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Interaction of degraded iota carrageenans with plasma membranes: Sedimentation of erythrocytes of different species
Life Sciences Vol. 17, pp . 969-974 Printed in the U .S .A .
Pergamon Press
INTERACTION OF DEGRADED IOTA CARRAGEENANS WITH PLASMA MEMBRANES: SED72~II1~TfATION OF ERYTHROCYTES OF DIFFERENT SPECIES E .P . Pittz, L . Golberg and F. Coulaton
Summary
Center of Experimental Pathology and Toxicology Albany Medical College Albany, New York 12208 (Received in final form August 22, 1975)
The sedimentation of erythrocytes by polydieperse fractions of iota carrageenan has been studied . For human erythrocytes, it is found that their sedimentation is dependent oa the number average molecular weight of the carrageenan fraction, the sedimentation rate increasing generally as the molecular weight of the fraction increases until a maximum value is reached, after which the sedimentation rate decreases. The increase in sedimentation of erythrocytes can be reversed by removal of the carrageenan . Addition of EDTA to the carrageenan-erythrocyte medium does not effect the increase is erythrocyte sedimentation rate by carrageenan . The carrageenan-facilitated sedimentation of erythrocytes eahibita wide species differences . The mechanism by which iota carrageenans enhance erythrocyte sedimentation is discussed. INTRODUCTION Carrageenans are high molecular weight sulfated polysaccharides extracted from marine algae (1) . They are widely used as food additives . The cytotoxicity of carrageenan has been studied .fn v.itJCO (2,3) . Under certain conditions a form of degraded iota carrageenan used as a therapeutic agent has been found to be ulcerogenic .En vfvo (4-7) . In order to gain an understanding of the pathogenesis of intestinal ,ulceration elicited by carrageenan fractions, it is necessary to determine whether or not carrageenane can penetrate and cross membranes of non-phago cytic cells . Little is known concerning the sites of interaction of carrageenans and other sulfated polysaccharides with cell surfaces . However, it is recognized that carrageenan and other sulfated polysaccharides can alter membrane mediated biological functions such as cell division (8) and cell fertilization (8), can suppress delayed hypersensitivity (9), can inhibit the complement system (9,10) and alter cell fragility (11) . A study of the mechanism of interaction of carrageenan with cell surfaces was undertaken, using erythrocytes as the initial source of membranes . MATERIALS AND METHODS Human blood was obtained from the Albany Medical Center Hospital Blood Bank and used immediately after being drawn. Blood was collected from the following laboratory animals: guinea pig (Hartley strain, average weight 360g), rat (Sprague-Dawley, average weight 230g), monkey (Rhesus, MQCLLCIL mu.Pattlt, average weight 7 kg) and mouse (Charles River strain, average weight 30g) . Chicken blood was obtained from a local farm . All blood was drawn in citrate, the erythrocytes washed three times with isotonic saline (five volumes) in phosphate buffer, pH 7 .4, and used immediately after washing . The degraded polydieperse fractions of iota carrageenan, prepared by mild acid hydrolysis were obtained from Marine Colloids, Inc ., Rockland, Maine . The counterion in these fractions was predominantly sodium, with 0 .75 - 1 .60X calcium by weight of the fraction . Number average molecular 969
g7p
Sedimentation of Erythrocytes
Vol. 17, No . 6
