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Effect of phenylalanine-or tryptophan-loaded liposomes on the rheological properties of AA and SS erythrocytes
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
38,
246-2.51 (1987)
Effect of Phenylalanine- or Tryptophan-Loaded Liposomes on the Rheological Properties of AA and SS Erythrocytes’ JAYALAKSHMI
KUMPATI
Department of Pediatrics, Section of Hematology/Oncology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262 Received March 10. 1987
Certain aromatic amino acids such as phenylalanine (Phe) or tryptophan (Trp) and short peptides containing Phe have been shown to inhibit polymerization of deoxy hemoglobin (Hb)S in solution (l-4). Permeability studies with 14C-labeled peptides showed that these compounds do not readily penetrate the intact red blood cell membrane under the usual conditions of incubation (1,4,5). However, the author has developed a liposomal transport system (6,7) to transfer aromatic amino acids effectively across the red blood cell membrane, and has shown that the incorporation of Phe or Trp into sickle (SS) red blood cells (RBCs) via liposomal transport system markedly inhibits and reverses the in vitro sickling of deoxy Hb S (6,7). Furthermore, normal and SS RBCs loaded with Phe or Trp did not exhibit significant changes in osmotic fragility, mechanical fragility, autohemolysis, and glycolysis when compared to untreated RBCs (7). In addition, the oxygen affinity measured as the Ps,, and concentrations of 2,3-DPG and ATP were not affected by the incorporation of Phe or Trp into normal (AA) or SS RBCs (7). In the present study, the effect of Phe or Trp incorporation on the rheological properties of AA and SS RBCs are evaluated. MATERIALS
AND METHODS
Preparation of Phe or Trp loaded RBCs. Liposomes loaded with or without amino acids (Phe or Trp) were prepared by the method of Kumpati (7). Venous blood was obtained in CPDA (Fenwal Labs, Deerfield, IL) or heparin as anticoagulant from patients with homozygous sickle cell anemia and from normal, healthy volunteers. RBCs were prepared as described previously (7). Equal volumes of RBCs and liposome suspensions were mixed and incubated at 37°C for 30-60 min. After incubation, the RBC suspensions were centrifuged at 800g for 10 min. After removing the supematant, the RBCs were washed twice with 3 vol of 310 mM (isotonic) phosphate buffer, pH 7.45, and were resuspended in the same buffer to a final hematocrit (HCT) of 40% (7). ’ Part of this work was presented in poster form at the American Society of Hematology Meeting (December 5-9, 1985), and the American Pediatric Society Meeting (May 5-9, 1986). 246 08%4505/87 Copyright All rights
$3.00
Q 1987 by Academic Press, Inc. of reproduction in any form reserved.
Phe- AND Trp-LOADED
LIPOSOMES’
EFFECT
ON ERYTHROCYTES
247
o Untreated l Treated
;; 2i
& 20 = I!
01
I 60
I
1
40
20
HEMATOCRIT
!%)
FIG. 1. Viscosity in centipoise at various speeds for buffered, liposome-treated, (Phe or Trp)-incorporated, oxy and deoxy AA and SS RBCs.
and amino acid
Rheology. Viscosity measurements were carried out by the method of Chien et al. (8) and Leasberg and Hedley-Whyte (9) with a Wells-Brookfield LVT cone and plate viscometer (Brookfield Engineering Labs, Stoughton, MA) using 1.5 ml each of buffered RBCs, liposome-treated, and amino acid (Phe or Trp)incorporated RBCs (both AA and SS). To keep the RBCs in the deoxy state, the sample cup of the viscometer was equipped with a purge setting so that air could be flushed out and nitrogen could be introduced into the chamber. The viscometer was calibrated against ASTM viscosity standard, S-2 oil, Lot 71202 (Canon Instruments, PA). RESULTS The viscosities in centipoise at various speeds for buffered, liposome-treated, and amino acid (Phe or Trp)-incorporated, oxy and deoxy AA and SS RBCs are illustrated in Figs. lA-D. Figure 1A demonstrates the markedly increased viscosity of oxygenated SS RBCs in buffer which is further elevated with deoxygenation. AA RBCs showed no change with deoxygenation. Viscosity patterns for both oxy and deoxy AA and SS RBCs after treatment by liposome without amino acid incorporation appeared similar to that for untreated RBCs (B). With incorporation of Phe, there was a decrease in viscosity of both oxygenated and deoxygenated SS RBCs to levels observed with AA RBCs. Similar results were seen when Trp was incorprated into SS RBCs. Figure 2 shows the viscosity at different sheer rates for deoxygenated AA and SS RBCs. The increased viscosity of deoxygenated SS RBCs in buffer and after treatment by liposomes without amino acids is most pronounced at the lower sheer rates (Figs. 2A and B). Incorporation of Phe and Trp restored normal viscosity to deoxygenated SS RBCs even at low sheer rates (Figs. 2C and D). The viscosity at different HCT levels for deoxy AA and SS RBCs treated with liposomes alone and after Phe incorporation is presented in Fig. 3. The increased viscosity of deoxy SS cells
248
JAYALAKSHMI 0 W-AA . w-.%3
24 -
KLJMPATI
24 -
A
0 L&AA l Lip.-SS
B
20 -
0
46n
92
138
184
230
o-
’ 46
SHEER RATE/Sac
o Pha-AA . PhcSS
R-
c
’ ’ 92 138 SHEER RATE/Sac
S-
’ 184
a 230
OTWAA
D
l
20
46
92
13R
1111
230
2O
SHEER RAlE/Ssc
FIG.
