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ARTIFICIAL CELLS, BLOOD SUBSTITUTES, AND BIOTECHNOLOGY

Vol. 32, No. 2, pp. 243262, 2004

Hemoglobin Polymerized with a Naturally

Occurring Crosslinking Agent as a Blood

Substitute: In Vitro and In Vivo Studies

Wen-Hsiang Chang,1 Yen Chang,2 Yi-Chien Chen,1

and Hsing-Wen Sung1,*

1Department of Chemical Engineering, National Tsing Hua

University, Hsinchu, Taiwan, R.O.C.2Division of Cardiovascular Surgery, Veterans General Hospital-

Taichung and College of Medicine, National Yang-Ming University,Taipei, Taiwan, R.O.C.

ABSTRACT

A naturally occurring crosslinking agent, genipin, extracted from the

fruits of Gardenia jasminoides Ellis was used by our group to

chemically modified biomolecules. Genipin and its related iridoid

glucosides have been widely used as an antiphlogistic and cholagogue

in herbal medicine. Our previous study showed that the cytotoxicity

of genipin is significantly lower than glutaraldehyde. The study was to

investigate the feasibility of using genipin to polymerize hemoglobin

*Correspondence: Hsing-Wen Sung, Department of Chemical Engineering,

National Tsing Hua University, Hsinchu, Taiwan, 30013; Fax: 886-3-572-6832;

E-mail: [email protected].

243

DOI: 10.1081/BIO-120037830 1073-1199 (Print); 1532-4184 (Online)

Copyright &2004 by Marcel Dekker, Inc. www.dekker.com

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as a blood substitute. The results indicated that the rate of

hemoglobin polymerization by glutaraldehyde was significantly

faster than that by genipin and it readily produced polymers with

molecular masses greater than 500,000 Da. It was found that the

maximum degree of hemoglobin polymerization by genipin was

approximately 40% if over-polymerization is to be prevented. With

increasing the reaction temperature, hemoglobin concentration, and

genipin-to-hemoglobin molar ratio, the duration taken to achieve the

maximum degree of hemoglobin polymerization by genipin became

significantly shorter. The P50 value of the unmodified hemoglobin

was 9 mmHg, while that of the genipin-polymerized PLP-hemoglobin

increased to 21 mmHg. It was found in a rat model that the genipin-

polymerized PLP-hemoglobin resulted in a longer circulation time

than the unmodified hemoglobin. In conclusion, the results of thestudy indicated that the genipin-polymerized hemoglobin solution

has a lower oxygen affinity and a longer vascular retention time than

the unmodified hemoglobin solution.

Key Words: Stroma-free hemoglobin; Hemoglobin polymerization;

Genipin; Blood substitute; Exchange transfusion.

INTRODUCTION

Hemoglobin has been used as raw materials for manufacturing blood

substitutes (Chang, 1992; Everse and Hsia, 1997; Powanda and Chang,

2002). However, because of its high oxygen affinity and short vascular

retention time, limitations on hemoglobin as a blood substitute in

clinical therapy have been reported in the literature (Bakker et al., 1992;

MacDonald and Pepper, 1994; Nelson et al., 1992). To decrease its

oxygen affinity, hemoglobin has been modified by pyridoxylation and

followed by polymerization with glutaraldehyde (De Venuto and Zegna,

1983; Feola et al., 1983; Lee et al., 1989; Marini et al., 1990; Sehgal et al.,

1983). It was reported that the polymerized hemoglobin showed a P50value of 1922 mmHg. Nevertheless, the reaction rate of hemoglobin

with glutaraldehyde is too fast to control its molecular weight distri-

bution (MacDonald and Pepper, 1994). Hence, polymerization of

hemoglobin by glutaraldehyde is usually undertaken at 4C. Additio-

nally, the glutaraldehyde-polymerized hemoglobin is relatively unstableand may release glutaraldehyde residues during storage or sterilization

(MacDonald and Pepper, 1994). It was reported that glutaraldehyde is

cytotoxic even at low doses (MacDonald and Pepper, 1994). This may

impair the biocompatibility of the polymerized products.

