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Polyamines in sickle cell disease
BIOCHEMICAL
MEDICINE
2%
144-149
(1980)
Polyami nes in Sickle Cell Disease CLAYTON L. NATTA,* ARLENE A. MOTYCZKA,~AND LEON T. KREMZNER~ Departments of *Medicine and Pathology and TNeurology, Columbia of Physicians & Surgeons, and Harlem and Columbia-Presbyterian New York, New York 10032 Received
May
University, College Hospitals,
9, 1979
Sickle cell anemia, a chronic hemolytic disease characterized by severe pain and premature death, affects to varying degrees 1% of the world’s black population. Although a number of biochemical alterations have previously been associated with this anemia (l), the recent report of a five- to tenfold elevation in blood polyamine levels (2) is especially noteworthy because the polyamines can alter the normal electrokinetic properties of the red blood cell membrane (3). These observations may help explain the abnormalities of the erythrocyte membrane and decreased red cell deformability associated with the sickling phenomenon. The principal polyamines present in eukaryotes are, respectively, the di-, tri-, and tetramines-putrescine, spermidine, and spermine. Their biological function, which is related to their unique charge distribution, is linked to cell growth, division, and differentiation (4). The polyamines also exert direct effects on membrane stability and permeability (5), as well as modifying the activity of membrane-bound enzymes including acetylcholinesterase (6) and Na,K-ATPase (7). In the present study it is shown that the erythrocyte stroma fraction, obtained from sickle cells, has associated with it abnormally large amounts of spermine. Stroma binding appears to be selective and not directly dependent on either plasma or erythrocyte lysate polyamine concentration, MATERIALS
AND METHODS
Heparinized or EDTA blood was collected from patients of known genotype and the plasma was removed by centrifugation at SOOgfor 10 min; the btiy coat was carefully removed, and the cells were washed three times with normal saline. A l.O-ml volume of packed red cells was 144 OW6-2944/80/020144-06$02.00/O Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
POLYAMINES
AND
SICKLING
145
lysed with 40 vol of 1 mM, EDTA, pH 7.2 (8). The stroma was recovered by centrifugation at 31,OOQgfor 20 min, followed by exhaustive washing with a large excess of buffer. Finally, the packed stroma was acidified with 1.0 ml of 0.4 N perchloric acid. After 30 min on ice, the extract was centrifuged at 25,OOOgfor 10 min and the clear supematant removed. Protein content of the stroma was determined by solubilization of the perchloric acid precipitate with 4.0 ml of 0.1 N NaOH, and estimation of the amount of protein calorimetrically (9). The red cell lysate volume was recorded, and a 2.0-ml aliquot was removed, to which was added 0.2 ml of 4 N perchloric acid; it was processed as above. Polyamine content of the perchloric acid extract of stroma and lysate was determined with an automatic amino acid analyzer, using Hamilton Company HPAN-90 cationic resin, column dimensions 0.5 x 7 cm. The initial buffer was sodium citrate, 0.35 M, pH 5.25 + 0.02, containing 2% l-propanol, followed (at 30 min) by the above buffer made 2.35 M with respect to sodium chloride. The pumping rate was 45 ml/hr and the column temperature 43.5”C. Amino acids and amines were quantitated with 0-phthaldehyde reagent (10) and fluorescence detection (Gilson Corp.; SpectroGlo fluorometer); the sensitivity of the method was such that 25 pmole of authentic polyamine could be quantitated. RESULTS
The chromatographic retention times, separations, and fluorescent response of authentic polyamines and the amines isolated from the human red blood cell stroma fraction, prepared as described under Materials and Methods, are shown in Fig. 1. In addition to the polyamines putrescine, spermidine, and spermine present in the stroma fraction, a distinct shoulder adjacent to spermidine, at 65 min, is evident in this specimen. The elution time of this unidentified amine does not correspond to the elution position of authentic cadaverine or histamine, amines known to occur in mm (s). Comparison of the polyamine analysis of the extensively washed red cell stroma and lysate shows statistically