46
92 130 WEEK UlE/k
-
Trp-ss
184
230
2. Viscosity at different sheer rates for deoxygenated AA and SS RBCs (n = 4).
Speed
Crpm)
FIG. 3. Viscosity versus hematocrit levels for deoxy AA and SS RBCs treated with liposomes alone, and after Phe incorporation.
Phe-
AND
Trp-LOADED
LIPOSOMES’
EFFECT
is the most marked at the higher hematocrits, HCT levels after Phe incorporation.
ON
ERYTHROCYTES
but is reduced to normal
249
at all
DISCUSSION
A therapeutic agent for the treatment of sickle cell disease needs to satisfy many criteria. It must prevent sickling, act at low concentrations, and should not interfere with erythrocyte function and metabolism. The incorporation of the amino acids Phe or Trp into SS RBCs by use of liposomes to transport them across the red blood cell membrane has recently been shown by Kumpati to totally inhibit and reverse sickling of Hb S in vitro (67). Further, the incorporation of Phe or Trp into RBCs did not have any adverse effect on cell function or metabolism (7). The oxygen afIinity, concentrations of 2,3-DPG and ATP, glycolytic activity, osmotic and mechanical fragilities, and autohemolysis of both AA and SS RBCs were not altered by either liposome or amino acid (Phe or Trp) incorporation (7). It was therefore important to determine whether this technique has any beneficial or adverse effect on the rheological properties of RBCs. The most noteworthy effect of the incorporation of Phe or Trp into SS RBCs was the marked decrease in blood viscosity to the normal range for both deoxygenated and oxygenated SS RBCs. Factors affecting blood viscosity include hematocrit, aggregation of red cells, the internal viscosity of red cells, osmolarity, red cell ATP, membrane flexibility or rigidity (RBC deformability), and the shape of the red cells (10-14). The increased viscosity of deoxy SS RBCs in buffer or treated with liposomes alone (without Phe or Trp) was most likely related to changes in both cell shape and membrane since 100% of the SS cells were “sickled” in this sytsem (13). It has been shown that the membranes of SS RBCs become rigid upon deoxygenation (11,13) with associated loss of elasticity which contributes to the marked increase in viscosity (10). These membrane changes have been postulated to be secondary to polymerization of Hb S (11,13). In hemolysates, Phe or Trp are known to inhibit polymerization (3,4). Recent proton nuclear magnetic resonance studies (15) show that the binding of Phe/Trp to deoxy Hb S is comparable to Hb A. One of the binding sites common to all of the antisickling compounds (including Phe or Trp) investigated (15), is at or near the heme pockets in the a and p chain of both deoxy Hb A and Hb S. The other binding site is located at or near the /3” position, containing the mutation in Hb S, p6 Glu + Val (15). The binding of the antisickling compound to the latter site induces conformational changes in the amino terminal derivatives of the j3 chains (1.5). Introduction of Phe or Trp into the intact deoxygenated SS RBCs by the liposomal transport system has been previously shown by Kumpati (67) to prevent sickling of intact sickle red cells. This antipolymerization effect may be the one mechanism that is responsible for normalization of the viscosity of these deoxygenated SS RBCs. The increased viscosity of oxygenated SS RBCs was confirmed in this study. It has previously been shown to be due primarily to the altered rheology of irreversibly sickled cells (ISCs) and to a lesser extent to non-ISCs (13). Ninety percent of the oxygenated SS RBCs in the present system were not sickled, suggesting increased rigidity of the membrane. The complete normalization of
250
JAYALAKSHMI
KUMPATI
