Biomaterials, Artificial Cells and Immobilization Biotechnology
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effect of Polymerization on Clearance and Degradation of Free Hemoglobin Wim K. Bleeker, Guy A. M. Berbers, Piet J. den Boer, Jacques Agterberg, Gemma Rigter & Joachim C. Bakker To cite this article: Wim K. Bleeker, Guy A. M. Berbers, Piet J. den Boer, Jacques Agterberg, Gemma Rigter & Joachim C. Bakker (1992) Effect of Polymerization on Clearance and Degradation of Free Hemoglobin, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 747-750, DOI: 10.3109/10731199209119713 To link to this article: http://dx.doi.org/10.3109/10731199209119713
Published online: 11 Jul 2009.
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Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 23 April 2016, At: 07:35
BIOMAT., ART. CELLS b IMMOB. BIOTECH., 20(2-4), 747-750 (1992)
Downloaded by [McMaster University] at 07:35 23 April 2016
EFFECT OF POLYMERIZATION ON CLEARANCE AND DEGRADATION OF FREE HEMOGLOBIN. Wim K.Bleeker, Guy A.M.Berbers, Piet J.den Boer, Jacques Agterberg, Gemma Rigter, and Joachim C.Bakker. Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. ABSTRACT In the present study we investigated the mechanism of prolongation of the plasma retention of free hemoglobin by polymerization. Polymerization of intramolecularly crosslinked hemoglobin with glutaraldehyde yields a mixture of large polymers, small polymers and monomers. In exchange transfusion experiments in rats we analyzed plasma samples by gel filtration to determine the clearance of polymers of different size. A positive correlation was found between polymer size and vascular retention. Furthermore, the clearance of large polymers appeared to be highly dose-dependent: after 20% and 70% exchange transfusions, we observed for large polymers a plasma half-life of 12 and 26 hours, respectively, whereas the half-life for 64kD monomers was 4 hours in both cases. The degradation of hemoglobin was followed by measuring the bilirubin excretion. The infused heme was recovered as bilirubin within 72 hours. The delay between the disappearance of free hemoglobin from the plasma and the recovery as bilirubin was about six hours and was not affected by polymerization or dose. We conclude that polymerization prevents the operation of certain clearance mechanisms, while still allowing a route of clearance that is easily saturated. The intracellular degradation of heme into bilirubin is not affected by the modifications of hemoglobin and is not easily saturated.
INTRODUCTION The vascular retention time of a hemoglobin solution is an important factor for its usefulness as blood substitute. To prolong vascular retention, we have modified human hemoglobin by intra- and intermolecular crosslinking with NFF'LP and glutaraldehyde, respectively. Previously, we have reported that intramolecular crosslinking (HbNFPLP) prolongs the vascular retention by eliminating the kidney excretion (1). After nephrectomy, there was no difference in clearance rate between HbNFPLP and unmodified hemoglobin, indicating that the other clearance mechanisms were not influenced. The present study was directed to the mechanism of further prolongation of the vascular retention by polymerization of HbNFPLP (polyHbNFPLP). In exchange transfusion experiments in rats with different doses of polyHbNFPLP we analyzed plasma samples to follow the clearance of polymers of different size. The degradation of plasma hemoglobin was studied by measuring the bilirubin excretion.
747 Copyright 0 1992 by Marcel DsLker, Inc.
7 48
BLEEKER ET AL.
30
h
Ul
M
25
DOSE
20
1.2 g/kg
3 0 5:
v
2.0 g/kg
15
M
3.8 g/kg
0
Downloaded by [McMaster University] at 07:35 23 April 2016
2
10 5
0
POLY
TE/TRI
DI
MONO
Figure 1 . Dose dependency o f half-disappearance time (T50%) of polyHbNFPLP polymers o f d i f f e r e n t s i z e s (mean 2 SD, n = 3 ) .