weights of the degraded fractions were estimated by Marine Colloids, using viscosity and electrophoresis (13) . A degraded fraction of iota carrageenan (Mn - 9,700) was provided by LCibohA,to .iJc¢.6 O~u.zo, Paris, France . All Calcium chloride (reagent carrageenan fractions were used as received . grade) was obtained from Fisher Scientific, Fair Lawn, New Jersey, and EDTA from J.T . Baker Co ., Phillipaburg, New Jersey . Sedimentation experiments were carried out in Wintrobe tubes (ClaySuspensions in isotonic Adams, Inc ., New Jersey) at ambient temperature . saline pH 7 .4, were adjusted to a standard hematocrit of 57X . Hematocrits of 27 .8 or 28 .5X were used since these give erythrocyte sedimentation rates that can be accurately and conveniently measured . Carrageenan stock solutions in isotonic saline, pH 7 .4 were prepared immediately before use . One ml aliquots of each carrageenaa stock solution were mixed with 1 ml of erythrocyte suspension and kept at room temperature for 20 min before addition to the Wintrobe tube . Reversibility experiments were carried out by incubating carrageenan with erythrocytes for 20 min, measuring the sedimentation rate in the presence and absence (control) of carrageenan, and then washing both sample and control erythrocytes three times with isotonic saline, pH 7 .4 (5 volumes), and again measuring the sedimentation of sample and control erythrocytes . RESULTS Figure 1 illustrates the time course of sedimentation of human erythrocytes by the degraded polydisperse fractions of iota carrageenan (ICG) 1 . It can be seen that the higher molecular weight fractions of ICG (M >39,000) greatly enhance the sedimentation rate of human erythrocy~es in contrast to the lower molecular weight fractions, which It ie also have only a alight effect on the sedimentation rate . noted from this figure that the highest sedimentation rate of h~an erythrocytes is attained in the presence of ICG having M 145,000 ; the fraction with 250,000 has less effect on the sedimentation ~ rate . The viscosity of ICG increases with increasing molecular weight . Thus, taking the viscosity of the solutions into consideration, the increased sedimentation rate of erythrocytes by the higher Mil fractions would be greater than shown in Figure 1. When human erythrocytes which were previously incubated with ICG times with isotonic saline, pH 7 .4 (5 volumes (~n 145,000) were washed three per wash) to remove ICG, the sedimentation rate returned to control levels . The addition of EDTA (0 .66 mM) to the medium containing h~an eryOn the throcytes and ICG (Mn 145,000) did not alter the sedimentation rate . other hand, addition of calcium to the system caused extensive visible ag-
gregation of the erythrocytes . Species differences in the sedimentation rate of erythrocytes in the presence of ICG (Mn 145,000) are presented in Fig . II . Monkey and human erythrocytes sediment at about the same rate while guinea pig and rat erythro cytes sediment at a slightly but significantly lower rate . Mouse and chicken erythrocytes exhibit sedimentation characteristics distinctly different from those of the other species studied; the mouse erythrocytes had about the same sedimentation rate as controls (no ICG) while with chicken erythroIn cytes there was a long delay before appreciable sedimentation commenced. fact the erythrocytes of all species studied exhibited some delay in sedimentation, but with chicken erythrochtes sedimentation was delayed for 100 min compared to about 20 min for four of the other species studied . Abbreviations : ICG, polydiaperae number average molecular weight iota carrageenan ; EDTA, dieodium ethylenediamine tetraacetic acid .
Vol. 17, No . 6
Figure 1.
DISCUSSION
Sedimentation of Erythrocytes
Sedimentation of human erythrocytes in the presence and absence of degraded polydieperee fractions of iota carrageenan . tlematocrit 27 .8X ; carrageenaa concentration 0 .66 mg/ml . The M value of each fraction is indicated at the end of the corresp$nding curve.
The sedimentation of erythrocytes by polymers has been well documented (13-17) . Dextran, which ie used as a plasma expander, has been studied eatenaively (14-18) . However, from observation of the hematocrit used, sedimentation rates, molecular weight and concentration of the polymers, it appears that ICG is three hundred times more efficient than dextran in sedimenting erythrocytes (17) .
972
Sedimentation of Erythrocytes
20
40
60
80
100
120
Vol. 17, No . 6
140
160
1A0
200
TIME (min .)
Figure II
Sedimentation of erythrocytes from different species in the presence of iota carrageenan (fraction having 145,000) . Hematocrit, â 28 .5X ; carrageenan concentration, 2 mg/ml .
