 weeks were allowed for recovery from splenectomy before initiation of the experimental protocol. On the day of the efficacy study, sheep were again anesthetized as described. By means of the Seldinger technique, an 8.5F vascular introducer was inserted into a jugular vein and a thermodilution cardiac output catheter was inserted and flowdirected into the pulmonary artery. With the use of aseptic technique, a branch of the superficial femoral artery was dissected out and cannulated with a 16-gauge arterial catheter. The catheter was advanced into the abdominal aorta, exteriorized, and flushed with heparinized saline solution. The animals were allowed to recover from anesthesia in a metabolic cage for 1 hour. For the subsequent study, animals were awake and breathing room air with access to food and potable water ad libitum. The data collected included standard hemodynamic monitoring of arterial, pulmonary capillary wedge, and central venous pressures. Cardiac output was determined by the thermodilution technique with cold injectate. Arterial and mixed venous blood samples were analyzed for blood gases and pH by standard electrodes. Oxygen content was determined by an oxygen-specific fuel cell (Lex-Oj-Con, Lexington Instruments, Lexington, Mass.). Hematocrit value was determined by capillary tube centrifugation. Blood urea nitrogen and creatinine determinations were made by an AutoAnalyzer (Technicon SMAC AutoAnalyzer, Smith Kline BioScience Laboratories, Cambridge, Mass.). In separate studies, it was shown that the presence of plasma hemoglobin did not interfere with these determinations. Baseline hemodynamic parameters were recorded and arterial and mixed venous blood samples were drawn for blood gas and oxygen content determinations. Additional blood samples were drawn for baseline hematology and renal function studies. Since the starting hematocrit value among animals varied, an initial hemodilution with Ringer's solution was done to reduce and to equalize the starting hematocrit value in all sheep to approximately 20%. Blood was removed via the femoral artery catheter and warm (37° C) Ringer's lactate solution was infused via the central venous sheath to replace the blood removed. The amount infused was titrated to bring hemodynamic parameters back to baseline. After .a l O-minute equilibration period, hemodynamic parameters and arterial and mixed venous blood gas and oxygen content determinations were made. Subsequently, the exchange process was repeated in eight of the 14 animals with the polymerized bovine hemoglobin solution at a concentration of 11 gm/dl. The specifications of this material are summarized in Table I. Bags containing this material were stored frozen and were prepared for use by thawing and equilibration in a 37° C water bath. Again, the amount infused was adjusted to bring

Volume 100 Number 3 September 1990

New blood substitute

38 1

A

' ......

'2'<," o Control

'2............ ;1;... ~

• Blood Substitute

. . --..'2

Q'---_---l-_---'_ _-'---_-----'-_ _.l.---_---l..-_-----l_-...l

8

Q'--_----'-_----'_ _--'-----_---L_ _L--_---L-_---'_-----'

Baseline

17-22

13-17

Ovine

10-13

~-4

nemotocnt (%)

Fig 1. Oxygen content data for blood substitute recipients (solid circles) and for hetastarch controls (open circles). Values shown are at various stages in the exchange protocol. Asterisks indicate statistically significant differences between control and blood substitute recipients within each hematocrit group (t test, P < 0.05). A, Arterial oxygen content; B, venous oxygen content

Table II. Hemodynamic parameters before (baseline), after initial exchange with crystalloid, and after final exchange with bovine hemoglobin blood substitute Baseline

Hematocrit (%) Heart rate (min-I) Mean arterial pressure (torr) Cardiacoutput (Ljmin) Pulmonary capillarywedge pressure (torr)

28.4 ± 120 ± 97 ± 4.8 ± 11.6 ±

2.3 33 14 1.1

4.8

After crystalloid exchange

18.1 ± 126 ± 81 ± 4.6 ± 9.8 ±

4.3* 35 14 0.9 3.2

After final exchange

2.4 ± 101 ± 101 ± 3.9 ± 13.6 ±

0.5* 21 13 0.6 4.5

Data shown are mean ± standard deviation; n = 8. Statistical analysis by one-way analysis of variance with repeated measures and the Newman-Keuls test: versus baseline. 'p < 0.001.

hemodynamic parameters to baseline. Hematocrit value, hemodynamic parameters, and oxygen contents were determined after a I O-minuteequilibration period after each exchange. This exchange process was repeated serially with the polymerized bovinehemoglobin preparation until the final hematocrit value was less than 4%. After completion of the final exchange, the

condition of the sheep was allowed to equilibrate for I hour. Final hemodynamic parameters, blood gas determinations, and oxygen content determinations were then performed. After this exchange transfusion protocol, anesthesia was again induced as previously described. All catheters were removed, and all catheterization sites were surgically closed by

The Journal of Thoracic and Cardiovascular Surgery

3 8 2 Vlahakes et al.