the viscosity of these predominantly unsickled oxygenated cells after amino acid incorporation might suggest normalization of certain factors affecting the cell membrane in addition to the antipolymerizing effect on hemoglobin S. While a beneficial effect was noted in the viscosity of SS RBCs after amino acid incorporation, no adverse effect was found on cell function or metabolism (7). The oxygen affinity, concentrations of ATP and 2,3-DPG, glycolytic activity, mechanical fragility, and autohemolysis of both AA and SS RBCs were not altered by either liposome or amino acid incorporation (7). No change was demonstrated in osmotic fragility with treatment although as previously described (10) increased resistance of SS RBCs was noted. In conclusion, these data suggestno adverse effect in vitro from the incorporation of liposomes, the amino acids Phe or Trp, on the rheology, the membrane, the metabolism, or the oxygen affinity of erythrocytes from normal adults or from persons with homozygous sickle cell anemia. On the contrary, the abnormal viscosity of both oxy and deoxy erythrocytes from sickle cell disease patients was restored to normal. This confirms the original finding that sickling can be prevented by the incorporation of Phe or Trp into intact RBCs by a liposomal transport system (6,7) and further shows that certain rheological abnormalities are inhibited. SUMMARY
Phenylalanine or tryptophan was incorporated into AA and SS red blood cells by a liposomal transport system which was previously shown by Kumpati to inhibit and reverse sickling of intact SS red blood cells in vitro. In the present study, the effect of phenylalanine or tryptophan incorporation on the rheological properties was evaluated. The incorporation of phenylalanine or tryptophan into red blood cells decreased the viscosity of deoxy SS red blood cells which reached a level close to that for normal red blood cells due to the antisickling effect. These results demonstrate that this liposomal transport system which transferred phenylalanine or tryptophan into intact red cells and did not have any adverse effect on red cell metabolism or function did correct the viscosity of deoxy SS red cells by its antisickling effect. This method may have significant therapeutic implications in the treatment of sickle cell disease. ACKNOWLEDGMENTS The author thanks Drs. D. Ambruso and J. Githens for review of the manuscript, Donna Dixon, R.N., MS., for obtaining blood samples, and Kathleen Hoyer for its preparation. This work was supported in part by Grant HL 31383 from the National Institutes of Health, the Bonfils Blood Center, and the Colorado Sickle Cell Treatment and Research Center.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Gore&i, Kubota, Noguchi, Noguchi,
M., Votano, J. R., and Rich, A., Biochemistry 19, 1564 (1980). S., and Yang, J. T., Proc. Natl. Acad. Sci. USA 74, 5431 (1977). C. T., and Schechter, A. N., Biochem. Biophys. Res. Commum. 74, 637 (1977). C. T., and Schechter, A. N., Biochemistry 17, 5455 (1978). votano, J. R., Gorecki, M., and Rich, A., Science 196, 1216 (1977). Kumpati, J., Biochem. Biophys. Res. Commun. 105, 482 (1982). Kumpati, J., Biochem. Med. Metab. Biol. 38, 170 (1987).
Phe- AND Trp-LOADED
LIPOSOMES’
EFFECT ON ERYTHROCYTES
251
8. Chien, S., Mccomis, W., Tejada, C., and Kurland, G., J. Appl. Physiol. 18, 1217 (1963). 9. Hans- Leasberg, L., and Hedley-Whyte, .I., J. Appl. Physiol. 35, 837 (1973). 10. Harris, J. W., Brewster, H. H., Ham, T. H., and Castelo, W. E., A.M.A. Arch. Intern. Med. 95, 145 (1955). 11. Bertles, J. F., and Dobler, J., Blood 33, 884 (1969). 12. Charache, S., and Conley, C. L., Blood 24, 25 (1964). 13. Chien, S. C., Usami, S., and Bertles, J., J. C&n. Invest. 49, 623 (1970). 14. Wells, R., and Schmidt-Schonbein, H., J. Appl. Physiol. 27, 213 (1969). 15. Russu, I. M., Allison, K. L. C., and Yang, C., and Ho, C., Biochemistry 25, 1808 (1986).