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In an attempt to overcome the aforementioned problems, a naturally

occurring crosslinking agent, genipin, was used by our group to

polymerize hemoglobin. Genipin and its related iridoid glucosides

extracted from the fruits of Gardenia jasminoides Ellis have been

widely used as an antiphlogistic and cholagogue in herbal medicine

(Akao et al., 1994). Additionally, it is known that genipin can

spontaneously react with amino acids or proteins to form dark-blue

pigments (Touyama et al., 1994a,b) These dark-blue pigments have been

used in the fabrication of food dyes. The cytotoxicity of genipin was

previously studied by our group in vitro using 3T3 fibroblasts (Sung et al.,

1999). Glutaraldehyde was used as a control. The results indicated that

genipin is significantly less cytotoxic than glutaraldehyde. Additionally,

the genotoxicity of genipin was tested in vitro using Chinese hamsterovary (CHO-K1) cells (Tsai et al., 2000). The results hinted that glutar-

aldehyde may produce a weakly clastogenic response in CHO-K1 cells. In

contrast, genipin does not cause clastogenic response in CHO-K1 cells.

The biocompatibility of the genipin-crosslinked biological tissue

was studied in a growing rat model subcutaneously (Chang et al.,

2002). It was noted that the degree of inflammatory reaction for the

genipin-crosslinked tissue was significantly less than its glutaraldehyde-

crosslinked counterpart. This implied that genipin may form biocom-

patible crosslinked products.

The present study was to investigate the rate of hemoglobin

polymerization by genipin. Glutaraldehyde was used as a control.

Additionally, the effects of temperature, hemoglobin concentration, and

genipin-to-hemoglobin molar ratio on the degree of hemoglobin

polymerization by genipin were examined. Subsequently, the in vitro

characteristics of the unmodified hemoglobin and genipin-polymerized

hemoglobin solutions used for exchange transfusion were determined.

Finally, the in vivo performance of the unmodified hemoglobin and

genipin-polymerized hemoglobin solutions was tested in a rat model.

MATERIALS AND METHODS

Preparation of Stroma-Free Hemoglobin Solution

Porcine stroma-free hemoglobin solution was prepared by theaqueous two-phase system described in the literature (Hart and Bailey,

1991; Middaugh and Lawson, 1980). The porcine blood was collected

from a local slaughterhouse into glass bottles containing sodium citrate

solution (3.7 g/dl). The bottles were kept on ice to minimize the formation

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of methemoglobin. Upon return, the plasma was removed via centrifuga-

tion at 5,000g for 10 min. The red blood cells were washed three times

with normal saline (1:3 v/v) and lysed by treatment with three volumes DI

water, a hypotonic solution, over night. Subsequently, the cell membrane

remnants were removed via centrifugation at 15,000g for 1h.

The separation and purification of stroma-free hemoglobin was

performed by the aqueous two-phase system. The resulting solution

was dialyzed three times against a 0.05 M phosphate buffered saline

(PBS, pH 7.4), and the hemoglobin was concentrated by ultrafiltration

to 10 grams per deciliter. The solution was subsequently sterilized by

filtration through a 0.22-mm Millipore filter. The purity of stroma-free

hemoglobin was checked by electrophoresis in SDS-polyacrylamide gels

(PhastSystemTM, Pharmacia Biotech, Uppsala, Sweden) and by the gelfiltration analysis using a high-performance liquid chromatographer

(HPLC) equipped with a TSK G3000SWXL column (Tosoh Corp.,

Tokyo, Japan).

Pyridoxylation

Pyridoxylation of hemoglobin was achieved by the method of Benesch

et al. (1972). For a typical preparation of pyridoxal-50-phosphate-

hemoglobin (PLP-Hb), 9.3mmol (6g/dL) of stripped hemoglobin in

10 mL of 0.1M Tris buffer (pH 7.5 at 4C) was deoxygenated by bubbling

nitrogen through the solution, which contained 20 mL of caprylic alcohol

to prevent foaming in the subsequent steps. Subsequently, 37.2 mmol of

PLP was added. After 30 min under nitrogen, 186mmol of sodium

borohydride in 0.5 mL of 103 M NaOH was introduced for 30 min and

then was dialyzed against isotonic PBS to remove the excess PLP and

sodium borohydride. All operations were conducted at 4C. Finally, the

obtained PLP-Hb solution was concentrated by ultrafiltration to 20 g

per deciliter.