MATERIALS AND METHODS Hemoglobin solutions were prepared as described earlier (2). In brief, hemoglobin was isolated from human red blood cells by hypotonic lysis and tangential flow filtration. Intramolecular crosslinkingof the S-chainswas accomplished with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP). After purification with ion-exchange chromatography, HbNFPLP contained less than 5% non-crosslinked hemoglobin. Subsequent polymerhation (polyHbNFpLP) was achieved with glutaraldehyde to the desired molecular weight distribution: 10-20% polymers of >300 kD, 20-30% 64-kD monomers and virtually no dissociable hemoglobin. Exchange transfusions were performed in conscious female Wistar rats, weighing 200-250 g, placed in metabolic cages. One day prior to the exchange transfusion, a permanent cannula was introduced under anesthesia in the left carotid artery. In some rats, the common bile duct was also cannulated. Exchange transfusions were performed through the arterial cannula in steps of 2 ml. Blood samples were taken from the arterial cannula. Bile was collected in fractions of several hours, shielded from light, and stored at -2OOC. Hemoglobin concentration was measured photometrically as cyanomethemoglobin. The polymer size distribution of PolyHbNFPLP was determined by gel filtration analysis on a Superose-12 column (HR 10/30; Pharmacia). Total bilirubin was measured by the diazo reaction (Monotest 10 Bilirubin, Boehringer, Germany); plasma samples were pretreated with DMSO to precipitate interfering hemoglobin.
RESULTS AND DISCUSSION The plasma retention of polyHbNFPLP was found to be dose dependent, as has also been observed by other investigators for other polymerized Hb preparations (3).
Downloaded by [McMaster University] at 07:35 23 April 2016
EFFECT OF POLYMERIZATION
0
10
749
20
TIME -0-
HEMOGLOBIN CLEARANCE
40
30
50
80
(HOURS) -0-
RECOVERY A S BILIRUBIN
Figure 2 . Cumulative curves of hemoglobin clearance from plasma and o f r e c o v e r y of t h e i n f u s e d heme a s b i l i r u b i n , a f t e r a 70% excharge t r a n s f u s i o n w i t h polyHbNFPLP ( n e t dose 3 . 3 g / k g ) (mean + S D , n=3)
.
After exchange transfusions of 20, 35 and 70 % of the blood volume, giving a net dose of 1.2, 2 and 3.8 g/kg, respectively, plasma half-disappearance times were found of 8, 10 and 13 hours, respectively. Gel filtration analyses of plasma samples permitted the separate determination of the plasma clearance of polymers of different size. A distinction was made between: a monomer peak (64kD), a dimer peak (130 kD), a trihetramer peak (190-250 kD) and polymers (void volume, >300 kD).The results (figure 1) showed a positive correlation between plasma half-disappearance time and polymer size. Furthermore, it appeared that only the plasma retention of the polymers was dose dependent. The time courses of the clearance curves were different for monomers and polymers. The curves for monomers were exponential, both at low and high doses, whereas those for polymers were almost linear, especially at high dose. The results indicate that the clearance of the 64-kD monomer fraction is similar to that of non-polymerized HbNFPLP, which has been studied previously (4). Thus, the modification of lysine residues by the treatment with glutaraldehyde, which occurs randomly in all polymers and monomers, does not influence the clearance rate. The larger size of the polymers seems to be crucial for the prolongation of vascular retention. Since the clearance mechanism of the polymers was saturated and since their presence did not affect the monomer clearance, we conclude that monomers are cleared by another mechanism than polymers. Thus, the clearance mechanism that is responsible for the rapid clearance of the monomers is unable to remove the larger polymers. This leads
Downloaded by [McMaster University] at 07:35 23 April 2016
7 50
BLEEKER ET AL.
