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©2002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY

Vol. 31, No. 1, pp. 1–17, 2003

The Intravascular Persistence andMethemoglobin Formation of Hemolinkk

(Hemoglobin Raffimer) in Dogs

David Wicks,1 Lawrence T. Wong,1 Reena Sandhu,1,#

Richard K. Stewart,2,z and George P. Biro1,*

1Hemosol Inc., Mississauga, Ontario, Canada2ITR Laboratories Canada, Inc., Baie d’Urfe, Quebec, Canada

ABSTRACT

Hemoglobin raffimer (HEMOLINKk, Hemosol Inc, Mississauga,

Canada) is an o-raffinose cross-linked, purified human hemoglobin-

based oxygen therapeutic that is currently being evaluated in late stage

clinical trials. It is composed of several molecular weight (MW) species,

comprising principally of stabilized tetramers (34–42%) and oligomers

(54–62%). The objective of this study was to determine the in vivo

circulating half-life (T1/2) of hemoglobin raffimer (Hb raffimer) and of

its individual MW components in dogs subjected to a topload infusion of

25% of the estimated blood volume (18 mL/kg). Sampling was done over

a 64-hour period that was expected to be equivalent to approximately

two-and-half to three half-lives. Methemoglobin (MetHb) levels were

also measured at intervals over the same period. The mean circulating

#Current address: Apotex Research Inc., Toronto, ON, Canada.zCurrent address: Cato Research Canada, St. Laurent, Quebec, Canada.*Correspondence: George P. Biro, Hemosol Inc., 2585 Meadowpine Blvd.,

Mississauga, Ontario, L5N 8H9 Canada; E-mail: [email protected].

1

DOI: 10.1081/BIO-120018000 1073-1199 (Print); 1532-4184 (Online)

Copyright D 2003 by Marcel Dekker, Inc. www.dekker.com



half-life of Hb raffimer was 25.4 ± 3.9 hours. The T1/2 for the individual

MW components (determined by size exclusion chromatography) of Hb

raffimer was 11 ± 2 hours for the cross-linked tetramer and 35 ± 7 hours

for the fraction of oligomers. The apparent volume of distribution for Hb

raffimer was estimated at 78 mL/kg. There was no difference in the

apparent volumes of distribution of the tetrameric and oligomeric com-

ponents of Hb raffimer. Throughout the course of the experiment (in

which MetHb could be measured), plasma MetHb concentration, ex-

pressed as a percentage of the total plasma hemoglobin concentration,

remained at 10% or less, and the mass concentration of MetHb in plasma

remained at about 1 g/L. Thus, in the dog subjected to an estimated 25%

topload infusion, the T1/2 of the infused Hb raffimer is approximately

one day with the intravascular retention of the individual Hb raffimer

components being dependent on the MW. Furthermore, oxidation of Hb

raffimer to MetHb is limited under these conditions.

Key Words: Red cell substitute; Oxygen therapeutic; Hemoglobin

raffimer; Hemoglobin solution; Modified hemoglobin solution;

Intravascular persistence; Half-life; Methemoglobin formation; Heme

iron oxidation; Plasma hemoglobin.

INTRODUCTION

Cell-free preparations of hemoglobin of human or bovine origin are

being developed to replace allogeneic red cell transfusions in various cli-

nical indications (Cohn, 2002). To make these preparations clinically use-

able, extensive purification and chemical cross-linking procedures have

been developed, to remove red cell stromal fragments which have toxic

properties, and to enlarge the molecules to improve their intravascular

retention. When large amounts of free hemoglobin are present in the

plasma, it spontaneously dissociates from its tetrameric state to alpha-beta

dimers (MW 32 KDa) which are rapidly excreted. Covalent cross-linking

stabilizes the molecule in the tetrameric or oligomeric form, thereby

enlarging the molecule and rendering it less likely to be filtered, thus pro-

longing its intravascular retention and physiological efficacy as an oxygen

transporter, or an ‘‘oxygen therapeutic’’. Other modifications are also re-

quired to offset the increase in oxygen affinity.