Polymerization

In the study, the rate of hemoglobin polymerization by genipin

(Challenge Bioproducts Co., Taichung, Taiwan) was investigated.Glutaraldehyde was used as a control. Additionally, the effects of

temperature (4, 10, 25C), hemoglobin concentration (PLP-Hb concen-

tration in 2, 6, 10 g/dl), and genipin-to-hemoglobin molar ratio (3/1, 5/1,

7/1, 10/1) on the degree of hemoglobin polymerization by genipin

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(GP-PLP-Hb) were investigated. The degree of hemoglobin polymeriza-

tion by genipin was monitored by the gel filtration analysis with a TSK

G3000SWXL column. In order to prevent the formation of polymeric

Hb (GP-PLP-Hb) with a molecular weight greater than 500,000 Da, the

polymerization reaction was terminated by the addition of glycine. It

is known that genipin can spontaneously react with glycine (Fujikawa

et al., 1987). Consequently, the effect of using glycine at a variety of

concentrations (in glycine-to-hemoglobin molar ratio) on the termination

of hemoglobin polymerization by genipin was examined.

Subsequently, the polymerized GP-PLP-Hb solution was dialyzed

to eliminate any unreacted genipin, glycine, and polymerized glycine.

Finally, the obtained GP-PLP-Hb solution was concentrated by

ultrafiltration to 10 grams per deciliter. Total hemoglobin and methe-moglobin concentrations were measured as per the methods described by

Crosby and co-workers and Evelyn and Malloy, respectively (Crosby

et al., 1954; Evelyn and Malloy, 1938).

Removal of Unpolymerized Hemoglobin

A final consideration for clinical products is the need to reduce the

residual unpolymerized hemoglobin to a minimum (MacDonald and

Pepper, 1994). In the study, the removal of unpolymerized hemoglobin

was carried out by an ion-exchange column (DEAE cellulose column,

Sigma Chemical Co., St. Louis, Missouri, USA) or a gel-filtration

column (Sephadex, G-100-120, Sigma Chemical Co.).

Characteristics of Test Solutions for Exchange Transfusion

The characteristics of the unmodified Hb and GP-PLP-Hb solutions

used for exchange transfusion in the rat were determined as follows: the

sodium and potassium concentrations by an atomic absorption spectro-

photometer (Model AA-100, Perkin Elmer Inc., Norwalk, Connecticut,

USA) and the osmolality values by an osmometer (Advanced Micro

Osmometer, Model 3300, Norwood, Massachusetts, USA). Hemoglobinoxygen affinity measurements were obtained using the biotonometry

technique reported by Neville (1974). The distributions in particle size for

the unmodified Hb and GP-PLP-Hb solutions (n 3) were analyzed by a

light-scattering method (ZetaSizer, Trekintal Corp., Taipei, Taiwan).

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Animal Studies

Animal studies were conduced in two groups of male Sprague-Dawley

rats. The first group of experiments was to examine the survival of the rats

at an approximately 50% blood-volume-exchange transfusion with the

GP-PLP-Hb solution. The unmodified Hb and PBS were used as controls.

Six rats (200250 g) were used for each exchange transfusion study. The

animals were anesthetized and the femoral vein was cannulated with a

polyethylene catheter connected by a three-way sterile stopcock to two

syringes (Lee et al., 1989), one used for phlebotomy and the other for

infusion of sample solution (De Venuto et al., 1977). Exchange transfusion

was done by removal one ml of blood and immediate infusion of one ml of

sample solution, repeating the process until the desired exchange level wasachieved. The percentage of blood-volume-exchange was calculated

assuming that the rat blood volume was 10% of its body weight.