10 r - - - - - - - - - - - - - - - - - - - - - - - , o Control • Blood Substitute A

8 6

4

2

o'------'---'----_-'-__'----_--'-

L-_---'-_---'

1Or----------------------,

B 8 6

2

4-6

i-4

Ovine Hematocrit (%) Fig 2. Statistical differences indicated as in Fig. 1. A, Cardiac output during the exchange protocol for blood substitute recipients (solid circles) and hetastarch controls (open circles). Note relative constancy of cardiac output in the blood substitute recipients, compared with the initial compensatory rise and subsequent fall in the control group. B, Arteriovenous oxygen content difference (AVd02) for blood substitute recipients (solid circles) and for hetastarch controls (open circles). Asterisks indicate statistically significant differences between control and blood substitute recipients within each hematocrit group (t test, P < 0.05).

aseptic technique. After recovery from anesthesia, sheep were returned to the animal care facility, where they had continuous access to food and potable water. Venous blood for hematology and renal function studies was drawn daily for 10 days and then every fifth day until completion of the study at 25 days, by which time the hematocrit value had returned to baseline levels. Because this bovine hemoglobin preparation was cleared from the circulation before animals had sufficient time to resynthesize enough erythrocytes, an additional infusion of 450 ml of the polymerized hemoglobin solution was given on days 2 and 4 after the exchange transfusion protocol. The remaining six splenectomized sheep were studied as control animals with the use of 6% hetastarch (Hespan, DuPont Critical Care, Wilmington, Del.) during the exchange protocol. These animals were instrumented as previously described and underwent serial blood exchange with warm (37 0 C) hetastarch

solution by means of the same exchange transfusion protocol. Hemodynamic parameters, as well as arterial and mixed venous blood gases and oxygen content, were determined during progressive hemodilution. Control sheep were studied under sedation with ketamine to prevent distress to the animals. The animals were immediately killed by barbiturate overdose when early signs of tachypnea or acidosis developed. None of the control animals could be exchanged to the same degree as the blood substitute recipients because of distress that might appear with extreme hemodilution.

Data analysis Arteriovenous oxygen content difference was calculated as the difference between arterial and mixed venous oxygen contents. Oxygen consumption was calculated as the product of

Volume 100 Number 3

New bloodsubstitute 383

September 1990

-

-..... ~

~O

O 2 Content =0041 ( Hct) + 0.35

8

~

'.......

6

"'c::::" (3

4

~ ~

~ ~

A

r 2 = 0.98

2

o

5

15

iO

25

20

Ovine Hematocfl~ (%) .~

fOO

.-::: ~:---.. 'r.:: ~ 80

~~ a c:::: ~

.~ ~

60

~c:::: ~ ~

40

~G

~ ~

:t::~ It)

.S:: ~ s;:

B

20

~

Ovine Hematocrtl (%) Fig. 3. A, Regression of oxygencontent (carried by ovineerythrocytes + plasma phase) on hematocrit, generated

from progressive hemodilution in the controlsheepduring roomair breathing. B, Calculatedcontributionto oxygen contentof bovine bloodsubstitute.This iscalculatedas indicatedin the appendix, withthe useof the regression shown in A.