us to the hypothesis that several cell types can remove free hemoglobin from the circulalation (e.g.: macrophages, hepatocytes and endothelial cells), but that only part of these cells (with a limited capacity) are able to remove polymerized hemoglobin. The degradation of polyHbNFPLP was followed by measuring the increase in bilirubin production. PolyHbNFPLP was given in three different doses in 15,30 or 65% exchange transfusions (n=3), resulting in a net heme dose of 14, 26 and 52 pmollrat, respectively. In all cases, an increase in bilirubin excretion into the bile was already observed in the first sample interval (0 - 2 hours): 0.2 - 0.4 pmoVhour (control: 0.1 pmofiour). The maximum values for excretion were observed in the sample interval from 4 - 8 hours after transfusion: 0.75,0.95 and 1.6 prnofiour, respectively. Thereafter, bilirubin excretion gradually diminished, and after 48 hours it had returned to values close to normal. For comparison, we administered also HbNFPLP, which has a much shorter plasma retention. After a net dose of 25 pmol the maximum value for bilirubin excretion was higher than after a comparable dose of polyHbNFPLP: 1.2 pmolhour. The total bilirubin concentration in plasma showed a moderate increase up to 100 pmoI/l. Since this was accompanied by excretion into the urine, part of it probably concerns conjugated bilirubin. Thus, accumulation in the plasma of the potentially toxic, unconjugated bilirubin seems not to be a point of major concern after administration of hemoglobin solutions. After 48 hours 98 & 20% (n= 12) of the infused heme was recovered as bilirubin in bile and urine. The time lag between the disappearance of free Hb from the circulation and recovery as bilirubin was about 6 - 8 hours (see figure 2). The recovery was expressed as a percentage of the expected amount, based on a conversion of 1 heme group to 1 bilirubin molecule. This time lag was about the same for all doses of polyHbNFPLP and also for HbNFPLP. Ostrow et al. (5). found at much lower doses of unmodified hemoglobin (12 to 60 mgkg), a similar value of 3 hours for the delay between extravascular sequestration of hemoglobin and recovery as bilirubin. Their experiments gave also evidence that the delay is mainly due to the breakdown of hemoglobin and conversion to bilirubin and not to the excretion of bilirubin. We conclude from our experiments on degradation that: 1/ the intracellular degradation of hemoglobin is not affected by the intramolecular crosslinkingwith NFPLP and subsequent polymerization with glutaraldehyde. 2/ the rate of disappearance of hemoglobin from the circulation is rate limiting for the bilirubin excretion. 3/ the mechanism of degradation is not easily saturated. ACKNOWLEDGEMENTS We wish to thank Dr D. Roos for critically reading the manuscript.
REFERENCES 1. Bleeker WK,van der Plas J, Feitsma RU, et al., J. Lab. Clin. Med. 113, 151-161 (1989). 2. Berbers GAM. Bleeker WK, Slekkinger P, Agterberg J, Rigter G & Bakker JC, J. Lab. Clin. Med. 117, 157-165 (1991). 3. Keipen PE & Chang TMS, Vox Sang. 53,7-14 (1987). 4. Bleeker WK, van der Plas J, Agterberg J. Rigter G & Bakker JC, J. Lab. Clin. Med. 108, 448-455 (1%). 5. Ostrow JD.Jandl JH & Schmid R. J. Clin. Invest. 41, 1628-1637 (1962).
ISSN: 1055-7172 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ianb18
Effect of Polymerization on Clearance and Degradation of Free Hemoglobin Wim K. Bleeker, Guy A. M. Berbers, Piet J. den Boer, Jacques Agterberg, Gemma Rigter & Joachim C. Bakker To cite this article: Wim K. Bleeker, Guy A. M. Berbers, Piet J. den Boer, Jacques Agterberg, Gemma Rigter & Joachim C. Bakker (1992) Effect of Polymerization on Clearance and Degradation of Free Hemoglobin, Biomaterials, Artificial Cells and Immobilization Biotechnology, 20:2-4, 747-750, DOI: 10.3109/10731199209119713 To link to this article: http://dx.doi.org/10.3109/10731199209119713
Published online: 11 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ianb18 Download by: [McMaster University]
Date: 23 April 2016, At: 07:35
BIOMAT., ART. CELLS b IMMOB. BIOTECH., 20(2-4), 747-750 (1992)
Downloaded by [McMaster University] at 07:35 23 April 2016
EFFECT OF POLYMERIZATION ON CLEARANCE AND DEGRADATION OF FREE HEMOGLOBIN. Wim K.Bleeker, Guy A.M.Berbers, Piet J.den Boer, Jacques Agterberg, Gemma Rigter, and Joachim C.Bakker. Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. ABSTRACT In the present study we investigated the mechanism of prolongation of the plasma retention of free hemoglobin by polymerization. Polymerization of intramolecularly crosslinked hemoglobin with glutaraldehyde yields a mixture of large polymers, small polymers and monomers. In exchange transfusion experiments in rats we analyzed plasma samples by gel filtration to determine the clearance of polymers of different size. A positive correlation was found between polymer size and vascular retention. Furthermore, the clearance of large polymers appeared to be highly dose-dependent: after 20% and 70% exchange transfusions, we observed for large polymers a plasma half-life of 12 and 26 hours, respectively, whereas the half-life for 64kD monomers was 4 hours in both cases. The degradation of hemoglobin was followed by measuring the bilirubin excretion. The infused heme was recovered as bilirubin within 72 hours. The delay between the disappearance of free hemoglobin from the plasma and the recovery as bilirubin was about six hours and was not affected by polymerization or dose. We conclude that polymerization prevents the operation of certain clearance mechanisms, while still allowing a route of clearance that is easily saturated. The intracellular degradation of heme into bilirubin is not affected by the modifications of hemoglobin and is not easily saturated.