The second group of experiments was to test the half-life of the

GP-PLP-Hb in blood circulation. The unmodified Hb was used as a

control. The experimental preparation and procedure were the same as

aforementioned. Samples of blood were obtained from the tail vein at

various intervals during the posttransfusion hours.

Statistical Analysis

Statistical analysis for the determination of differences in the

measured properties between groups was accomplished using one-way

analysis of variance and determination of confidence intervals, performed

with a computer statistical program (Statistical Analysis System,

Version 6.08, SAS Institute Inc., Cary, North Carolina, USA). All data

are presented as a mean value with its standard deviation indicated

(meanSD).

RESULTS

Stroma-Free Hemoglobin Solution

The purified stroma-free hemoglobin solution was obtained by theaqueous two-phase system. A typical elution pattern from the TSK

G3000SWXLcolumn for the purified stroma-free hemoglobin solution is

shown in Fig. 1a. The stroma-free hemoglobin solution was eluted from

the column as a single peak corresponding to a molecular weight of

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64,000Da. Electrophoresis in SDS-page of the purified stroma-free

hemoglobin solution yielded a single band corresponding to the 16,000-

dalton monomer (Fig. 1b). These results demonstrated that the stroma-

free hemoglobin solution obtained by the aqueous two-phase system was

highly pure.

Polymerized Hemoglobin Solution

A typical HPLC molecular weight profile following 4 h after initiation

of polymerization of hemoglobin (10 g/dL of PLP-Hb) by genipin (7/1

molar ratio of GP/Hb ) at 15C is shown in Fig. 2a. The unpolymerized

fraction (PLP-Hb) was approximately 60% and there was very little

polymeric GP-PLP-Hb with a molecular weight greater than 500,000Da.

In comparison, a typical HPLC molecular weight profile following 30 min

after initiation of polymerization of hemoglobin by glutaraldehyde at the

same reaction conditions is show in Fig. 2b. As indicated in the figure, the

rate of hemoglobin polymerization by glutaraldehyde was significantlyfaster than that by genipin (P< 0.05) and it readily produced polymers

with molecular masses greater than 500,000 Da.

It was found that the maximum degree of hemoglobin polymeriza-

tion by genipin was approximately 40% if the formation of polymeric

Figure 1. (a) A typical elution pattern from the TSK G3000SWXL column for

the purified stroma-free hemoglobin solution; (b) Electrophoresis in SDS-page of

the purified stroma-free hemoglobin solution.

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(a)

(b)

mv

5

10

15

20

25

30

35

40

45

5 10 15 20 25

minutes

5

10

15

20

25

30

35

40

45

mv

5

10

15

20

5

10

15

20

5 10 15 20 25

minutes

mv mv

over polymerization

Figure 2. (a) A typical HPLC molecular weight profile following 4 h after

initiation of polymerization of hemoglobin by genipin; (b) A typical HPLC

molecular weight profile following 30 min after initiation of polymerization of

hemoglobin by glutaraldehyde at the same reaction conditions.

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GP-PLP-Hb with a molecular weight >500,000 Da is to be prevented.

This maximum degree of hemoglobin polymerization by genipin could

be achieved at different reaction conditions at distinct durations. The

durations taken for various reaction conditions (temperature, hemoglo-

bin concentration, and genipin-to-hemoglobin molar ratio) to achieve

the maximum degree of hemoglobin polymerization by genipin (40%)

were listed in Table 1. As shown in the table, all reaction conditions

investigated significantly influenced the rate of hemoglobin polymeriza-

tion by genipin. With increasing the reaction temperature, hemoglobin

concentration, and genipin-to-hemoglobin molar ratio, the duration

taken to achieve the maximum degree of hemoglobin polymerization by

genipin became significantly shorter (P< 0.05).

Table 2 presents the percentage of methemoglobin produced at

each corresponding reaction condition investigated after the maximum

degree of hemoglobin polymerization by genipin was achieved. The

results indicated that the percentages of methemoglobin observed at all

investigated conditions were minimal, even at room temperature (25C).