cardiac output and arteriovenous oxygen content difference. The oxygen extraction fraction was calculated as the ratio of arteriovenous oxygen content difference to arterial oxygencontent. In this study the total measured oxygen content of arterial blood is the sum of the oxygencarried by three differentcompartments: (1) erythrocytes (ovine hemoglobin), (2) plasma(by dissolution), and (3) bovine hemoglobin blood substitute. The bovine hemoglobin solution's contribution to the total oxygen contentwascalculatedas the difference betweenthe total measured oxygen content and the oxygencontent carried by both ovine erythrocytes and plasma. The latter was estimated from a regression analysis of arterial oxygen content against hematocritgeneratedfrom the controlsheepdata (see Fig. 3, A). Formulasfor the calculation of these oxygen transport parameters are summarized in the appendix. For the purposes of analysis and comparisons, data were

organizedintodifferentgroupsaccordingto hematocrit ranges. This was necessary because, after each successive step in the exchange transfusion protocol, the hematocrit values obtained werenot all exactlyequal acrossthe eight sheepstudied.Statistical analyseswere performedby one-way analysisof variance with repeated measures and the Newman-Keuls test and by Student's t test where appropriate. Data are presented as mean ± standard deviation.

Results The eight sheep exchange transfused with the bovine hemoglobin solution and breathing room air all survived long term (> 1 month) without clinically evident adverse reactions or distress. A mean final hematocrit value of 2.4% ± 0.5% was achieved (range of 1.5% to 4.5%) by

The Joumal of Thoracic and Cardiovascular Surgery

3 8 4 Vlahakes et al.

Table III. Oxygen transport parameters before (baseline). after initial exchange with crystalloidsolution. and after final exchange with bovine hemoglobin blood substitute Hematocrit (%) Plasma Hb (gm/dl) Arterial P02 (torr) Arterial PC02 (torr) Arterial pH Arterial 02 content (vol%) Venous O 2 content (vol%) AV02 diff. (vol%) Extraction fraction (%) O 2 consumption (ml 02/min/kg)

Baseline

After crystalloid exchange

28.4 ± 2.3

18.1 ± 4.3

o

86 36 7.45 10.7 7.1 3.0 33.7 7.2

± 4 ± 4 ± 0.03 ± 1.4 ± 1.4 ± 0.9 ± 7.8 ± 2.6

o

85 37 7.44 7.5 2.8 4.7 62.4 9.3

± 10 ± 3 ± 0.02 ± 0.8 ± 1.2 ± 1.3 ± 16.0 ± 3.7

After hemoglobin exchange 2.4 6.1 85 32 7.46 7.6 3.1 4.5 58.4 7.0

± 0.5* ± 1.6* ± 8 ± 4 ± 0.09 ± 2.1 ± 1.4 ± 1.3 ± 10.2 ± 2.6

Values shown are mean ± standard deviation. Hb, Hemoglobin; Po2, oxygen tension; PC02, carbon dioxide tension; O 2, oxygen; AV O 2 diff., arteriovenous oxygen content difference; n = 8. Statistical comparisons made by one-way analysis of variance with repeated measures and Newman-Keuls test comparing values obtained after the final exchange with hemoglobin polymer with values obtained after crystalloid exchange: • p < 0.001.

end of the exchange protocol. This required repeated exchanges, with the hemoglobin solution totaling an average of 2.0 ± 0.4 L per sheep, which represents nearly two blood volumes given the size of the sheep used in this study. In contrast, all six control sheep exchanged with 6% hetastarch exhibited initial physiologic compensatory mechanisms to progressive normovolemic anemia. With further hemodilution, however, all control animals developed cardiovascular and respiratory decompensation necessitating that they be put to death before the exchange protocol could be completed. Hemodynamic data are shown in Table II. In the group exchanged with this blood substitute, heart rate, mean arterial pressure, pulmonary capillary wedge pressure, and cardiac output all remained stable and did not change significantly throughout the experiment. The oxygen transport data are presented in Table III and in Figs. I and 2. With progressive exchange transfusion, the recipients ofthe bovine hemoglobin solution were able to support systemic oxygen needs despite the loss of ovine erythrocytes. As plasma bovine hemoglobin concentration increased from 0 gmf dlto a mean of 6.1 ± 1.6 gmfdl, oxygen consumption was supported atbaseline levels (Table III) without any increase in cardiac output (Table II and Fig. 2, A). This final value for plasma hemoglobin concentration resulted in a decrease in the final arterial oxygen content (7.6 ± 2.1 vol%) compared with baseline value (10.7 ± 1.4 vol%). Because the intravascular half-life of pure, polymerized bovine hemoglobin is approximately 48 hours, additional 450 ml intravenous infusions of blood substitute were given on days 2 and 4 after exchange to support the animal until the ovine hematocrit value was sufficient to support life. 'The initial hemodilution with crystalloid solution decreased arterial oxygen content from a mean of