INTRODUCTION The vascular retention time of a hemoglobin solution is an important factor for its usefulness as blood substitute. To prolong vascular retention, we have modified human hemoglobin by intra- and intermolecular crosslinking with NFF'LP and glutaraldehyde, respectively. Previously, we have reported that intramolecular crosslinking (HbNFPLP) prolongs the vascular retention by eliminating the kidney excretion (1). After nephrectomy, there was no difference in clearance rate between HbNFPLP and unmodified hemoglobin, indicating that the other clearance mechanisms were not influenced. The present study was directed to the mechanism of further prolongation of the vascular retention by polymerization of HbNFPLP (polyHbNFPLP). In exchange transfusion experiments in rats with different doses of polyHbNFPLP we analyzed plasma samples to follow the clearance of polymers of different size. The degradation of plasma hemoglobin was studied by measuring the bilirubin excretion.
747 Copyright 0 1992 by Marcel DsLker, Inc.
7 48
BLEEKER ET AL.
30
h
Ul
M
25
DOSE
20
1.2 g/kg
3 0 5:
v
2.0 g/kg
15
M
3.8 g/kg
0
Downloaded by [McMaster University] at 07:35 23 April 2016
2
10 5
0
POLY
TE/TRI
DI
MONO
Figure 1 . Dose dependency o f half-disappearance time (T50%) of polyHbNFPLP polymers o f d i f f e r e n t s i z e s (mean 2 SD, n = 3 ) .
MATERIALS AND METHODS Hemoglobin solutions were prepared as described earlier (2). In brief, hemoglobin was isolated from human red blood cells by hypotonic lysis and tangential flow filtration. Intramolecular crosslinkingof the S-chainswas accomplished with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP). After purification with ion-exchange chromatography, HbNFPLP contained less than 5% non-crosslinked hemoglobin. Subsequent polymerhation (polyHbNFpLP) was achieved with glutaraldehyde to the desired molecular weight distribution: 10-20% polymers of >300 kD, 20-30% 64-kD monomers and virtually no dissociable hemoglobin. Exchange transfusions were performed in conscious female Wistar rats, weighing 200-250 g, placed in metabolic cages. One day prior to the exchange transfusion, a permanent cannula was introduced under anesthesia in the left carotid artery. In some rats, the common bile duct was also cannulated. Exchange transfusions were performed through the arterial cannula in steps of 2 ml. Blood samples were taken from the arterial cannula. Bile was collected in fractions of several hours, shielded from light, and stored at -2OOC. Hemoglobin concentration was measured photometrically as cyanomethemoglobin. The polymer size distribution of PolyHbNFPLP was determined by gel filtration analysis on a Superose-12 column (HR 10/30; Pharmacia). Total bilirubin was measured by the diazo reaction (Monotest 10 Bilirubin, Boehringer, Germany); plasma samples were pretreated with DMSO to precipitate interfering hemoglobin.
RESULTS AND DISCUSSION The plasma retention of polyHbNFPLP was found to be dose dependent, as has also been observed by other investigators for other polymerized Hb preparations (3).
Downloaded by [McMaster University] at 07:35 23 April 2016
EFFECT OF POLYMERIZATION
0
10
749
20
TIME -0-
HEMOGLOBIN CLEARANCE
40
30
50
80
(HOURS) -0-
RECOVERY A S BILIRUBIN
Figure 2 . Cumulative curves of hemoglobin clearance from plasma and o f r e c o v e r y of t h e i n f u s e d heme a s b i l i r u b i n , a f t e r a 70% excharge t r a n s f u s i o n w i t h polyHbNFPLP ( n e t dose 3 . 3 g / k g ) (mean + S D , n=3)
.