The high oxygen environment to which the hemoglobin in the red cell

is exposed favours the oxidation of the heme iron to the ferric form, re-

sulting in the formation of methemoglobin (MetHb) (Brooks, 1935). In this

form, the pigment is not able to bind oxygen reversibly and is, therefore,

physiologically inactive. An important mechanism preventing this loss of

2 Wicks et al.



functionality is the MetHb reductase activity within the red cell (Scott

et al., 1965), which, with the generation of NADPH through the hexose

monophosphate shunt, reduces the heme iron to the ferrous state, restoring

its functionality. When the hemoglobin is liberated from the protective

environment of the red cell, it is no longer protected from oxidation by this

mechanism. Hence when administered in vivo, the maintenance of the

physiological efficacy and functionality of the oxygen therapeutics requires

that MetHb formation be minimal.

Hemoglobin raffimer (HEMOLINKk, Hemosol, Inc, Mississauga,

Ontario, Canada) is a cell-free human hemoglobin solution under devel-

opment as an oxygen therapeutic. It is manufactured from outdated human

red cell units collected and approved for transfusion, by a method that

involves lysis, pasteurization, chromatographic purification, filtration and

cross-linking by o-raffinose, which is derived from a naturally occurring

trisaccharide. The cross-linking occurs both within the hemoglobin mole-

cule to stabilize it in the tetrameric form, as well as inter-molecularly,

generating oligomers. Hemoglobin raffimer (Hb raffimer) is prepared from

purified hemoglobin which contains > 99% hemoglobin A0. After cross-

linking, Hb raffimer is comprised of approximately 34–42% cross-linked

tetramer (MW 64 kDa) and 54–62 % oligomer (MW 64–500 kDa). The

intravascular persistence, volume of distribution and rate of heme oxidation

in vivo were estimated in an experiment on beagle dogs given Hb raffimer

by intravenous infusion.

METHODS

The experiments were performed by ITR Laboratories, Montreal

Quebec a Clinical Research Organization in compliance with Good La-

boratory Practices. Beagle dogs were used, after approval by an Animal

Care Committee, and the animals were treated in all respects in compliance

with the Guidelines of the Canadian Council on Animal Care. The dogs

(8.2–12.7 kg body weight) were housed individually in a controlled en-

vironment and fed a standard certified commercial dog chow and given

water ad libitum. A clinical veterinarian carried out a detailed physical

examination and blood analysis to ensure good health before dogs were

included in the study.

Characteristics of the Hb Raffimer Solution

Hb raffimer is supplied at a concentration of 10 g/dL and is dissolved

in lactated Ringer’s solution. The Hb raffimer supplied for this study was

Hemolinkk in Dogs 3



packaged in the de-oxy form and can be stored refrigerated, and is in use

for the current clinical trials. Its physicochemical characteristics include:

pH 7.5, kinematic viscosity 1.11 cSt, colloid osmotic pressure 30 mmHg,

MetHb content 8.6% and endotoxin < 0.06 EU/mL; these are within ac-

ceptable limits for release.

Experimental Procedures

After three days of acclimation to the experimental environment and

with the dogs standing in a Pavlov sling, the experiment proper com-

menced. An indwelling catheter was inserted in the cephalic vein and Hb

raffimer, warmed to room temperature (approx. 25�C), was infused by a

pump at a rate of 10 mL/min, to a total dose of 18 mL/kg (approx. 25% of

the estimated blood volume). Blood samples were collected from the ju-

gular vein immediately prior to the start of the infusion, and at 0.25, 0.5,

0.75, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 44, 52, 60 and 64 hours after the

end of the infusion. These times were chosen in the anticipation that the

intravascular half-life of Hb raffimer would be about 24 hours, and would

thus provide at least three time points in each of three consecutive half-

lives, as well as sufficient additional time points during the early ‘‘post-

mixing’’ period.