significant differences in the amine composition according to patient genotype (Table 1). Relative to the clinical normal (AA), spermidine and spermine are markedly elevated in the stroma and lysate of sickle cell anemic (SS) subjects. In contrast, in the sickle-hemoglobin C disease (SC), a clinically less severe hemoglobinopathy than the SS form, spermine is signi!?cantly elevated only in the red cell lysate, but not the stroma. Comparison of the polyamine content of the stroma and lysate of the SS subjects to that of the SC shows that spermidine (P < 0.005) and spermine (P < 0.0025) are elevated in the stroma, and spermine in the lysate (P < 0.05). Analysis of the stroma and lysate of B-thalassemic subjects, characterized by a chronic hemolytic
NATTA,
146
MOTYCZKA,
AND KREMZNER
100 90 F
1
Y aoz
M 702 2 60i 2 50 2 40. 2 E 30: !g 20-
34
10 t. OL'
A
'
24
48
'
0
5
'
'
'
'
72
'
'
0
96
24
48
72
96
TIME IN MINUTES FIG. 1. Analysis of authentic mixture of amino acids and polyamines (left) and perchioric acid extract of erythroctye stroma fraction (right). The authentic mixture consisted of the following compounds (in picomoles): (1) +&utine, 507; (2) GABA, 525; (3) ornithiie, 500; (4) homocarnosine, 675; (5) putreanine, 973; (6) arginine, buffer artifact, 320; (7) putrescine, 249; (8) cadaverine, 2140; (9) spermidine, 500; (10) spermine, 752. The stroma fraction (figure on right) contains putrescine (48 min), spermidine (60 min), and spermine (88 mitt). TABLE
1
POLYAMINE LEVELSINREDCELLS Subject genotype
N
Putrescine
AA SS SC Thal
7 13 6 4
0.05 (0.07) 0.13 (0.20) 0.06 (0.06) N.Q.
AA ss SC Thal
7 13 6 4
1.08 (2.58) 0.44 (0.54) 0.37 (0.78) N.Q.
Spermidine
Spermine
stroma o.55b (* 1.57@(k 0.36 (rt 0.11 (a
0.55) 0.95) 0.41) 0.06)
1.13’ 3.W 0.91 0.51
(2 (2 (2 (”
0.54) 1.47) 0.62) 0.29)
Red cell lysate 3.21 (2 1.82) 11.2tP (2 6.91) 9.31 (k 16.98) 0.41b (k 0.42)
0.53 4.7Bc 2.41” 0.19
(k (.c (2 (2
0.75) 2.51) 1.94) 0.38)
a Sign&W at the P < 0.05 probability level. b Siicant at the P > 0.01 probability level. c Signiticant at the P < 0.W probability level. Note. Stroma polyamine values are expressed on a milligram stroma protein basis; lysate values are expressed on the basis of lysate obtained, under standardized conditions, from the cells equivalent to 1.O mg of stroma protein. N.Q., not quantifiable; either not present or present at concentrations less than 25 to 50 pmole/mg protein.
PGLYAMINES
147
AND SICKLING
anemia due to decreased synthesis of B-chains, shows what appears to be a reduction in the concentration of all three of the polyamines relative to AA subjects. However, only spermine in the lysate is significantly reduced. Putrescine, the metabolic precursor of both spermidine and spermine, was not signifkantly altered in either stroma or lysate in the anemias studied. Red cell lysate putrescine values in the three anemic states appear to be somewhat lower than in the nonanemic reference group. Analysis of the polyamine content of the red cell stroma and lysate of sickle hereditary persistent fetal hemoglobin (SHPFH), a hereditary condition characterized by the absence of anemia and reticulocytosis, Table 2, shows values similar to those in SC subjects, i.e., somewhat elevated relative to AA subjects and below those in SS subjects. Patient B shows an unusually high stroma spermine concentration; this subject has been hematologically characterized (11) and reticulocytosis has been ruled out. DISCUSSION
This study shows the red blood cell stroma and lysate polyamines are signifkantly elevated in sickle cell anemia, relative to a reference group of nonanemic subjects. The alterations in polyamine concentration appear to be related both qualitatively and quantitatively to severity of the anemic state and patient genotype. These studies co&m and extend the observations of Rennert and Shukla (12) who originally reported that whole blood and erythrocyte polyamine content was elevated in sickle cell anemia. One interpretation of these findings is that the polyamines are elevated as the result of the anemic state and the attendant reticulocytosis. This TABLE
2
POLYAMINE CONCENTRATIONS IN SICKLE HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (CONCENTRATION IN nmole/mg STROMA PROTEIN)
Patient
Genotype
Putrescine
A B
SHPFH SHPFH
N.Q. N.Q.