MEDICINE
AND
METABOLIC
BIOLOGY
38,
246-2.51 (1987)
Effect of Phenylalanine- or Tryptophan-Loaded Liposomes on the Rheological Properties of AA and SS Erythrocytes’ JAYALAKSHMI
KUMPATI
Department of Pediatrics, Section of Hematology/Oncology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, Colorado 80262 Received March 10. 1987
Certain aromatic amino acids such as phenylalanine (Phe) or tryptophan (Trp) and short peptides containing Phe have been shown to inhibit polymerization of deoxy hemoglobin (Hb)S in solution (l-4). Permeability studies with 14C-labeled peptides showed that these compounds do not readily penetrate the intact red blood cell membrane under the usual conditions of incubation (1,4,5). However, the author has developed a liposomal transport system (6,7) to transfer aromatic amino acids effectively across the red blood cell membrane, and has shown that the incorporation of Phe or Trp into sickle (SS) red blood cells (RBCs) via liposomal transport system markedly inhibits and reverses the in vitro sickling of deoxy Hb S (6,7). Furthermore, normal and SS RBCs loaded with Phe or Trp did not exhibit significant changes in osmotic fragility, mechanical fragility, autohemolysis, and glycolysis when compared to untreated RBCs (7). In addition, the oxygen affinity measured as the Ps,, and concentrations of 2,3-DPG and ATP were not affected by the incorporation of Phe or Trp into normal (AA) or SS RBCs (7). In the present study, the effect of Phe or Trp incorporation on the rheological properties of AA and SS RBCs are evaluated. MATERIALS
AND METHODS
Preparation of Phe or Trp loaded RBCs. Liposomes loaded with or without amino acids (Phe or Trp) were prepared by the method of Kumpati (7). Venous blood was obtained in CPDA (Fenwal Labs, Deerfield, IL) or heparin as anticoagulant from patients with homozygous sickle cell anemia and from normal, healthy volunteers. RBCs were prepared as described previously (7). Equal volumes of RBCs and liposome suspensions were mixed and incubated at 37°C for 30-60 min. After incubation, the RBC suspensions were centrifuged at 800g for 10 min. After removing the supematant, the RBCs were washed twice with 3 vol of 310 mM (isotonic) phosphate buffer, pH 7.45, and were resuspended in the same buffer to a final hematocrit (HCT) of 40% (7). ’ Part of this work was presented in poster form at the American Society of Hematology Meeting (December 5-9, 1985), and the American Pediatric Society Meeting (May 5-9, 1986). 246 08%4505/87 Copyright All rights
$3.00
Q 1987 by Academic Press, Inc. of reproduction in any form reserved.
Phe- AND Trp-LOADED
LIPOSOMES’
EFFECT
ON ERYTHROCYTES
247
o Untreated l Treated
;; 2i
& 20 = I!
01
I 60
I
1
40
20
HEMATOCRIT
!%)
FIG. 1. Viscosity in centipoise at various speeds for buffered, liposome-treated, (Phe or Trp)-incorporated, oxy and deoxy AA and SS RBCs.
and amino acid
Rheology. Viscosity measurements were carried out by the method of Chien et al. (8) and Leasberg and Hedley-Whyte (9) with a Wells-Brookfield LVT cone and plate viscometer (Brookfield Engineering Labs, Stoughton, MA) using 1.5 ml each of buffered RBCs, liposome-treated, and amino acid (Phe or Trp)incorporated RBCs (both AA and SS). To keep the RBCs in the deoxy state, the sample cup of the viscometer was equipped with a purge setting so that air could be flushed out and nitrogen could be introduced into the chamber. The viscometer was calibrated against ASTM viscosity standard, S-2 oil, Lot 71202 (Canon Instruments, PA). RESULTS The viscosities in centipoise at various speeds for buffered, liposome-treated, and amino acid (Phe or Trp)-incorporated, oxy and deoxy AA and SS RBCs are illustrated in Figs. lA-D. Figure 1A demonstrates the markedly increased viscosity of oxygenated SS RBCs in buffer which is further elevated with deoxygenation. AA RBCs showed no change with deoxygenation. Viscosity patterns for both oxy and deoxy AA and SS RBCs after treatment by liposome without amino acid incorporation appeared similar to that for untreated RBCs (B). With incorporation of Phe, there was a decrease in viscosity of both oxygenated and deoxygenated SS RBCs to levels observed with AA RBCs. Similar results were seen when Trp was incorprated into SS RBCs. Figure 2 shows the viscosity at different sheer rates for deoxygenated AA and SS RBCs. The increased viscosity of deoxygenated SS RBCs in buffer and after treatment by liposomes without amino acids is most pronounced at the lower sheer rates (Figs. 2A and B). Incorporation of Phe and Trp restored normal viscosity to deoxygenated SS RBCs even at low sheer rates (Figs. 2C and D). The viscosity at different HCT levels for deoxy AA and SS RBCs treated with liposomes alone and after Phe incorporation is presented in Fig. 3. The increased viscosity of deoxy SS cells
248
JAYALAKSHMI 0 W-AA . w-.%3
24 -
KLJMPATI
24 -
A
0 L&AA l Lip.-SS
B
20 -
0
46n
92
138
184
230
o-
’ 46
SHEER RATE/Sac
o Pha-AA . PhcSS
R-
c
’ ’ 92 138 SHEER RATE/Sac
S-
’ 184
a 230
OTWAA
D
l
20
46
92
13R
1111
230
2O
SHEER RAlE/Ssc
FIG.