Blood Sampling and Analytical Procedures

At each blood collection time point, approximately 1 mL of blood

was drawn into each of three tubes (one containing lithium and heparin,

and two containing EDTA). The tube containing the heparin was used to

determine MetHb concentration in plasma and total hemoglobin concen-

tration in whole blood. The first EDTA tube was used to determine

plasma hemoglobin concentration and the second EDTA tube was frozen

and sent to Hemosol Inc. for analysis of MW distribution by size ex-

clusion chromatography (S.E.C.). An additional 10 mL sample of ‘‘blank’’

canine plasma and whole blood from a spare dog was also obtained

and used by Hemosol, Inc as blank canine hemoglobin in chromatogra-

phic analysis.

Following the infusion urine was collected over 12-hour periods from

the cage pans and samples were frozen for the subsequent determination of

hemoglobin concentration.

Total hemoglobin and MetHb concentrations in samples of whole blood

and separated plasma were determined with an OSM3 Hemoximeter.

Samples were retained frozen for subsequent manual analysis (Evelyn-

4 Wicks et al.



Malloy method) if total hemoglobin concentration in the plasma was < 10

g/L, at which concentration the automated instrument loses accuracy. Whole

blood and plasma MetHb concentrations were not reported when plasma Hb

concentration was < 10 g/L, as the measurement is not accurate below this

level. Hemoglobin concentration in urine was determined by a Monarch 2000

automated biochemistry analyzer.

Size Exclusion Chromatography

Plasma samples were analyzed by size exclusion chromatography under

conditions that dissociate all non-covalently cross-linked hemoglobin into

ab dimers (Guidiotti, 1967). Twenty mL aliquots of the thawed plasma

samples were diluted with Milli-Q water (to a 300 mL final volume), sealed

and briefly vortexed. The plasma samples were analyzed using a Beckman

System Gold HPLC system. Samples (100 mL) were injected onto a

Superdex1 200 Column (Pharmacia) and eluted with 0.5 M MgCl2 in 25

mM Tris-HCl, pH 7.2 at a flow rate of 0.4 mL/min. Needle washes

occurred between all samples to avoid cross-contamination. Eluant was

monitored at 280 and 414 nm to assist in the identification of hemoglobin

(absorbance at 414 > 280) versus non-heme (absorbance at 280 > 414)

proteins. Additionally, absorbance surface data from 220–600 nm were

collected on the first replicate of each specific sample throughout the

chromatographic run.

Pharmacokinetic analysis was performed using WinNonlin software

(Pharsight Corp. Mountainview, California) for non-compartmental ana-

lysis for constant intravenous infusion, yielding circulating half-life for

hemoglobin by the least squares method. Additional pharmacokinetic

analysis for the individual MW components of Hb raffimer, as determined

by size exclusion chromatography (see above), was performed using Grafit

software (Erithacus Software, U.K.). The volume of distribution for total

Hb raffimer as well as the tetrameric (64 kDa) and oligomeric ( > 64 kDa)

fraction were calculated using the concentration of Hb raffimer injected

and the plasma concentration of total hemoglobin, the 64 kDa fraction and

the > 64 kDa fractions, respectively.

Reverse Phase Chromatography

Small but variable amounts of dimeric (32 kDa) hemoglobin were

detected at each time point after infusion. Reverse phase chromatographic

Hemolinkk in Dogs 5



analysis was performed to identify whether the dimeric hemoglobin was of

human or canine origin since canine hemoglobin, originating from small

amounts of lysed canine red cells, could have potentially contaminated the

samples, whereas dimeric hemoglobin derived from Hb raffimer would be

identifiable as human. The post-infusion sample with the highest measured

concentrations of 32kDa ‘‘dissociable’’ hemoglobin was fractionated by

size exclusion chromatography on a Superdex1 75 column (Pharmacia) to

isolate the 32kDa Hb fraction. The post-infusion fraction was then ex-

haustively dialyzed (3� ) against Milli-Q water in ‘‘Slide-a-Lyzer1’’