10.7 ± 1.4 vol% to 7.5 ± 0.8 vol%. As shown in Fig. 1, A, with subsequent exchanges performed with bovine hemoglobin solution, arterial oxygen content was maintained close to this latter value while animals were breathing room air. With relatively constant cardiac output (Fig. 2, A), oxygen consumption was maintained by a relatively constant arteriovenous oxygen content difference (Fig. 2, B). The extraction fraction increased from 33.7% ± 7.8% in the baseline state to 62.4% ± 16% after crystalloid exchange. The extraction fraction decreased slightly to 58.4% ± 10.2% after the final exchange with hemoglobin solution. Venous oxygen content decreased from 7.1 ± 1.4 vol% in the baseline state 1!J 2.8 ± 1.2 vol% after the initial hemodilution with crystalloid. Subsequently, after exchange with the hemoglobin solution, venous oxygen content increased slightly to 3.1 ± 1.4 vol% (Fig. 1, B) and was higher than in sheep exchanged with hetastarch. In contrast, in control animals with progressive hemodilution to the level achieved, arterialoxygen content decreased to approximately 30% of the starting value (Fig. 1, A). As the arterial oxygen content decreased from a baseline of 7.8 ± 1.2 vol% to the nadir achieved at 2.4 ± 0.4 vol%, the control animals extracted an increasing percentage of the arterial oxygen content until very little oxygen remained in the venous blood. At a hematocrit range of 6.0% to 8.5%,0.6 ± 0.2vol%oxygen content remained in the venous blood, as determined just before the animal was put to death. Although the extraction fraction progressively increased to the final peak achieved at 87.0% ± 7.2%, the arteriovenous oxygen content difference decreased beyond the level needed to support oxygen consumption, even with an increase in cardiac output (Fig. 2); thus, decompensation occurred. With the use of the oxygen content data obtained from control animals, the contribution of plasma and ovine

Volume 100 Number 3 September 1990

New bloodsubstitute 385

-..

A

30

~

'-

......

i:::

'-l

~ t3

20

~ :::t Cl)

.~ ::=.;

iO

C)

O ' - - - - - - ' - - - - - L - - - J . - - - - - J . -_ _--J.-_ _. L - _ - - - - l

B

10

20

30

Time Following Exchange Protocol (days) Fig. 4. A, Recovery of hematocrit in the days after exchange with bovine hemoglobin polymer. Note that additional blood substitute was administered on days 2 and 4. B, Reticulocytosis after exchange with blood substitute.

erythrocytes to oxygen content can beestimated from the regression of oxygen content on hematocrit (Fig. 3, A, and appendix). By extrapolation to the vertical axis of the regression shown in Fig. 3, A, the amount of oxygen carried by dissolution in plasma can be estimated. In these animals breathing room air, the plasma phase carried approximately 0.35 vol% of oxygen. These data can be used to calculate the contribution of bovine hemoglobin to total arterial oxygen content. This was calculated at various stages of exchange with bovine hemoglobin, and data are shown in Fig. 3, B. At the mean final ovine hematocrit value of 2.4%±0.5%, the bovine hemoglobin blood substitute contributed 81.6% ± 4.2% of the total oxygen content. In addition to transporting and releasing oxygen, this study demonstrates that this bovine hemoglobin prepara-

tion transports carbon dioxide. In recipients of this blood substitute, arterial carbon dioxide tension (PC02) and pH remained normal throughout the experiment (Table III). The postexchange follow-up data are shown in Fig. 4. All sheep that received the bovine hemoglobin polymer tolerated the mean final ovine hematocrit value of 2.4% ± 0.5% extremely well with no adverse reactions or clinical signs of distress, even at the lowest hematocrit value achieved (1.5%). All of these animals exhibited a brisk reticulocytosis during the first postexchange week, with a mean peak reticulocyte count on day 7 of 22.5% ± 4.3% (Fig. 4, B). Consequently by day 10 the average hematocrit value had increased to 20.0% ± 6.0% and by day 25 all animals returned to baseline levels,with anaveragehematocritvalueof27.3% ± 3.2%(Fig.4,A). Fig. 5 demonstrates the daily blood urea nitrogen

386

The Journal of Thoracic and Cardiovascular

Vlahakes et al.