Experimental evidence has been put forward to explain the mechanism by which neutral polymers, such as dextran, enhance the sedimentation of erythrocytes (13-18) . Polymers of molecular weight greater than a certain value (20,000 - 40,000) are long enough to straddle the electrical double layers of two cells and thus cause cellular aggregation . In contrast to dextran and the other neutral polymers known to aggregate and thus sediment erythrocytes, ICG is a highly negatively charged polyelectrolyte that theoretically should not interact strongly with the net negatively charged erythrocyte surface . The fact that erythrocytes incubated with ICG sediment at a rate 300 times that of erythrocytes incubated with dextraa suggests that ICG undergoes a strong interaction with the For a polyelectrolyte such ae ICG to interact powererythrocyte surface. fully with the erythrocyte surface, electrostatic forces would appear to be the prominent influence involved in the aggregation phenomenon . The fact that EDTA does not interfere in the aggregation process indicates that either divalent cations are not necessary for ICG to interact with erythrocytes or that the erythrocyte surface has a much greater affinity for Cam than does EDTA . On the other hand, Cam greatly enhances the aggregation of erythrocytes by high molecular weight ICG . This observation is consistent with the theory that ICG must straddle the electrical double layer of cells in order to enhance aggregatioa . That ie, Cam can act to charge screen the net negative charges on the electrical double layers of the
Vol. 17, No . 6
Sedimentation of Erythrocytes
973
cell surface (11) and can also form bridges between the sulfate groups of ICG and the negatively charged groups on the erythrocyte surface . Sulfated mucopolyeaccharides are known to play a role in inhibiting cell division and are believed to do so by masking activator sites on cell surfaces (8) . Thus, as understanding of the toxicological effects of ICG on living organisms may provide clues to the physiological mechanisms by which sulfated mucopolyeaccharides, in general, interact with cellular surfaces . The fact that ICG affects the sedimentation of erythrocytes from various species of animals in a differnt manner indicates that there are qualitative as well as quantitative differences in the interaction of ICG with different types of cell surfaces . Research is presently under way to determine the physical 88 well as the chemical nature of the site of interaction of ICG with plasma membranes . Acknowledgements : Supported by research grant 2P01-ES00226-08 from the National Institute of Environmental Sciences and by National Institutes of Health training grant 2T01-ES00703-08 . The authors are grateful to Marine Colloids, Inc ., Rockland, Maine for the gift of the carrageenan fractions and to Albany Medical Center Hospital Blood Bask for the human whole blood .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
13 . 14 . 15 . 16 . 17 . 18 .
K .B . Guiaeley, Seaweed Colloids . Encyclopedi a of Chemical Technology , 763-784 (1968) . P .J . Catanzaro, H.G . Schwartz and R.C . Grahm, Am . J . Path . 64, 387-399 (1971) . A.C . Allison, J .G . Harringtoa and M. Birbeck, J . Ezptl . Med . 124, 141153 (1966) . R. Abraham, R.J . Fabien, L . Golberg and F. Coulaton, Gaetroenterol . 67, 1169-1181 (1974) . R.-F . Benitz, L . Golberg and F . Coulaton, Fd . Cosmet . Toxicol . 11, 569579 (1973) . J . Watt and R. Marcus, _Gut 14, 506-510 (1973) . P . Grasso, M. Sharratt, F.M .B . Carpanini aad S .D . Gangopi, Fd . Cosmet . Toxicol . 11, 555-564 (1973) . M. Lippman, Traps . N .Y . Aced . Sci . 27, 342-360 (1964) . T . Boron, H .T . Rapp and C .J . Crisler, J . Immunol., 94, 662-673 (1965) G .E . Davier, Immuaology 8, 291-299 (1965) . E .P . Pittz, L . Golberg and F. Coulaton, Toxicol . Appl . Pharmacol . In press (1975) . N .F . Stanley aad D .W . Renn, Proç. VIII Int . Seaweed Symposium. Bangor, In press (1975) . S . Chien and K.M . Jan, J. Supramolecular Struct . _1, 385-409 (1975) . K .M . Jan and S . Chien, J. Gen . Phyeiol . _61, 638-654 (1973) . K.M . Jan and S . Chien, J . Gen. Phyeiol . _61, 655-668 (1973) . A. Larcan, F . Streiff, B . Genstet and A. Peters, Bibl . Haematol ., No . 33, 330-337 (1969) . Jan, Ref . 15, page 659 . K . Hummel, Bibl . Haematol . No . 33, 322-329 (1969) .