The conditions used to polymerize hemoglobin by genipin for the

following studies were: a hemoglobin (PLP-Hb) concentration of 10 g/dL,

a genipin-to-hemoglobin molar ratio of 7/1, and a reaction temperature

at 15C. The corresponding HPLC molecular weight profile for the

genipin-polymerized hemoglobin (GP-PLP-Hb) solution under such

conditions is already shown in Fig. 2a.

Table 1. The durations taken (h) for various reaction conditions (temperature,hemoglobin concentration, and genipin-to-hemoglobin molar ratio) to achieve

the maximum degree of hemoglobin polymerization by genipin (40%).

Genipin/Hb

(molar ratio) 3/1 5/1 7/1 10/1

25C Hb conc.

10 g/dL 3 2 2 1

6 g/dL 3.5 2.5 2 2

2 g/dL 8.5 5.5 4.5 3

15C Hb conc.

10 g/dL 6 4 4 2

6 g/dL 12 8 4 4

2 g/dL 24 16 12 12

10C Hb conc.

10 g/dL 12 6 4 4

6 g/dL 14 10 8 6

2 g/dL 32 24 18 14

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In order to prevent over polymerization (the formation of polymeric

GP-PLP-Hb with a molecular weight >500,000 Da), the hemoglobin

polymerization by genipin has to be terminated. In the study, the

hemoglobin polymerization by genipin was terminated by the addition ofglycine. The effect of using glycine at various concentrations (in glycine-

to-hemoglobin molar ratio) on the termination of hemoglobin polymer-

ization by genipin was shown in Fig. 3. The molecular weight profile

of the genipin-polymerized hemoglobin (GP-PLP-Hb) shifted slightly

towards higher molecular weight (compared with the result presented in

Fig. 2a), an indication of over polymerization, when the glycine-to-

hemoglobin molar ratio used was 20/1 or 50/1. This phenomenon in over

polymerization could be effectively prevented if the glycine-to-hemoglo-

bin molar ratio was greater than 100/1. Under this condition, there was

no further buildup of higher molecular weight polymers.

Removal of Unpolymerized Hemoglobin

The results of removal of the unpolymerized hemoglobin (PLP-Hb)

from the GP-PLP-Hb carried out by an ion-exchange column or a

Table 2. Percentages of methemoglobin produced at various reaction conditions

investigated (temperature, hemoglobin concentration, and genipin-to-hemoglobin

molar ratio) after the maximum degree of hemoglobin polymerization by genipin

was achieved.

Genipin/Hb

(molar ratio) 3/1 5/1 7/1 10/1

25C Hb conc.

10 g/dL 1.0 0.2 2.6 0.1 2.30.3 3.2 0.1

6 g/dL 2.6 0.3 1.5 0.1 1.20.2 1.2 0.3

2 g/dL 1.2 0.2 1.0 0.1 0.80.1 1.2 0.1

15C Hb conc.

10 g/dL 3.2 0.3 1.2 0.1 1.30.1 1.1 0.26 g/dL 1.1 0.2 1.2 0.1 1.60.3 2.4 0.2

2 g/dL 2.1 0.2 1.7 0.1 1.60.2 1.1 0.2

10C Hb conc.

10 g/dL 1.4 0.1 2.7 0.3 1.80.2 2.5 0.4

6 g/dL 1.4 0.2 1.4 0.1 1.60.2 3.3 0.4

2 g/dL 1.5 0.1 1.0 0.1 1.40.2 1.6 0.2

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gel-filtration column were presented in Fig. 4. As shown in the figure,

after passage through the ion-exchange column, the fraction of residual

unpolymerized hemoglobin was reduced from 60% (Fig. 4a) to 27%

(Fig. 4b). In contrast, there was very little unpolymerized hemoglobin

left in its HPLC molecular weight profile after passage through thegel-filtration column (Fig. 4c). This indicated that removal of the

unpolymerized hemoglobin from the genipin-polymerized hemoglobin by

the gel-filtration column was significantly more effective than by the ion-

exchange column (P< 0.05).