A B
SHPFH SHPFH
N.Q. N.Q.
Spermidine
Spermine
0.43 1.0
0.59 2.3
1.1 4.5
1.9 2.8
Stroma
Lysate
Note. Stroma polyamine values are expressed on a mill&am stroma protein basis; lysate values are expressed on the basis of lysate obtained, under standardized conditions. from the cells equivalent to 1 .Omg of stroma protein. N.Q., not quantifiable; either not present or present at concentrations less than 25 to 50 pmoldmg protein.
148
NATTA,
MOTYCZKA,
AND
KREMZNER
interpretation would appear to be consistent with experimental data (12) which shows putrescine, spermidine, and spermine elevated in the top fraction (reticulocyte rich) of density fractionated erythrocytes, relative to the bottom cell fraction which contains relatively few reticulocytes. Reticulocyte polyamines are probably associated with the ribosomal fraction: As polycations, at physiological pH’s, the major portion of the cellular polyamines are bound electrostatically to both DNA and RNA (13).
In the laboratory preparation of the red cell stroma and lysate fractions a secondary redistribution of the polyamines could and possibly does occur; although it is not clear why the red cell stroma would bind polyamines from the 30-fold diluted cell lysate, during laboratory preparation, and not under physiological conditions in which the polyamines are more concentrated. In as much as the stroma and lysate analysis, Tables 1 and 2, does not show an elevation of all three polyamines in either the stroma or lysate fraction in any anemic condition, it is probably that anemic reticulocytosis is not the principal and/or only interpretation of these data. Further, the elevation in polyamines when observed appears to be characteristic of the genotypic state, and the most significant elevation is that in spermine, approximately ninefold. In contrast, analysis of the reticulocyte-rich fraction (12) showed that spermidine was elevated to the greatest extent. That a simple reticulocytosis is not an adequate explanation of these findings is also supported by the analysis of the red cell stroma and lysate of the B-thalassemic subjects; in this condition there is an increased number of nucleated cells in the peripheral blood (although not of the magnitude found in SS subjects). The polyamine concentration is not elevated, but reduced to a significant degree in lysate spermidine. It is interesting to note that both putrescine and spermidine binding appear to be independent of spermine binding to stroma. This implies either a specificity of binding sites, not dependent on charge alone, or differing accessibility of stroma binding sites. The polyamines, as polycations, may be associated with negatively charged ligands such as calmodulin, glycophorin, or sialic acid. Perhaps putrescine and spermidine binding occur principally on the external stroma surface sites exposed to plasma whereas spermine binding occurs on internal sites in contact with hemoglobin solution (lysate). This suggestion would be compatible with our knowledge of plasma polyamine concentrations in which the spermidine concentrations are greater than putrescine, or spermine, the latter amine being hardly detectable. Perhaps in the sickle state the red blood cell membrane is genetically altered resulting in a selective increased binding of the available polyamines, especially under the circumstances of the greater availability of the polyamines as the result of the anemic state.