Surgery

,,

Additional Infusions

A

*

o

L--_----'-_ _.l...--_ _- - ' -_ _- ' - - - _ - - ' -_ _...L-_--'

,,

B

Additional Infusions

*

10

20

30

Time Following Exchange Protocol (days) Fig 5. Serial determinations of renal function after exchange with bovine blood substitute. Asterisks indicate statistical significance when values are compared with baseline (one-way analysis of variance with repeated measures . and the Newman-Keuls test, p < 0.01). A, Blood urea nitrogen; B, creatinine.

(BUN) and serum creatinine data. Both panels demonstrate transient increases (P < 0.01) in both BUN and creatinine by day 2. BUN increased from a baseline of 26.9 ± 4.2 mg/dl to a peakof 35.8 ± 4.5 mg/dl on day 2. Similarly, serum creatinine increased from a baseline of0.9 ± 0.1mg/dlto a smallpeakat 1.1 ± 0.1mg/dl on day 2. BUN and creatinine returnedto baseline levels by the next day. BUN and creatinine remained at baseline levels for the remainderof the study,despite the infusion of additional blood substituteon days 2 and 4. Discussion .The resultsof this study clearlydemonstrate that this ultrapure,polymerized bovine hemoglobin preparation is extremely efficacious in oxygen transport,oxygen release,

carbondioxide transport, and blood volume maintenance. The exchange withthisoxygen-carrying blood substitute allowed at least 95% of the circulating ovine erythrocytes to be removed while maintaining the animals' systemic oxygen utilization at or above baseline levels. In the presenceof a final mean plasma bovine hemoglobin concentration of 6.1 ± 1.6 gm/dl, all animals survived while awake' and breathingroom air. The mean ovine hematocrit value measured at that time was 2.4% ± 0.5%, which byitselfisinsufficient tosupportlife. Indeed, inthis study, none of the controlanimals survived to reach this level of ovine hematocrit. In addition to supporting oxygendynamics, thispolymerized bovine hemoglobin preparation effectively supported circulating blood volume, thus maintaining hemodynamics at baseline levels.

Volume 100 Number 3 September 1990

All bovine hemoglobin recipients survived long term and exhibited no evidence of distress even at an ovine hematocrit level as low as 1.5%. There was no clinical evidence of any acute allergic reactions or any clinicalor laboratoryevidence of severerenal toxicity.A brisk reticulocytosis was observedby postexchange day 3. Starting with the mean final ovine hematocrit value of 2.4% ± 0.5%,the hematocrit value of all animals returned to 20.0% ± 6.0% by postexchange day 10, thus permitting long-termsurvivalwithout further replacement therapy. All sheep that had exchange transfusion with blood substitute are long-term survivors (> 1 month). In this study we examined a new, highly purifiedpolymerized bovine hemoglobin preparation. There are several unique and specialreasonsto use bovinehemoglobin. Bovine bloodisa more disease-free sourceof hemoglobin, and it also avoids the supply constraints inherent with human hemoglobin-derived preparations. Bovine hemoglobin has the additional physiologic advantage of not requiring 2,3-diphosphoglycerate (2,3-DPG) to lower its oxygen affinity. Human hemoglobinmust be modifiedby pyridoxylation to allow it to release oxygen in a stromafree, and hence 2,3-DPG-free, environment. This increases the risk of contamination during chemical preparation. In contrast, bovinehemoglobin does not require modification since it utilizes chloride ion to lower its oxygenaffinity.l? Fortuitously,the chlorideionconcentration of human plasma is adequate to decrease the oxygen affinity of bovine hemoglobin to a satisfactory level (Pso = 28 torr before polymerization).13 Becauseof these factors this polymerized bovine hemoglobin preparation may be a very promising blood substitute material. Interest in free hemoglobin solutions as blood substitutes is over 100 years 01d. 14 Many prior studies with stroma-freehemoglobin preparations have demonstrated significant toxicity. Traditionally, stroma-free modified hemoglobin has been prepared with techniques that utilizeacid precipitation,centrifugation, and ultrafiltration. Despite these techniques, the major obstacle that has plaguedthese preparations has been the inability to purify hemoglobin free of stromal phospholipid, endotoxin, and other contaminants responsible for the observedtoxicity.In this study we have introduced an ultrapure, polymerized bovine hemoglobin preparation that appears to have overcome many of these past obstacles. A unique purification process involving ultrafiltration, combined with large-volume, high-performance absorption and ion exchangechromatography, produces a hemoglobinpreparation with the purity specifications outlined in Table I. The purity specifications achieved with this preparation exceed those achieved by previous investigators.'> The oxygen transport properties of this bovine hemoglobinpreparation are similar to those of the native ovine