Glycine/Hb 20/1 50/1

100/1 200/1

20

10

30

40

50

60

70

80

20

10

30

40

50

60

70

80

20

10

30

40

50

60

70

80

20

10

30

40

50

60

70

80

20

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40

50

60

70

80

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60

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80

20

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50

60

20

10

30

40

50

60

5 10 15 20 25minutes

5 10 15 20 25minutes

5 10 15 20 25minutes

5 10 15 20 25minutes

mv mv mv mv

mvmvmvmv

over polymerizationover polymerization

Figure 3. Effects of using glycine at various concentrations (in glycine-to-

hemoglobin molar ratio) on the termination of hemoglobin polymerization by

genipin.

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Characteristics of Test Solutions for Exchange Transfusion

The characteristics of the unmodified Hb and GP-PLP-Hb solutions

used for exchange transfusion in the rat are shown in Table 3. The two

solutions were essentially the same in their compositions except for the

parameter of P50 value. The P50 values of the Hb and GP-PLP-Hb

solutions were 9 mmHg and 21 mmHg, respectively. The distribution

curves in particle size for the unmodified Hb and GP-PLP-Hb solutions

determined by a light-scattering method are presented in Fig. 5. As

shown in the figure, the distribution in particle size for the GP-PLP-Hbsolution was significantly wider than that for the unmodified Hb

solution. Additionally, the averaged particle size for the GP-PLP-

Hb solution (32.0 7.4 nm) was significantly larger than the unmodified

Hb solution (6.8 0.3 nm,P < 0.05).

5 5

10 10

15 15

20 20

25 25

30 30

35 35

40 40

45 45

mv

5 10 15 20 25

minutes

Unpolymerized

PLP-Hb (60%)

45 45

50 50

55 55

60 60

65 65

5 10 15 2520

minutes

mv

Unpolymerized

PLP-Hb (27%)

5 10 15 20 25

minutes

5 5

7.5 7.5

10 10

mv

(a) (b)

(c)

Figure 4. Results of removal of the unpolymerized hemoglobin (PLP-Hb)

from the genipin-polymerized hemoglobin (GP-PLP-Hb) carried out by an

ion-exchange column (b) or a gel-filtration column (c).

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Animal Studies

Test solutions for exchange transfusion in the rat were sterilized by

filtration through a 0.22-mm Millipore filter. The results of the first group

of experiments in examining the survival of the rats at an approximately

50% blood-volume-exchange transfusion with the unmodified Hb, PBS,

or GP-PLP-Hb solution are summarized in Table 4. It was found that

Unmodified Hb GP-PLP-Hb

32 7.4 nm6.8 0.3 nm

Figure 5. Particle-size distribution curves for the unmodified Hb and GP-PLP-

Hb solutions determined by a light-scattering method. (View this art in color at

www.dekker.com.)

Table 3. Characteristics of the unmodified Hb and GP-PLP-Hb solutions used

for exchange transfusions in the rat.

Hb GP-PLP-Hb

Concentration (g/dL) 10 10

MetHb (%) 3.3 3.0

Na (meq) 151 136

K (meq) 4.6 4.4

Osmolality (mOsm) 287 289

PH 7.4 7.4

P50 (mmHg) 9 21

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the results for the animals treated with the GP-PLP-Hb solution were

significantly superior to the other control groups (P< 0.05). In this

specific test group, all test animals (n6) after treatment with the GP-

PLP-Hb solution survived and remained healthy more than 3 months. In

contrast, only one of six rats survived for the control groups treated with

the unmodified Hb or PBS solution, while the other test animals in the

corresponding groups died in about 5 h after exchange transfusion.

The results of the second group of experiments in testing the half-life

durations of the unmodified Hb and GP-PLP-Hb solutions in circulation

are given in Fig. 6. As shown in the figure, the disappearance of

hemoglobin was significantly faster in the animals treated with the

unmodified Hb solution than their counterparts treated with the GP-

PLP-Hb solution (P< 0.05). The half-life duration, that is the time

0. 0 2. 5 5. 0 7. 5 10. 0 1 2. 5 1 5. 0 1 7. 5 2 0. 0 2 2. 5 2 5. 0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

HbGP-PLP-Hb

(n = 6)

Time (hours)

PlasmaHbConcentration(g/dl)

Figure 6. Disappearance of plasma hemoglobin for the animals treated with theunmodified Hb or GP-PLP-Hb solution.