POLYAMINES
AND SICKLING
149
Although the data are open to interpretation, the elevations are significant. Perhaps the increased polyamine binding contributes to an increased red blood cell stiffness and other membrane alterations associated with sickle cell anemia. ACKNOWLEDGMENTS We thank Drs. Reinhold and Ruth Benesch for helpful discussions, and Ms. Madeline Moshel for secretarial assistance. This study was supported by Grants HL 19324, AG 00276, and NS 11766.
REFERENCES 1. Bunn, H. F., Forget, B. G., and Ranney, H. M., “Sickle Cell Anemia and Related Disorders.” Saunders, Philadelphia, 1977. 2. Chun, P. W., Rennert, 0. M., Saffem, E. E., and Taylor, W. J., Biochem. Biophys. Res. Commun. 69, 1096 (1976). 3. Chun, P. W., Saffem, E. E., Ditore, R. J., Rennert, 0. M., and Weinstein, N. H., Biophys. Gem. 6, 321 (1977). 4. Cohen, S. S., “Introduction to the Polyamines.” Academic Press, New York, 1971. 5. Bachrach, U., “Function of Naturally Occurring Polyatnines.” Academic Press, New York, 1973. 6. Kossorotow, A., Wolf, H. U., and Seiler, N., Eiochem. J. 144, 21 (1974). 7. Heinrich-Hirsch, B., Ahlers, J., and Peter, H. W., Enzyme 22, 235 (1977). 8. Bank, A., Meats, S., Weiss, R., O’Donnell, J. V., and Natta, C.,J. Clin. Invest. 54,805 (1974). 9. Lowry, O., Rosebrough, N., Farr, N., and Randall, R., J. Bid. Gem. 193,265 (1951). 10. Roth, M., Anal. Chem. 43, 880 (1971). 11. Natta, C. L., Niazi, G. A., Ford, S., and Bank, A., J. Clin. Invest. 54, 433 (1974). 12. Rennet% 0. M., and Shukla, J. B., in “Advances in Polyamine Research” (R. A. Campbell et al., Eds.), Vol. 2, p. 195. Raven Press, New York, 1978. 13. McCormick, F., J. CeN Physiol. 93, 285 (1977).
MEDICINE
2%
144-149
(1980)
Polyami nes in Sickle Cell Disease CLAYTON L. NATTA,* ARLENE A. MOTYCZKA,~AND LEON T. KREMZNER~ Departments of *Medicine and Pathology and TNeurology, Columbia of Physicians & Surgeons, and Harlem and Columbia-Presbyterian New York, New York 10032 Received
May
University, College Hospitals,
9, 1979
Sickle cell anemia, a chronic hemolytic disease characterized by severe pain and premature death, affects to varying degrees 1% of the world’s black population. Although a number of biochemical alterations have previously been associated with this anemia (l), the recent report of a five- to tenfold elevation in blood polyamine levels (2) is especially noteworthy because the polyamines can alter the normal electrokinetic properties of the red blood cell membrane (3). These observations may help explain the abnormalities of the erythrocyte membrane and decreased red cell deformability associated with the sickling phenomenon. The principal polyamines present in eukaryotes are, respectively, the di-, tri-, and tetramines-putrescine, spermidine, and spermine. Their biological function, which is related to their unique charge distribution, is linked to cell growth, division, and differentiation (4). The polyamines also exert direct effects on membrane stability and permeability (5), as well as modifying the activity of membrane-bound enzymes including acetylcholinesterase (6) and Na,K-ATPase (7). In the present study it is shown that the erythrocyte stroma fraction, obtained from sickle cells, has associated with it abnormally large amounts of spermine. Stroma binding appears to be selective and not directly dependent on either plasma or erythrocyte lysate polyamine concentration, MATERIALS
AND METHODS
Heparinized or EDTA blood was collected from patients of known genotype and the plasma was removed by centrifugation at SOOgfor 10 min; the btiy coat was carefully removed, and the cells were washed three times with normal saline. A l.O-ml volume of packed red cells was 144 OW6-2944/80/020144-06$02.00/O Copyright All rights
@ 1980 by Academic Press. Inc. of reproduction in any form reserved.