New blood substitute

387

hemoglobin. Table III demonstrates that, at a mean baseline hematocrit of 28.4%,a mean oxygen content of. 10.7 vol% was obtained. This corresponds to approximately 1.1 vol% oxygen carried per gram of native ovine hemoglobin. By means of the data in Table III and the regressionshown in Fig. 3, A, this parameter can be calculated for bovine hemoglobin in this model. At a mean final ovine hematocrit of 2.4%, there is 1.3 vol%oxygen carried by the remaining ovine erythrocytes. Given that the total arterial oxygen content after hemoglobin exchange was 7.6 vol%, this leaves 6.3 vo1% oxygen carried by the bovinehemoglobinin the plasma phase. This correspondsto 1.0vol% oxygencarried per gram of bovine hemoglobin, which is very similar to the oxygen-carrying capacity estimated for the native ovineerythrocyte hemoglobin. Thus the oxygen transport characterisitic of this purifiedbovinehemoglobinsolution is nearly identical to that of the native ovine hemoglobin. Furthermore, the data suggest that even with a P so value of approximately 20 torr, release of oxygen at the tissue levelis still within physiologic limits. This is demonstrated by the fact that a mean oxygenextraction fraction of 58.4%was achieved and that systemicoxygen consumption was supported at baselinelevels (Table III) with normal acid-base parameters. Another essentialaspectof an effectivebloodsubstitute is the transport of carbon dioxide. The Pco, and pH remained within normal limits throughout the study. This indicatesthat the purificationand polymerizationprocess did not interfere with the capacity of the hemoglobin solution to participate in the transport of carbon dioxide. Historically, renal toxicity has been a major impediment to the use of hemoglobinblood substitutes." 16 In a recent study from our laboratory, 17 we have administered this hemoglobinpreparation at doses far exceeding those used by any prior investigators and did not observe any significant irreversible renal toxicity. Although the present study was not designed to examine nephrotoxicity per se, we did make serial determinations of BUN and creatinine. The exchange protocol resulted in the administration of a large amount of hemoglobin,amounting to an average of 220 gm per sheep (8.9 gmjkg). This is in excessof ten times that utilizedin prior hemoglobinblood substitute nephrotoxicity studies. Yet there was only a small, transient rise in serum creatinine and BUN on day 2. Despite the administration of additional blood substitute on days 2 and 4, the BUN and creatinine levels remained normal. Thus no clinically significant nephrotoxicity was observed in this study. In addition to its Use in situations of traditional blood replacement, this pure, polymerized bovine hemoglobin preparation may be used in new modes of therapy. Without the requirement of typing and crossmatching, this

388

The Journal of Thoracic and Cardiovascular Surgery

Vlahakes et al.