dialysis cassettes (0.5–3 mL capacity) having a molecular weight cut-off

(MWCO) of 10,000 and subsequently concentrated using Centriconk 10

concentrators to reduce the total volume to 200 mL. To serve as a true

control for the reverse phase chromatography, a canine hemoglobin solu-

tion, free of cellular fragments, was prepared from a blood sample obtained

from a control beagle dog (i.e. a dog not administered Hb raffimer). In

addition to this control canine hemoglobin sample, additional control sam-

ples were also run consisting of the 32 kDa fraction of Hb raffimer as well

as a sample of purified human HbA0. All samples were diluted to a total

final concentration of 0.08% with Milli-Q water and run on reverse phase

using neat 20 mL injections. Reverse phase chromatography was performed,

using a Beckman System Gold HPLC system. Chromatography conditions

followed several linear gradients of varying acetonitrile:water ratios (from

39.2% to 52% acetonitrile) at a constant 0.1% trifluoroacetic (TFA) acid

concentration. These conditions were chosen as they provide baseline re-

solution between the a and b globin chains of normal human hemoglobin.

A 0.46� 025 cm Vydac C4 column (The Separations Group, Heperia, Ca-

lifornia) was used, on the HPLC. Flow rate was 0.9 mL/min and eluant was

monitored at 220 nm and 414 nm.

RESULTS

Three dogs were given Hb raffimer infusion. All tolerated the

procedures well, were in good clinical condition throughout the duration

of the experiment and were returned to the animal colony on completion of

the study.

The time course of the disappearance of hemoglobin from plasma is

shown in Figure 1. The intravascular half-life of Hb raffimer as expressed

by the total hemoglobin concentration in the plasma phase was 25.4 ± 3.9

(SD) hours. The WinNonlin software also provided the parameters

6 Wicks et al.



60483624120

28

26

24

22

20

18

16

14

12

10

8

6

4

2

0

Time (hrs)

Pla

sma

TH

b (g

/L)

Mean (of 3)

Mean MetHb (of 3)

T1/2 = 25.4+ 3.9 hr

Figure 1. Plasma clearance and half-life (T1/2) of total Hb raffimer (THb) ad-

ministered to dogs subjected to a single topload infusion of 18 mL/kg. Total hemog-

lobin values are shown by the filled circles (.). Mean plasma methemoglobin

(MetHb) levels are given by the (*) symbol. All values are presented as mean ± SD.

Hemolinkk in Dogs 7



Cmax and Tmax, but these are of little value in this case, since the initial

samples were drawn 15 minutes after the infusion was completed, prior to

which time the maximum concentration would have already been reached.

Hence, these parameters are not reported.

Size exclusion chromatography identified and quantified the various

molecular weight species. This yielded intravascular half-lives for the

various molecular weight fractions which is summarized in Figure 2. The

relative percentage of the various hemoglobin moieties in the plasma over

time is summarized in Figure 3. Hemoglobin dimers were present in small

but variable amounts. However, as shown in Figure 4, it was determined by

reverse phase chromatography that the dimers were consistent with those of

canine, not human hemoglobin.

From the maximal concentration of hemoglobin measured in plasma at

15-min post-infusion (23 ± 1.7 g/L), the apparent volume of distribution was

estimated at 78 mL/kg. This is approximately 24% larger than the

‘‘expected’’ plasma volume, taking into account the enlargement of the

plasma volume by the 18 mL/kg topload infusion. The apparent volumes of

distribution of the 64-kDa and > 64-kDa fractions of Hb raffimer were

estimated at 76.6 and 74.6 mL/kg. Hence there appears to be no difference

in the apparent volumes of distribution of the tetrameric and oligomeric

components of Hb raffimer.

The timecourse of MetHb found in plasma is shown in Figure 5.