After exchange transfusions of 20, 35 and 70 % of the blood volume, giving a net dose of 1.2, 2 and 3.8 g/kg, respectively, plasma half-disappearance times were found of 8, 10 and 13 hours, respectively. Gel filtration analyses of plasma samples permitted the separate determination of the plasma clearance of polymers of different size. A distinction was made between: a monomer peak (64kD), a dimer peak (130 kD), a trihetramer peak (190-250 kD) and polymers (void volume, >300 kD).The results (figure 1) showed a positive correlation between plasma half-disappearance time and polymer size. Furthermore, it appeared that only the plasma retention of the polymers was dose dependent. The time courses of the clearance curves were different for monomers and polymers. The curves for monomers were exponential, both at low and high doses, whereas those for polymers were almost linear, especially at high dose. The results indicate that the clearance of the 64-kD monomer fraction is similar to that of non-polymerized HbNFPLP, which has been studied previously (4). Thus, the modification of lysine residues by the treatment with glutaraldehyde, which occurs randomly in all polymers and monomers, does not influence the clearance rate. The larger size of the polymers seems to be crucial for the prolongation of vascular retention. Since the clearance mechanism of the polymers was saturated and since their presence did not affect the monomer clearance, we conclude that monomers are cleared by another mechanism than polymers. Thus, the clearance mechanism that is responsible for the rapid clearance of the monomers is unable to remove the larger polymers. This leads
Downloaded by [McMaster University] at 07:35 23 April 2016
7 50
BLEEKER ET AL.
us to the hypothesis that several cell types can remove free hemoglobin from the circulalation (e.g.: macrophages, hepatocytes and endothelial cells), but that only part of these cells (with a limited capacity) are able to remove polymerized hemoglobin. The degradation of polyHbNFPLP was followed by measuring the increase in bilirubin production. PolyHbNFPLP was given in three different doses in 15,30 or 65% exchange transfusions (n=3), resulting in a net heme dose of 14, 26 and 52 pmollrat, respectively. In all cases, an increase in bilirubin excretion into the bile was already observed in the first sample interval (0 - 2 hours): 0.2 - 0.4 pmoVhour (control: 0.1 pmofiour). The maximum values for excretion were observed in the sample interval from 4 - 8 hours after transfusion: 0.75,0.95 and 1.6 prnofiour, respectively. Thereafter, bilirubin excretion gradually diminished, and after 48 hours it had returned to values close to normal. For comparison, we administered also HbNFPLP, which has a much shorter plasma retention. After a net dose of 25 pmol the maximum value for bilirubin excretion was higher than after a comparable dose of polyHbNFPLP: 1.2 pmolhour. The total bilirubin concentration in plasma showed a moderate increase up to 100 pmoI/l. Since this was accompanied by excretion into the urine, part of it probably concerns conjugated bilirubin. Thus, accumulation in the plasma of the potentially toxic, unconjugated bilirubin seems not to be a point of major concern after administration of hemoglobin solutions. After 48 hours 98 & 20% (n= 12) of the infused heme was recovered as bilirubin in bile and urine. The time lag between the disappearance of free Hb from the circulation and recovery as bilirubin was about 6 - 8 hours (see figure 2). The recovery was expressed as a percentage of the expected amount, based on a conversion of 1 heme group to 1 bilirubin molecule. This time lag was about the same for all doses of polyHbNFPLP and also for HbNFPLP. Ostrow et al. (5). found at much lower doses of unmodified hemoglobin (12 to 60 mgkg), a similar value of 3 hours for the delay between extravascular sequestration of hemoglobin and recovery as bilirubin. Their experiments gave also evidence that the delay is mainly due to the breakdown of hemoglobin and conversion to bilirubin and not to the excretion of bilirubin. We conclude from our experiments on degradation that: 1/ the intracellular degradation of hemoglobin is not affected by the intramolecular crosslinkingwith NFPLP and subsequent polymerization with glutaraldehyde. 2/ the rate of disappearance of hemoglobin from the circulation is rate limiting for the bilirubin excretion. 3/ the mechanism of degradation is not easily saturated. ACKNOWLEDGEMENTS We wish to thank Dr D. Roos for critically reading the manuscript.
REFERENCES 1. Bleeker WK,van der Plas J, Feitsma RU, et al., J. Lab. Clin. Med. 113, 151-161 (1989). 2. Berbers GAM. Bleeker WK, Slekkinger P, Agterberg J, Rigter G & Bakker JC, J. Lab. Clin. Med. 117, 157-165 (1991). 3. Keipen PE & Chang TMS, Vox Sang. 53,7-14 (1987). 4. Bleeker WK, van der Plas J, Agterberg J. Rigter G & Bakker JC, J. Lab. Clin. Med. 108, 448-455 (1%). 5. Ostrow JD.Jandl JH & Schmid R. J. Clin. Invest. 41, 1628-1637 (1962).