blood substitute could be infused immediately at the scene of trauma. By replacing multiple units of whole blood with such an oxygen carrier, participation in preoperative autologous blood donation may be extended, and the number of units donated may be further increased. This purified polymerized bovine hemoglobin preparation provides an exciting alternative to standard homologous blood transfusion. We thank Mr. J. Luis Guererro and Ms. Tracy Svizzero for their technical assistance and Ms. Jane McDermott for assistance in preparation of this manuscript. The purified, polymerized bovine hemoglobin polymer was kindly supplied by the Biopure Corporation, 68 Harrison Ave., Boston, MA 02111. REFERENCES I. Human immunodeficiency virus infection in transfusion recipients and their family members. MMWR 1987; 36:137-40. 2. Peterman TA, Lui K-J, Lawrence DN, Allen JR. Estimating the risks of transfusion-associated acquired immune deficiency syndrome and. human immunodeficiency virus infection. Transfusion 1987;27:371-4. 3. Ward JW, Holmberg SD, AllenJR, et al. Transmission of human immunodeficiency virus (HIV) by blood transfusions screened as negative for HIV antibody. N Engl J Med 1988;318:473-8. 4. Gould SA, Rosen AL, Sehgal LR, Sehgal HL, Moss GS. Is polyhemoglobin an effective O 2 carrier? J Trauma 1986; 26:903-8. 5. Friedman HI, Devenuto F, Schartz BD, Nemeth TJ. In vivo evaluation of pyridoxylated-polymerized hemoglobin solution. Surg Gynecol Obstet 1984;159:429-35. 6. Hobbhahn J, Voogel H, Kothe N, Brendel W, Peter K, Jesch F. Hemodynamics and oxygen transport after partial and total blood exchange with pyridoxylated polyhemoglobin in dogs. Acta Anaesthesiol Scand 1985;29:537-43. 7. Savitsky JP, Doczi J, Black J, Arnold JD. A clinical safety trial of stroma-free hemoglobin. Clin Pharmacol Ther 1978;23:73-80. 8. Rabiner SF, Hebert JR, Lopas H, Friedman LH. Evaluation of a stroma-free hemoglobin solution for. use as a plasma expander. J Exp Med 1967;126:1127-42. .

9. Cochin A, Das Gupta TK, DeWoskin R, Moss GS. Immunogenic properties of stroma vs. stroma-free hemoglobin solution. J Surg OncoI1974;4:19-26. 10. Birndorf NI, Lopas H. Effects of red cell stroma-free hemoglobin solution on renal function in monkeys. J Appl Physiol 1970;29:573-8. II. Moss GS, DeWoskin R, Cochin A. Stroma-free hemoglobin. I. Preparation and observations on in vitro changes in coagulation. Surgery 1973;74:198-203. 12. Benesch RE, Benesch R, Renthal RD, Maeda N. Affinity labelling of the polyphosphate binding site of hemoglobin. Biochemistry 1972;11:3576-82. 13. Fronticelli C, Bucci E, Orth C. Solvent regulation of oxygen affinity in hemoglobin. J Bioi Chem 1984;259: 10841-4. 14. Amberson WR, Jennings JJ, Rhode CM. Clinical experience with hemoglobin saline solutions. J Appl Physiol 1949;1:469-89. 15. Sehgal LR, Rosen AL, N oud G, et al. Large-volume preparation ofpyridoxylated hemoglobin with high P so. J Surg Res 1981;30:14-20. 16. Jaenike JR. The renal lesion associated with hemoglobinemia. I. Its production and functional evolution in the rat. J Exp Med 1966;123:523-35. 17. Lee R, Atsumi N, Jacobs EE, Austen WG, Vlahakes GJ. Ultra-pure, stroma-free, polymerized bovine hemoglobin solution:evaluation of renal toxicity. J Surg Res 1989;47: 407-11.

Appendix Arteriovenous oxygen content difference (vol,%) = (arterial oxygen content) - (venous oxygen content). . Oxygen consumption (rnl/min/kg) = (cardiac output) X (arteriovenous oxygen content difference). Oxygen extraction fraction (%) = ([arteriovenous oxygen content difference] + [arterial oxygen content]) X 100. Oxygen content carried by both ovine erythrocytes and plasma (vol%) = 0.41 X (hematocrit) + 0.35 (r 2 = 0.98) (derived from control animals; see Fig. 3, A). Oxygen content carried by bovine hemoglobin polymer (vol%) = (total oxygen content) - (ovine erythrocyte and plasma oxygen content). Bovine hemoglobin contribution to oxygen content (%) = ([bovine hemoglobin oxygen content] + [total arterial oxygen content]) X 100.