POLYAMINES
AND
SICKLING
145
lysed with 40 vol of 1 mM, EDTA, pH 7.2 (8). The stroma was recovered by centrifugation at 31,OOQgfor 20 min, followed by exhaustive washing with a large excess of buffer. Finally, the packed stroma was acidified with 1.0 ml of 0.4 N perchloric acid. After 30 min on ice, the extract was centrifuged at 25,OOOgfor 10 min and the clear supematant removed. Protein content of the stroma was determined by solubilization of the perchloric acid precipitate with 4.0 ml of 0.1 N NaOH, and estimation of the amount of protein calorimetrically (9). The red cell lysate volume was recorded, and a 2.0-ml aliquot was removed, to which was added 0.2 ml of 4 N perchloric acid; it was processed as above. Polyamine content of the perchloric acid extract of stroma and lysate was determined with an automatic amino acid analyzer, using Hamilton Company HPAN-90 cationic resin, column dimensions 0.5 x 7 cm. The initial buffer was sodium citrate, 0.35 M, pH 5.25 + 0.02, containing 2% l-propanol, followed (at 30 min) by the above buffer made 2.35 M with respect to sodium chloride. The pumping rate was 45 ml/hr and the column temperature 43.5”C. Amino acids and amines were quantitated with 0-phthaldehyde reagent (10) and fluorescence detection (Gilson Corp.; SpectroGlo fluorometer); the sensitivity of the method was such that 25 pmole of authentic polyamine could be quantitated. RESULTS
The chromatographic retention times, separations, and fluorescent response of authentic polyamines and the amines isolated from the human red blood cell stroma fraction, prepared as described under Materials and Methods, are shown in Fig. 1. In addition to the polyamines putrescine, spermidine, and spermine present in the stroma fraction, a distinct shoulder adjacent to spermidine, at 65 min, is evident in this specimen. The elution time of this unidentified amine does not correspond to the elution position of authentic cadaverine or histamine, amines known to occur in mm (s). Comparison of the polyamine analysis of the extensively washed red cell stroma and lysate shows statistically significant differences in the amine composition according to patient genotype (Table 1). Relative to the clinical normal (AA), spermidine and spermine are markedly elevated in the stroma and lysate of sickle cell anemic (SS) subjects. In contrast, in the sickle-hemoglobin C disease (SC), a clinically less severe hemoglobinopathy than the SS form, spermine is signi!?cantly elevated only in the red cell lysate, but not the stroma. Comparison of the polyamine content of the stroma and lysate of the SS subjects to that of the SC shows that spermidine (P < 0.005) and spermine (P < 0.0025) are elevated in the stroma, and spermine in the lysate (P < 0.05). Analysis of the stroma and lysate of B-thalassemic subjects, characterized by a chronic hemolytic
NATTA,
146
MOTYCZKA,
AND KREMZNER
100 90 F
1
Y aoz
M 702 2 60i 2 50 2 40. 2 E 30: !g 20-
34
10 t. OL'
A
'
24
48
'
0
5
'
'
'
'
72
'
'
0
96
24
48
72
96
TIME IN MINUTES FIG. 1. Analysis of authentic mixture of amino acids and polyamines (left) and perchioric acid extract of erythroctye stroma fraction (right). The authentic mixture consisted of the following compounds (in picomoles): (1) +&utine, 507; (2) GABA, 525; (3) ornithiie, 500; (4) homocarnosine, 675; (5) putreanine, 973; (6) arginine, buffer artifact, 320; (7) putrescine, 249; (8) cadaverine, 2140; (9) spermidine, 500; (10) spermine, 752. The stroma fraction (figure on right) contains putrescine (48 min), spermidine (60 min), and spermine (88 mitt). TABLE
1
POLYAMINE LEVELSINREDCELLS Subject genotype
N
Putrescine
AA SS SC Thal
7 13 6 4
0.05 (0.07) 0.13 (0.20) 0.06 (0.06) N.Q.