Pergamon Press
INTERACTION OF DEGRADED IOTA CARRAGEENANS WITH PLASMA MEMBRANES: SED72~II1~TfATION OF ERYTHROCYTES OF DIFFERENT SPECIES E .P . Pittz, L . Golberg and F. Coulaton
Summary
Center of Experimental Pathology and Toxicology Albany Medical College Albany, New York 12208 (Received in final form August 22, 1975)
The sedimentation of erythrocytes by polydieperse fractions of iota carrageenan has been studied . For human erythrocytes, it is found that their sedimentation is dependent oa the number average molecular weight of the carrageenan fraction, the sedimentation rate increasing generally as the molecular weight of the fraction increases until a maximum value is reached, after which the sedimentation rate decreases. The increase in sedimentation of erythrocytes can be reversed by removal of the carrageenan . Addition of EDTA to the carrageenan-erythrocyte medium does not effect the increase is erythrocyte sedimentation rate by carrageenan . The carrageenan-facilitated sedimentation of erythrocytes eahibita wide species differences . The mechanism by which iota carrageenans enhance erythrocyte sedimentation is discussed. INTRODUCTION Carrageenans are high molecular weight sulfated polysaccharides extracted from marine algae (1) . They are widely used as food additives . The cytotoxicity of carrageenan has been studied .fn v.itJCO (2,3) . Under certain conditions a form of degraded iota carrageenan used as a therapeutic agent has been found to be ulcerogenic .En vfvo (4-7) . In order to gain an understanding of the pathogenesis of intestinal ,ulceration elicited by carrageenan fractions, it is necessary to determine whether or not carrageenane can penetrate and cross membranes of non-phago cytic cells . Little is known concerning the sites of interaction of carrageenans and other sulfated polysaccharides with cell surfaces . However, it is recognized that carrageenan and other sulfated polysaccharides can alter membrane mediated biological functions such as cell division (8) and cell fertilization (8), can suppress delayed hypersensitivity (9), can inhibit the complement system (9,10) and alter cell fragility (11) . A study of the mechanism of interaction of carrageenan with cell surfaces was undertaken, using erythrocytes as the initial source of membranes . MATERIALS AND METHODS Human blood was obtained from the Albany Medical Center Hospital Blood Bank and used immediately after being drawn. Blood was collected from the following laboratory animals: guinea pig (Hartley strain, average weight 360g), rat (Sprague-Dawley, average weight 230g), monkey (Rhesus, MQCLLCIL mu.Pattlt, average weight 7 kg) and mouse (Charles River strain, average weight 30g) . Chicken blood was obtained from a local farm . All blood was drawn in citrate, the erythrocytes washed three times with isotonic saline (five volumes) in phosphate buffer, pH 7 .4, and used immediately after washing . The degraded polydieperse fractions of iota carrageenan, prepared by mild acid hydrolysis were obtained from Marine Colloids, Inc ., Rockland, Maine . The counterion in these fractions was predominantly sodium, with 0 .75 - 1 .60X calcium by weight of the fraction . Number average molecular 969
g7p
Sedimentation of Erythrocytes
Vol. 17, No . 6
weights of the degraded fractions were estimated by Marine Colloids, using viscosity and electrophoresis (13) . A degraded fraction of iota carrageenan (Mn - 9,700) was provided by LCibohA,to .iJc¢.6 O~u.zo, Paris, France . All Calcium chloride (reagent carrageenan fractions were used as received . grade) was obtained from Fisher Scientific, Fair Lawn, New Jersey, and EDTA from J.T . Baker Co ., Phillipaburg, New Jersey . Sedimentation experiments were carried out in Wintrobe tubes (ClaySuspensions in isotonic Adams, Inc ., New Jersey) at ambient temperature . saline pH 7 .4, were adjusted to a standard hematocrit of 57X . Hematocrits of 27 .8 or 28 .5X were used since these give erythrocyte sedimentation rates