Table 4. Results of the first group of experiments in examining the survival of

the rats at an approximately 50% blood-volume-exchange transfusion with the

unmodified Hb, PBS, or GP-PLP-Hb solution.

Percentage of

exchange

Survival

ratio (n 6)

Averaged

survival period (h)

Hb 53.3 0.9 1/6 5.7 1.3

PBS 54.3 6.5 1/6 3.6 0.4

GP-PLP-Hb 51.7 1.3 6/6 >3 months

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necessary for the plasma hemoglobin concentration to reach one half the

value observed at the end of exchange transfusion, was 1.5 h for the

control group treated with the unmodified Hb solution and 12.5 h for

the test group treated with the GP-PLP-Hb solution.

DISCUSSION

It is well known that high oxygen affinity and short vascular retention

time represent two important limitations for the stroma-free hemoglobin

solution in the blood-volume-exchange transfusion (De Venuto and

Zegna, 1983; Keipert and Chang, 1985). To overcome the former limita-

tion, an agent to intramolecularly crosslink stroma-free hemoglobin haslong been thought desirable. Intramolecular crosslinks between tetramer

subunits prevent dissociation into excretable dimmers (32,000 Da).

Benesch et al. demonstrated that PLP can be attached to the N-terminal

valine of hemoglobin chains by forming a Schiff base and that the

resulting PLP-Hb has lower oxygen affinity than the unmodified Hb

(Benesch et al., 1972). It was reported that pyridoxylation of hemoglobin

appeared to be necessary to maintain the cooperativity of the molecular

chains and the ability to reversibly bind oxygen when hemoglobin is

subsequently subjected to polymerization (De Venuto and Zegna, 1983).

To overcome the latter limitation, it was reported that intermolecular

polymerization of PLP-Hb can yield a product with a longer vascular

retention time and at the same time with a lower oxygen affinity than the

unmodified Hb (Bakker et al., 1992; Keipert and Chang, 1985). However,

carrying out intermolecular polymerization too far, producing polymers

with molecular masses> 500,000 Da, is undesirable because the large

aggregates may have altered surface charge characteristics (MacDonald

and Pepper, 1994). These alterations could lead to flow changes in the

microcirculation and may well acquire additional untoward toxicities,

such as reticuloendothelial system blockade or stimulation of an

immunological response to the altered aggregate surface (MacDonald

and Pepper, 1994).

Glutaraldehyde is a well-known crosslinking agent, producing rapid

intermolecular, as well as limited intramolecular, polymerization of

hemoglobin (MacDonald and Pepper, 1994). Due to its rapid reaction, a

mixture of glutaraldehyde-polymerized hemoglobin with a wide range ofmolecular weight is readily produced. Additionally, a reversible reaction

releasing free glutaraldehyde after hemoglobin polymerization is possible

(MacDonald and Pepper, 1994). It was reported that glutaraldehyde

is cytotoxic even at low doses (MacDonald and Pepper, 1994). The

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mechanism of polymerization of hemoglobin by glutaraldehyde can be

found in the literature (MacDonald and Pepper, 1994).

It was found in our previous study that genipin can react with the

free amino groups of lysine, hydroxylysine, or arginine residues within

biological tissues (Sung et al., 1998). Touyama et al. studied the

structures of the intermediates leading to a blue pigment produced

from genipin and methylamine, the simplest primary amine (Touyama

et al., 1994a, 1994b). The presumed mechanism for the formation of the

genipin-methylamine monomer and the blue-pigment polymers proposed

by the Touyamas group can be found in the literature (Touyama et al.,

1994a,b). Briefly speaking, the genipin-methylamine monomer is formed

through a nucleophilic attack by methylamine on the olefinic carbon at

C-3 of genipin, followed by opening of the dihydropyran ring and

attacked by the secondary amino group on the resulting aldehyde

group (Touyama et al., 1994a, 1994b). The blue-pigment polymers are

presumably formed through oxygen radical-induced polymerization and

dehydrogenation of several intermediary pigments. The results of the

aforementioned studies suggest that genipin may form intermolecular

polymerization of hemoglobin (Fig. 7) (Fujikawa et al., 1987, 1988).