AA ss SC Thal
7 13 6 4
1.08 (2.58) 0.44 (0.54) 0.37 (0.78) N.Q.
Spermidine
Spermine
stroma o.55b (* 1.57@(k 0.36 (rt 0.11 (a
0.55) 0.95) 0.41) 0.06)
1.13’ 3.W 0.91 0.51
(2 (2 (2 (”
0.54) 1.47) 0.62) 0.29)
Red cell lysate 3.21 (2 1.82) 11.2tP (2 6.91) 9.31 (k 16.98) 0.41b (k 0.42)
0.53 4.7Bc 2.41” 0.19
(k (.c (2 (2
0.75) 2.51) 1.94) 0.38)
a Sign&W at the P < 0.05 probability level. b Siicant at the P > 0.01 probability level. c Signiticant at the P < 0.W probability level. Note. Stroma polyamine values are expressed on a milligram stroma protein basis; lysate values are expressed on the basis of lysate obtained, under standardized conditions, from the cells equivalent to 1.O mg of stroma protein. N.Q., not quantifiable; either not present or present at concentrations less than 25 to 50 pmole/mg protein.
PGLYAMINES
147
AND SICKLING
anemia due to decreased synthesis of B-chains, shows what appears to be a reduction in the concentration of all three of the polyamines relative to AA subjects. However, only spermine in the lysate is significantly reduced. Putrescine, the metabolic precursor of both spermidine and spermine, was not signifkantly altered in either stroma or lysate in the anemias studied. Red cell lysate putrescine values in the three anemic states appear to be somewhat lower than in the nonanemic reference group. Analysis of the polyamine content of the red cell stroma and lysate of sickle hereditary persistent fetal hemoglobin (SHPFH), a hereditary condition characterized by the absence of anemia and reticulocytosis, Table 2, shows values similar to those in SC subjects, i.e., somewhat elevated relative to AA subjects and below those in SS subjects. Patient B shows an unusually high stroma spermine concentration; this subject has been hematologically characterized (11) and reticulocytosis has been ruled out. DISCUSSION
This study shows the red blood cell stroma and lysate polyamines are signifkantly elevated in sickle cell anemia, relative to a reference group of nonanemic subjects. The alterations in polyamine concentration appear to be related both qualitatively and quantitatively to severity of the anemic state and patient genotype. These studies co&m and extend the observations of Rennert and Shukla (12) who originally reported that whole blood and erythrocyte polyamine content was elevated in sickle cell anemia. One interpretation of these findings is that the polyamines are elevated as the result of the anemic state and the attendant reticulocytosis. This TABLE
2
POLYAMINE CONCENTRATIONS IN SICKLE HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (CONCENTRATION IN nmole/mg STROMA PROTEIN)
Patient
Genotype
Putrescine
A B
SHPFH SHPFH
N.Q. N.Q.
A B
SHPFH SHPFH
N.Q. N.Q.
Spermidine
Spermine
0.43 1.0
0.59 2.3
1.1 4.5
1.9 2.8
Stroma
Lysate
Note. Stroma polyamine values are expressed on a mill&am stroma protein basis; lysate values are expressed on the basis of lysate obtained, under standardized conditions. from the cells equivalent to 1 .Omg of stroma protein. N.Q., not quantifiable; either not present or present at concentrations less than 25 to 50 pmoldmg protein.
148
NATTA,
MOTYCZKA,
AND
KREMZNER
interpretation would appear to be consistent with experimental data (12) which shows putrescine, spermidine, and spermine elevated in the top fraction (reticulocyte rich) of density fractionated erythrocytes, relative to the bottom cell fraction which contains relatively few reticulocytes. Reticulocyte polyamines are probably associated with the ribosomal fraction: As polycations, at physiological pH’s, the major portion of the cellular polyamines are bound electrostatically to both DNA and RNA (13).