During the course of the experiment, representing approximately 2.5 half-

lives, plasma hemoglobin declined from 23 ± 1.7 g/L to approximately 3 g/

L, while total whole blood hemoglobin concentration increased from

120 ± 3.1 g/L at 15 min to 132 ± 5.7 g/L at 64 hours. Relative plasma

MetHb concentration during the experiment increased from 6.2 ± 0.3 to

10.4 ± 0.2 %, near the end of measurement at 32 hours. The apparent rate of

appearance was approximately 0.13 %/hr, when total hemoglobin

concentration in plasma declined nearly 2.5 fold over the same time

interval. As a result, the calculated MetHb mass-concentration in plasma

remained essentially stable, declining from 1.4 to 1.04 g/L. Expressed as a

percentage of the total hemoglobin mass-concentration in whole blood

(Figure 6), MetHb represented 1.1% of the total at the beginning of the

experiment and 0.8% at the end. Throughout the course of the entire

experiment whole blood MetHb concentration was about 1 % or less.

Hemoglobin determinations in urine excreted over 12-hour periods

showed that the concentration was very low (Table 1). Since accurate

determinations of the volume excreted during this period could not be

made, the amount of hemoglobin, either as absolute mass, or the fraction of

the injected dose, could not be estimated.

8 Wicks et al.



60483624120

12

8

4

0

64kD

a

60483624120

12

8

4

0

Time (hours)

>12

8kD

a

60483624120

12

8

4

0

128k

Da

42 + 7

25 + 5

11+ 2

T1/2 (hrs)P

lasm

a H

b F

ract

ion

Figure 2. Plasma clearance and half-lives (T1/2) of the different hemoglobin

fractions that make up Hb raffimer as determined by size exclusion chromatography

under dissociating conditions. The disappearance of the 64 kDa fraction (tetramer) is

shown in the top panel, the 128 kDa fraction in middle panel, and the fraction

composed of all molecular weight species > 128 kDa in the bottom panel. All values

are shown as mean ± SD for all animals.

Hemolinkk in Dogs 9



Figure 3. The relative percentage of the different hemoglobin fractions that make up

Hb raffimer in the plasma over time. Grey bars indicate the 32 kDa fraction, hatched

bars indicate the 64 kDa fraction and open bars indicate > 64 kDa fraction.

10 Wicks et al.



605040302010

Time (minutes)

Abs

orba

nce

(220

nm)

Heme BeagleMixedGlobins

HumanNativeβ Globin

HumanNativeα Globin

32kDa Fraction obtained from dog 52 hrspost-treatment (scale expanded x 2)

32D-HML(32kDa Hemolink™ Fraction)

Human HbA0 Control

Beagle Hb Control

Figure 4. Reverse phase chromatogram showing absorbance at 220 nM. Chro-

matograms presented are from a control beagle dog, human hemoglobin A0, the 32

kDa fraction of Hb raffimer and a 32 kDa fraction collected from a dog 52 hours after

treatment with Hb raffimer. For this sample the scale is expanded 2�.

Hemolinkk in Dogs 11



60483624120

15

12

9

6

3

0

24

20

16

12

8

4

0

Pla

sma

Met

hem

oglo

bin

(%)

Pla

sma

TH

b (g

/L)

60483624120

3

2.5

2

1.5

1

0.5

0

24

20

16

12

8

4

0

Time (hrs)

Pla

sma

Met

hem

oglo

bin

Mas

s (g

/L)

Pla

sma

TH

b (g

/L)

Mean Plasma metHb Mass (of 3)

Mean THb (of 3)

Mean Plasma %metHb (of 3)

Mean Plasma THb (of 3)

Figure 5. Plasma MetHb levels (.), expressed as a percentage of the total plasma

hemoglobin concentration (top panel) and also presented as the plasma mass

concentration in g/L (bottom panel). Levels are not reported beyond 32 hrs as

hemoglobin concentration has fallen below 10 g/L and measurement of MetHb is not

accurate at such low levels of total plasma hemoglobin. For comparison, total plasma

hemoglobin concentration (6), expressed in g/L is also shown on the right Y-axis. All

values presented are given as mean ± SD.