46
92 130 WEEK UlE/k
-
Trp-ss
184
230
2. Viscosity at different sheer rates for deoxygenated AA and SS RBCs (n = 4).
Speed
Crpm)
FIG. 3. Viscosity versus hematocrit levels for deoxy AA and SS RBCs treated with liposomes alone, and after Phe incorporation.
Phe-
AND
Trp-LOADED
LIPOSOMES’
EFFECT
is the most marked at the higher hematocrits, HCT levels after Phe incorporation.
ON
ERYTHROCYTES
but is reduced to normal
249
at all
DISCUSSION
A therapeutic agent for the treatment of sickle cell disease needs to satisfy many criteria. It must prevent sickling, act at low concentrations, and should not interfere with erythrocyte function and metabolism. The incorporation of the amino acids Phe or Trp into SS RBCs by use of liposomes to transport them across the red blood cell membrane has recently been shown by Kumpati to totally inhibit and reverse sickling of Hb S in vitro (67). Further, the incorporation of Phe or Trp into RBCs did not have any adverse effect on cell function or metabolism (7). The oxygen afIinity, concentrations of 2,3-DPG and ATP, glycolytic activity, osmotic and mechanical fragilities, and autohemolysis of both AA and SS RBCs were not altered by either liposome or amino acid (Phe or Trp) incorporation (7). It was therefore important to determine whether this technique has any beneficial or adverse effect on the rheological properties of RBCs. The most noteworthy effect of the incorporation of Phe or Trp into SS RBCs was the marked decrease in blood viscosity to the normal range for both deoxygenated and oxygenated SS RBCs. Factors affecting blood viscosity include hematocrit, aggregation of red cells, the internal viscosity of red cells, osmolarity, red cell ATP, membrane flexibility or rigidity (RBC deformability), and the shape of the red cells (10-14). The increased viscosity of deoxy SS RBCs in buffer or treated with liposomes alone (without Phe or Trp) was most likely related to changes in both cell shape and membrane since 100% of the SS cells were “sickled” in this sytsem (13). It has been shown that the membranes of SS RBCs become rigid upon deoxygenation (11,13) with associated loss of elasticity which contributes to the marked increase in viscosity (10). These membrane changes have been postulated to be secondary to polymerization of Hb S (11,13). In hemolysates, Phe or Trp are known to inhibit polymerization (3,4). Recent proton nuclear magnetic resonance studies (15) show that the binding of Phe/Trp to deoxy Hb S is comparable to Hb A. One of the binding sites common to all of the antisickling compounds (including Phe or Trp) investigated (15), is at or near the heme pockets in the a and p chain of both deoxy Hb A and Hb S. The other binding site is located at or near the /3” position, containing the mutation in Hb S, p6 Glu + Val (15). The binding of the antisickling compound to the latter site induces conformational changes in the amino terminal derivatives of the j3 chains (1.5). Introduction of Phe or Trp into the intact deoxygenated SS RBCs by the liposomal transport system has been previously shown by Kumpati (67) to prevent sickling of intact sickle red cells. This antipolymerization effect may be the one mechanism that is responsible for normalization of the viscosity of these deoxygenated SS RBCs. The increased viscosity of oxygenated SS RBCs was confirmed in this study. It has previously been shown to be due primarily to the altered rheology of irreversibly sickled cells (ISCs) and to a lesser extent to non-ISCs (13). Ninety percent of the oxygenated SS RBCs in the present system were not sickled, suggesting increased rigidity of the membrane. The complete normalization of
250
JAYALAKSHMI
KUMPATI