that can be accurately and conveniently measured . Carrageenan stock solutions in isotonic saline, pH 7 .4 were prepared immediately before use . One ml aliquots of each carrageenaa stock solution were mixed with 1 ml of erythrocyte suspension and kept at room temperature for 20 min before addition to the Wintrobe tube . Reversibility experiments were carried out by incubating carrageenan with erythrocytes for 20 min, measuring the sedimentation rate in the presence and absence (control) of carrageenan, and then washing both sample and control erythrocytes three times with isotonic saline, pH 7 .4 (5 volumes), and again measuring the sedimentation of sample and control erythrocytes . RESULTS Figure 1 illustrates the time course of sedimentation of human erythrocytes by the degraded polydisperse fractions of iota carrageenan (ICG) 1 . It can be seen that the higher molecular weight fractions of ICG (M >39,000) greatly enhance the sedimentation rate of human erythrocy~es in contrast to the lower molecular weight fractions, which It ie also have only a alight effect on the sedimentation rate . noted from this figure that the highest sedimentation rate of h~an erythrocytes is attained in the presence of ICG having M 145,000 ; the fraction with 250,000 has less effect on the sedimentation ~ rate . The viscosity of ICG increases with increasing molecular weight . Thus, taking the viscosity of the solutions into consideration, the increased sedimentation rate of erythrocytes by the higher Mil fractions would be greater than shown in Figure 1. When human erythrocytes which were previously incubated with ICG times with isotonic saline, pH 7 .4 (5 volumes (~n 145,000) were washed three per wash) to remove ICG, the sedimentation rate returned to control levels . The addition of EDTA (0 .66 mM) to the medium containing h~an eryOn the throcytes and ICG (Mn 145,000) did not alter the sedimentation rate . other hand, addition of calcium to the system caused extensive visible ag-
gregation of the erythrocytes . Species differences in the sedimentation rate of erythrocytes in the presence of ICG (Mn 145,000) are presented in Fig . II . Monkey and human erythrocytes sediment at about the same rate while guinea pig and rat erythro cytes sediment at a slightly but significantly lower rate . Mouse and chicken erythrocytes exhibit sedimentation characteristics distinctly different from those of the other species studied; the mouse erythrocytes had about the same sedimentation rate as controls (no ICG) while with chicken erythroIn cytes there was a long delay before appreciable sedimentation commenced. fact the erythrocytes of all species studied exhibited some delay in sedimentation, but with chicken erythrochtes sedimentation was delayed for 100 min compared to about 20 min for four of the other species studied . Abbreviations : ICG, polydiaperae number average molecular weight iota carrageenan ; EDTA, dieodium ethylenediamine tetraacetic acid .
Vol. 17, No . 6
Figure 1.
DISCUSSION
Sedimentation of Erythrocytes
Sedimentation of human erythrocytes in the presence and absence of degraded polydieperee fractions of iota carrageenan . tlematocrit 27 .8X ; carrageenaa concentration 0 .66 mg/ml . The M value of each fraction is indicated at the end of the corresp$nding curve.
The sedimentation of erythrocytes by polymers has been well documented (13-17) . Dextran, which ie used as a plasma expander, has been studied eatenaively (14-18) . However, from observation of the hematocrit used, sedimentation rates, molecular weight and concentration of the polymers, it appears that ICG is three hundred times more efficient than dextran in sedimenting erythrocytes (17) .
972
Sedimentation of Erythrocytes
20
40
60
80
100
120
Vol. 17, No . 6
140
160
1A0
200
TIME (min .)
Figure II
Sedimentation of erythrocytes from different species in the presence of iota carrageenan (fraction having 145,000) . Hematocrit, â 28 .5X ; carrageenan concentration, 2 mg/ml .