12 Wicks et al.



60483624120

2

1.5

1

0.5

0

170

160

150

140

130

120

110

100

Who

le B

lood

Met

hem

oglo

bin

(%)

Who

le B

lood

TH

b (g

/L)

Mean WB %metHb (of 3)

Mean WB THb (of 3)

60483624120

2

1.5

1

0.5

0

170

160

150

140

130

120

110

100

Time (hrs)

Who

le B

lood

Met

hem

oglo

bin

Mas

s (g

/L)

Who

le B

lood

TH

b (g

/L)

Mean WB metHb Mass (of 3)

Mean WB THb (of 3)

Figure 6. Whole blood methemoglobin levels (.), expressed as a percentage of the

total whole blood hemoglobin concentration (top panel) and also presented as the

whole blood mass concentration in g/L (bottom panel). For comparison, total whole

blood hemoglobin concentration (6), expressed in g/L is also shown on the right

Y-axis. All values presented are given as mean ± SD.




DISCUSSION

The results of this study demonstrate that Hb raffimer disappears from

plasma with a single-exponential decay, both for Hb raffimer overall as

well as for its constituent molecular weight components. Hb raffimer has

an overall intravascular half-life in the dog in the order of 25 hours, and

that the larger molecular weight oligomeric fraction has the longest persis-

tence. This dependence of disappearance rate on molecular weight is con-

sistent with the findings of the Phase I study conducted in healthy human

volunteers receiving Hb raffimer (Carmichael et al., 2000), in whom the

oligomers were found to have a longer plasma retention time than the

tetramers at each dose studied. Whereas unmodified stroma free hemog-

lobin solution is known to disappear from plasma rapidly, because of rapid

filtration of the small molecular weight dimers, renal loss of Hb raffimer

was minimal in the present experiments (Table 1). The longer intravascular

persistence of the oligomers may be due either to a lesser kinetic likelihood,

or reduced affinity of uptake into the principal site of clearance, presumed

to be the reticulo-endothelial system.

The half-lives determined in dogs of Hb raffimer infused at 18 mL/Kg

are substantially longer than the overall half-life of 14.6 ± 3 hrs, and

7.6 ± 2.8 hrs for the tetrameric species and 19.5 ± 3.2 hrs for the oligomers

in human subjects given 4–7 mL/kg (4–7 g/kg) (Carmichael et al., 2000).

The reasons for the longer persistence in dogs than in humans may be

related to the larger load in the former, but the experiments, in the absence

of comparable loads, do not permit a prediction of the half-life in humans at

this load.

Methemoglobin Formation

The in vivo formation of MetHb would reduce the oxygen transporting

capacity of cell-free hemoglobin based oxygen therapeutics, and would

thereby impair their physiological efficacy. Throughout the course of this

experiment, plasma MetHb concentration remained at about 10% or less of

Table 1. Mean urine hemoglobin concentrations (g/L).

Time 0–12 hrs 12–24 hrs 24–36 hrs 36–48 hrs 48–60 hrs

Hemoglobin (g/L) 0.02 ± 0.03 0.16 ± 0.26 0.01 ± 0.01 0.16 ± 0.18 0.00 ± 0.00

Values are presented as mean ± SD for all three dogs.

14 Wicks et al.



the total plasma Hb and the mass concentration of MetHb remained at

about 1 g/L, suggesting that excessive MetHb formation did not occur.

MetHb formation of Hb raffimer in vivo was previously determined in

rabbits by Caron et al. (2000). In these experiments, Hb raffimer was

administered as an exchange transfusion (13 mL/kg, or 1.3 g/kg) replacing

20% of the estimated blood volume, and animals were subsequently

followed for three hours. Over the three hour observation period, total

blood MetHb levels peaked at 2% in the Hb raffimer group and declined to

0.97 ± 0.37% at the end of the three hour observation period. Given that the

whole blood hemoglobin concentration at that time was 123 ± 9 g/L, the

MetHb mass-concentration can be estimated to be 1.2 g/L. If all of this is

assumed to be present as oxidized Hb raffimer in plasma, it would re-

present approximately 5% of the plasma Hb concentration of 22 g/L.