In the laboratory preparation of the red cell stroma and lysate fractions a secondary redistribution of the polyamines could and possibly does occur; although it is not clear why the red cell stroma would bind polyamines from the 30-fold diluted cell lysate, during laboratory preparation, and not under physiological conditions in which the polyamines are more concentrated. In as much as the stroma and lysate analysis, Tables 1 and 2, does not show an elevation of all three polyamines in either the stroma or lysate fraction in any anemic condition, it is probably that anemic reticulocytosis is not the principal and/or only interpretation of these data. Further, the elevation in polyamines when observed appears to be characteristic of the genotypic state, and the most significant elevation is that in spermine, approximately ninefold. In contrast, analysis of the reticulocyte-rich fraction (12) showed that spermidine was elevated to the greatest extent. That a simple reticulocytosis is not an adequate explanation of these findings is also supported by the analysis of the red cell stroma and lysate of the B-thalassemic subjects; in this condition there is an increased number of nucleated cells in the peripheral blood (although not of the magnitude found in SS subjects). The polyamine concentration is not elevated, but reduced to a significant degree in lysate spermidine. It is interesting to note that both putrescine and spermidine binding appear to be independent of spermine binding to stroma. This implies either a specificity of binding sites, not dependent on charge alone, or differing accessibility of stroma binding sites. The polyamines, as polycations, may be associated with negatively charged ligands such as calmodulin, glycophorin, or sialic acid. Perhaps putrescine and spermidine binding occur principally on the external stroma surface sites exposed to plasma whereas spermine binding occurs on internal sites in contact with hemoglobin solution (lysate). This suggestion would be compatible with our knowledge of plasma polyamine concentrations in which the spermidine concentrations are greater than putrescine, or spermine, the latter amine being hardly detectable. Perhaps in the sickle state the red blood cell membrane is genetically altered resulting in a selective increased binding of the available polyamines, especially under the circumstances of the greater availability of the polyamines as the result of the anemic state.
POLYAMINES
AND SICKLING
149
Although the data are open to interpretation, the elevations are significant. Perhaps the increased polyamine binding contributes to an increased red blood cell stiffness and other membrane alterations associated with sickle cell anemia. ACKNOWLEDGMENTS We thank Drs. Reinhold and Ruth Benesch for helpful discussions, and Ms. Madeline Moshel for secretarial assistance. This study was supported by Grants HL 19324, AG 00276, and NS 11766.
REFERENCES 1. Bunn, H. F., Forget, B. G., and Ranney, H. M., “Sickle Cell Anemia and Related Disorders.” Saunders, Philadelphia, 1977. 2. Chun, P. W., Rennert, 0. M., Saffem, E. E., and Taylor, W. J., Biochem. Biophys. Res. Commun. 69, 1096 (1976). 3. Chun, P. W., Saffem, E. E., Ditore, R. J., Rennert, 0. M., and Weinstein, N. H., Biophys. Gem. 6, 321 (1977). 4. Cohen, S. S., “Introduction to the Polyamines.” Academic Press, New York, 1971. 5. Bachrach, U., “Function of Naturally Occurring Polyatnines.” Academic Press, New York, 1973. 6. Kossorotow, A., Wolf, H. U., and Seiler, N., Eiochem. J. 144, 21 (1974). 7. Heinrich-Hirsch, B., Ahlers, J., and Peter, H. W., Enzyme 22, 235 (1977). 8. Bank, A., Meats, S., Weiss, R., O’Donnell, J. V., and Natta, C.,J. Clin. Invest. 54,805 (1974). 9. Lowry, O., Rosebrough, N., Farr, N., and Randall, R., J. Bid. Gem. 193,265 (1951). 10. Roth, M., Anal. Chem. 43, 880 (1971). 11. Natta, C. L., Niazi, G. A., Ford, S., and Bank, A., J. Clin. Invest. 54, 433 (1974). 12. Rennet% 0. M., and Shukla, J. B., in “Advances in Polyamine Research” (R. A. Campbell et al., Eds.), Vol. 2, p. 195. Raven Press, New York, 1978. 13. McCormick, F., J. CeN Physiol. 93, 285 (1977).