It was found that the stability of the genipin-crosslinked tissue during

storage was superior to its glutaraldehyde-crosslinked counterpart (Sung

et al., 2001). The differences in stability between the genipin- and

glutaraldehyde-crosslinked tissues during storage may be caused by their

N

C

OCH3O

CH3

H

CH

C

N+

H CH3

OH3CO

GP

GP

GP

C

COOCH3

NH

O

OH

..

CH3

H

COOCH3

OH

N

R

O

OHCH2

OH

C

OCH3O

+ NH2:1

7

11

35

9

10

Genipin (GP) hemoglobin

Figure 7. Presumable mechanism of hemoglobin polymerization by genipin.

(View this art in color at www.dekker.com.)

258 Chang et al.

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differences in crosslinking structure. It is known that glutaraldehyde may

react with free amino groups to form Schiff-bases. It was reported that

the reaction of Schiff-base is relatively unstable and reversible (Carey,

1992; Sung et al., 2001). On the other hand, genipin may react with free

amino groups and form a tertiary amine structure which is more stable

than Schiff-base (Fig. 7) (Carey, 1992).

The results shown in our study revealed that the rate of hemoglobin

polymerization by glutaraldehyde was significantly faster than that by

genipin (Figs. 2a and b). It was reported in the literature that

polymerization by glutaraldehyde is a rapid reaction (MacDonald and

Pepper, 1994). Therefore, it readily produced glutaraldehyde-polymerized

hemoglobin with molecular masses greater than 500,000 Da (Fig. 2b). In

contrast, there was very little polymeric GP-PLP-Hb with a molecularweight >500,000 Da observed (Fig. 2a).

The rate of hemoglobin polymerization by genipin may be influenced

by the conditions at which the reaction is run. It was noted in the study

that with increasing reaction temperature, hemoglobin concentration, and

genipin-to-hemoglobin molar ratio, the duration taken to achieve

the maximum degree of hemoglobin polymerization by genipin became

significantly shorter (Table 1). To prevent over polymerization, the

hemoglobin polymerization by genipin can be terminated by the addition

of glycine. It was found that once the addition of glycine-to-hemoglobin

molar ratio was greater than 100/1, no further buildup of higher molecular

weight polymers was observed (Fig. 3). The removal of the unpolymerized

hemoglobin (PLP-Hb) from the genipin-polymerized hemoglobin (GP-

PLP-Hb) can be effectively achieved by a gel-filtration column (Fig. 4).

It was found in the animal study that the GP-PLP-Hb solution

resulted in a longer circulation time (i.e., a greater half life in the

disappearance of plasma hemoglobin) than the unmodified Hb solution

(Fig. 6). It was reported that a main route of Hb clearance is through the

kidney (Bleeker et al., 1989; Lenz et al., 1991; Ning et al., 1992; Savitsky

et al., 1978). Renal glomeruli filter proteins under 65,000 Da out of the

circulation. Free unmodified Hb, with its tendency to dissociate into

dimmers (32,000 Da), is readily cleared from the blood and excreted into

the urine (Bunn et al., 1969). The averaged particle size of the GP-PLP-

Hb measured was significantly greater than the unmodified Hb (Fig. 5).

The large size of the GP-PLP-Hb may reduce its ability to pass into

extra-vascular spaces.In conclusion, the results of the study indicated that the genipin-

polymerized hemoglobin (GP-PLP-Hb) solution has a lower oxygen

affinity and a longer vascular retention time than the unmodified Hb

solution.

Genipin-Polymerized Hemoglobin 259

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ACKNOWLEDGMENTS

This work was supported partly by a grant from the National Science

Council of Taiwan, Republic of China (NSC91-2320-B-007-004) and

partly by another grant from the Veterans General Hospital, Tsing-Hua,

Yang-Ming Joint Research Program (VTY-92-P4-21).

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