Experimental evidence has been put forward to explain the mechanism by which neutral polymers, such as dextran, enhance the sedimentation of erythrocytes (13-18) . Polymers of molecular weight greater than a certain value (20,000 - 40,000) are long enough to straddle the electrical double layers of two cells and thus cause cellular aggregation . In contrast to dextran and the other neutral polymers known to aggregate and thus sediment erythrocytes, ICG is a highly negatively charged polyelectrolyte that theoretically should not interact strongly with the net negatively charged erythrocyte surface . The fact that erythrocytes incubated with ICG sediment at a rate 300 times that of erythrocytes incubated with dextraa suggests that ICG undergoes a strong interaction with the For a polyelectrolyte such ae ICG to interact powererythrocyte surface. fully with the erythrocyte surface, electrostatic forces would appear to be the prominent influence involved in the aggregation phenomenon . The fact that EDTA does not interfere in the aggregation process indicates that either divalent cations are not necessary for ICG to interact with erythrocytes or that the erythrocyte surface has a much greater affinity for Cam than does EDTA . On the other hand, Cam greatly enhances the aggregation of erythrocytes by high molecular weight ICG . This observation is consistent with the theory that ICG must straddle the electrical double layer of cells in order to enhance aggregatioa . That ie, Cam can act to charge screen the net negative charges on the electrical double layers of the
Vol. 17, No . 6
Sedimentation of Erythrocytes
973
cell surface (11) and can also form bridges between the sulfate groups of ICG and the negatively charged groups on the erythrocyte surface . Sulfated mucopolyeaccharides are known to play a role in inhibiting cell division and are believed to do so by masking activator sites on cell surfaces (8) . Thus, as understanding of the toxicological effects of ICG on living organisms may provide clues to the physiological mechanisms by which sulfated mucopolyeaccharides, in general, interact with cellular surfaces . The fact that ICG affects the sedimentation of erythrocytes from various species of animals in a differnt manner indicates that there are qualitative as well as quantitative differences in the interaction of ICG with different types of cell surfaces . Research is presently under way to determine the physical 88 well as the chemical nature of the site of interaction of ICG with plasma membranes . Acknowledgements : Supported by research grant 2P01-ES00226-08 from the National Institute of Environmental Sciences and by National Institutes of Health training grant 2T01-ES00703-08 . The authors are grateful to Marine Colloids, Inc ., Rockland, Maine for the gift of the carrageenan fractions and to Albany Medical Center Hospital Blood Bask for the human whole blood .
2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
13 . 14 . 15 . 16 . 17 . 18 .
K .B . Guiaeley, Seaweed Colloids . Encyclopedi a of Chemical Technology , 763-784 (1968) . P .J . Catanzaro, H.G . Schwartz and R.C . Grahm, Am . J . Path . 64, 387-399 (1971) . A.C . Allison, J .G . Harringtoa and M. Birbeck, J . Ezptl . Med . 124, 141153 (1966) . R. Abraham, R.J . Fabien, L . Golberg and F. Coulaton, Gaetroenterol . 67, 1169-1181 (1974) . R.-F . Benitz, L . Golberg and F . Coulaton, Fd . Cosmet . Toxicol . 11, 569579 (1973) . J . Watt and R. Marcus, _Gut 14, 506-510 (1973) . P . Grasso, M. Sharratt, F.M .B . Carpanini aad S .D . Gangopi, Fd . Cosmet . Toxicol . 11, 555-564 (1973) . M. Lippman, Traps . N .Y . Aced . Sci . 27, 342-360 (1964) . T . Boron, H .T . Rapp and C .J . Crisler, J . Immunol., 94, 662-673 (1965) G .E . Davier, Immuaology 8, 291-299 (1965) . E .P . Pittz, L . Golberg and F. Coulaton, Toxicol . Appl . Pharmacol . In press (1975) . N .F . Stanley aad D .W . Renn, Proç. VIII Int . Seaweed Symposium. Bangor, In press (1975) . S . Chien and K.M . Jan, J. Supramolecular Struct . _1, 385-409 (1975) . K .M . Jan and S . Chien, J. Gen . Phyeiol . _61, 638-654 (1973) . K.M . Jan and S . Chien, J . Gen. Phyeiol . _61, 655-668 (1973) . A. Larcan, F . Streiff, B . Genstet and A. Peters, Bibl . Haematol ., No . 33, 330-337 (1969) . Jan, Ref . 15, page 659 . K . Hummel, Bibl . Haematol . No . 33, 322-329 (1969) .