MetHb formation in vivo was also studied in rats that had 90% of their

blood volume replaced with Hb raffimer (Ning et al., 1998). Over a 3-hour

period, the plasma MetHb concentration in these rats rose from 6% at

baseline, to 8.5% at the peak. Thus, data from three species (rat, rabbit and

dog) with varying loads of Hb raffimer (20%, 25%, 90% of the blood

volume) suggest that MetHb in vivo formation is limited after Hb raffi-

mer administration.

In experiments with a large blood volume exchange in sheep (i.e. 90%

of the estimated blood volume) (Lee et al., 1995), hemoglobin gluta-

mer—250 [bovine] (Hemopure1, BioPure Corp, Cambridge MA), a bo-

vine hemoglobin-based oxygen carrier cross-linked with gluteraldahyde,

kept the animals alive and apparently maintained sufficient oxygen

transport. However, MetHb accumulated in vivo, to the extent that after

24 hours plasma MetHb concentration exceeded 40%, and remained at

that level, as the total plasma hemoglobin concentration declined by its

clearance. This was not the case in the experiments on Hb raffimer

reported here. MetHb concentration in the plasma of the dogs infused

with Hb raffimer remained a small fraction of the total both as a fraction

of the total pigment, and as mass-concentration. While a number of

possibilities may be suggested to account for what may be differences in

findings between experiments utilizing Hb raffimer and Hb glutamer, no

conclusions can be reached from the available evidence.

Interestingly, in a recently published paper, plasma hemoglobin and

MetHb concentrations were measured after infusions of large doses of

hemoglobin glutamer in bleeding surgical patients (Sprung et al., 2002). In

this study, plasma MetHb concentration rose in a dose dependent manner,

with peak concentrations on the third post-operative day, of 3% or less

when the dose was 1.5 g/kg (equivalent to 11.5 mL/kg) or less. The in-

crease was 6–7% when the dose was 2.0 or 2.5 g/kg (representing 15.4 and




19.2 mL/kg), declining thereafter to minimal levels on the seventh post-

operative day. This suggests that adequate reducing mechanisms are present

to prevent substantial increases in MetHb in humans.

In a review of the mechanisms promoting and preventing oxidation of

free hemoglobin in plasma, Faivre et al. (1994) noted the existence of non-

specific reducing agents, including ascorbic acid which is ‘‘cycled’’

between the red cell and plasma (McGown et al., 1990). In experiments in

which dextran-BTC-hemoglobin (Faivre et al., 1994) was infused in guinea

pigs, the fully reduced hemoglobin was oxidized to approximately 30–40%

MetHb, whereas when fully oxidized MetHb was infused, 40% was reduced

within 12 hours. These findings suggest that significant reducing capacity is

present in whole blood, which prevents MetHb formation in Hb free in the

plasma. This is also suggested by experiments conducted in vitro (Neya et

al., 1998) in which hemoglobin glutamer was exposed to continuous

oxygenation in cardiopulmonary bypass equipment for 5 hours in the

presence or absence of blood. MetHb formation was quite rapid under these

conditions, but the rate was reduced to about half in the presence of fresh

human blood. Interestingly, the results of continuous exposure to a high

oxygen concentration were reproduced in control experiments in which

after an initial 3-minute exposure, samples were held for 5 hours at 37�C.

MetHb formed at similar rates in these samples, and the effect of the presence

of fresh whole blood was also one of similar retardation of the oxidation.

The results of this study demonstrate that Hb raffimer does not exhibit

a rapid rate of oxidation of the heme iron in vivo in the dog. Supporting

literature reflects the same conclusion in rabbits and rats.

ACKNOWLEDGMENT

We gratefully acknowledge the staff of ITR Laboratories for their

expert completion of this study.

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Willan, A. R., Pierce, C. H., Greenburg, A. G. (2000). A phase I study

16 Wicks et al.



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Caron, A., Menu, P., Faivre-Fiorina, B., Labrude, P., Alayash, A., Vigneron,

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