the viscosity of these predominantly unsickled oxygenated cells after amino acid incorporation might suggest normalization of certain factors affecting the cell membrane in addition to the antipolymerizing effect on hemoglobin S. While a beneficial effect was noted in the viscosity of SS RBCs after amino acid incorporation, no adverse effect was found on cell function or metabolism (7). The oxygen affinity, concentrations of ATP and 2,3-DPG, glycolytic activity, mechanical fragility, and autohemolysis of both AA and SS RBCs were not altered by either liposome or amino acid incorporation (7). No change was demonstrated in osmotic fragility with treatment although as previously described (10) increased resistance of SS RBCs was noted. In conclusion, these data suggestno adverse effect in vitro from the incorporation of liposomes, the amino acids Phe or Trp, on the rheology, the membrane, the metabolism, or the oxygen affinity of erythrocytes from normal adults or from persons with homozygous sickle cell anemia. On the contrary, the abnormal viscosity of both oxy and deoxy erythrocytes from sickle cell disease patients was restored to normal. This confirms the original finding that sickling can be prevented by the incorporation of Phe or Trp into intact RBCs by a liposomal transport system (6,7) and further shows that certain rheological abnormalities are inhibited. SUMMARY
Phenylalanine or tryptophan was incorporated into AA and SS red blood cells by a liposomal transport system which was previously shown by Kumpati to inhibit and reverse sickling of intact SS red blood cells in vitro. In the present study, the effect of phenylalanine or tryptophan incorporation on the rheological properties was evaluated. The incorporation of phenylalanine or tryptophan into red blood cells decreased the viscosity of deoxy SS red blood cells which reached a level close to that for normal red blood cells due to the antisickling effect. These results demonstrate that this liposomal transport system which transferred phenylalanine or tryptophan into intact red cells and did not have any adverse effect on red cell metabolism or function did correct the viscosity of deoxy SS red cells by its antisickling effect. This method may have significant therapeutic implications in the treatment of sickle cell disease. ACKNOWLEDGMENTS The author thanks Drs. D. Ambruso and J. Githens for review of the manuscript, Donna Dixon, R.N., MS., for obtaining blood samples, and Kathleen Hoyer for its preparation. This work was supported in part by Grant HL 31383 from the National Institutes of Health, the Bonfils Blood Center, and the Colorado Sickle Cell Treatment and Research Center.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Gore&i, Kubota, Noguchi, Noguchi,
M., Votano, J. R., and Rich, A., Biochemistry 19, 1564 (1980). S., and Yang, J. T., Proc. Natl. Acad. Sci. USA 74, 5431 (1977). C. T., and Schechter, A. N., Biochem. Biophys. Res. Commum. 74, 637 (1977). C. T., and Schechter, A. N., Biochemistry 17, 5455 (1978). votano, J. R., Gorecki, M., and Rich, A., Science 196, 1216 (1977). Kumpati, J., Biochem. Biophys. Res. Commun. 105, 482 (1982). Kumpati, J., Biochem. Med. Metab. Biol. 38, 170 (1987).
Phe- AND Trp-LOADED
LIPOSOMES’
EFFECT ON ERYTHROCYTES
251
8. Chien, S., Mccomis, W., Tejada, C., and Kurland, G., J. Appl. Physiol. 18, 1217 (1963). 9. Hans- Leasberg, L., and Hedley-Whyte, .I., J. Appl. Physiol. 35, 837 (1973). 10. Harris, J. W., Brewster, H. H., Ham, T. H., and Castelo, W. E., A.M.A. Arch. Intern. Med. 95, 145 (1955). 11. Bertles, J. F., and Dobler, J., Blood 33, 884 (1969). 12. Charache, S., and Conley, C. L., Blood 24, 25 (1964). 13. Chien, S. C., Usami, S., and Bertles, J., J. C&n. Invest. 49, 623 (1970). 14. Wells, R., and Schmidt-Schonbein, H., J. Appl. Physiol. 27, 213 (1969). 15. Russu, I. M., Allison, K. L. C., and Yang, C., and Ho, C., Biochemistry 25, 1808 (1986).