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Lipid content of Plasmodium berghei-infected rat red blood cells
EXPERIMENTAL PARASITOLOGY 26, 181-186
Lipid
Content
(1969)
of Plasmodium Rat Red Blood
C. W.
Lawrence
and
Richard
berghei-Infected Cells J. Cenedella
Department of Pharmacology, West Virginia University Medical Center, Morgantown, West Virginia 26506 (Submitted
for publication
18 February
1969)
LAWFIENCE, C. W. AND CENEDELLA, R. J. 1969. Lipid content of Plasmodium berghei-infected rat red blood cells. Experimental ParasitoZogy 26, 181-186. The lipid extracts of rat red blood cells infected with the malarial parasite Plasmodium berghei contained increased amounts of total lipid when compared to extracts of cells from either normal or phenylhydrazine-treated rats. Total cholesterol increased approximately two-fold while the total lipid-P increased more than four-fold. The four-fold increase in phospholipid content of Plasmodium berghei parasitized over unparasitized blood cells largely represents a proportionate increase of all phospholipids studied rather than a selective increase of particular ones. However, the phospholipids of parasitized cells did contain a small but statistically significant higher percentage of phosphatidylethanolamine and also a significantly lower percentage of sphingomylins plus lysolecitbins than unparasitized cell phospholipids. These minor changes could reflect the phospholipid composition of the membranes characteristic of this malarial parasite. INDEX DESCRIPTORS: Plasmodium berghei, lipids; Red blood cells; Phosphohpids; Cholesterol; Phosphatidyl ethanolamine; Sphingomylein; Lysolecithin; Malaria; Metabolism; Phenylhydrazine.
Growth and multiplication of the malarial parasite in the mammalian red blood cells is accompanied by a marked increase in the lipid content of these cells (Ball et al., 1946; Lewert, 1952; Morrison and Jeskey, 1947). Since mammalian erythrocytes possess little ability to synthesize these lipids de nouo (Bishop and Surgenor, 1964), this increase in the lipid content of malarial-infected erythrocytes most reasonably reflects the lipid composition of the growing parasite. Although the increase in lipid content of malarial-infected blood cells is well documented, the identity of individual lipids producing this increase has been largely undetermined. The purpose of this investigation was to quantitate accurately the increase in lipid content of Plasmodium berghei infected rat red blood cells, to
determine the distribution of this lipid between neutral and phospholipid, and to identify and quantitate the major phospholipids. MATERIALS
AND METHODS
Blood from at least 10 male rats weighing 140-160 gm was collected by into 12 ml syringes cardiac puncture containing 1.2 ml of sodium citrate anticoagulant (3.69 gm tri-sodium citrate plus 1.13 gm citric acid/100 ml distilled water) and then pooled. The animals were of three types: ( 1) normal ( untreated) ; (2) Plasmodium berghei-parasitized and (3) phenylhydrazine-treated. PZusmodium berghei (strain KBG 173), obtained initially from Dr. William Trager of the Rockefeller University, was used for 181
182
LAWRENCE
AND CENEDELLA
these investigations. Parasites were maintained by blood passage from infected to healthy 6-week old male rats. In order to obtain uninfected rats with about the same reticulocytosis at the time of experimentation as the infected animals, normal rats were treated twice with phenylhydrazine ( 15 mg/kg body weight 5 and 3 days before being used). Total red cell counts were established with a hemocytometer; reticulocyte counts were made subsequent to staining with methylene blue N. Thin smears were made from infected blood pools, stained (Wright’s) and counted to determine the percentage of parasitized cells. Subsequently aliquots (26-40 ml) of the parasitized or unparasitized blood were centrifuged at about 1OOOg for 10 minutes at 5°C. After the plasma was removed and discarded, the cells were suspended to the original volume in Kreb’s phosphate buffer (pH 7.4, Ca2+ omitted) and centrifuged as above. This washing procedure was repeated twice. For preparation of free parasites, blood from parasitized rats was collected and the cells were centrifuged and washed as above. The washed erythrocytes were lysed by incubation at 37°C for 30 minutes in 40 volumes of a l:lO,OOO dilution of saponin in buffer according to Wallace et al. ( 1965). Lipid extraction of the red cells was carried out according to procedure III of Ways and Hanahan (1962). After extraction the lipid was transferred to tared vials, at weighed, and stored in chloroform -10°C. The phospholipid content of these total lipid extracts was measured by phosphorus analysis (Youngburg and Youngburg, 1930). Cholesterol content was determined by the method of Zlatkis, et al. ( 1953). Aliquots of each lipid sample were subjected to thin-layer chromatography on silica gel G with chloroform:methanol:acetic acid: water (65:25:8:4) (Skipski et al., 1962)
and phosphorus analyses on the individual phospholipid fractions recovered after chromatography were carried out after digestion of samples and blanks with 72% perchloric acid followed by treatment with ammonium molybdate solution and 10% ascorbic acid (Rouser et al., 1966). The plate was divided into five areas. Area A included the area from the solvent front down to the phosphatidyl ethanolamines. This area contains polyglycerol phosphatides, phosphatidic acid, and neutral lipids. Area B included the phosphatidyl ethanolamines down to the phosphatidyl serine area. Area C included the phosphatidyl serines as a minor component (Gier and VanDeenen, 1961) and lecithins down to the sphingomyelin area while area D included both sphingomyelin and lysolecithin (minor component according to Gier and van Deenen, 1961). Area E included material remaining at the origin. Values for total lipid-P applied to the plates was determined by taking the sum of these five areas since these included all the lipid originally applied. RESULTS
The results shown in Table I indicate that there was a marked increase in the total lipid weight (mg lipid/100 RBC’s) extracted from the parasitized erythrocytes when compared to the values obtained from either the normal or phenylhydrazinetreated animals. This increase progressed rather consistently from low parasitemias (12%) through the highest parasitemia studied (42%). In order to substantiate that the increase lipid content of the parasitized cells was attributable to the parasite and not to changes in the host cell, measurements were made of the total lipid content of free parasites isolated from infected cells. The data (Table II) indicate that the parasites freed from 100 infected cells would contain
LIPID
CONTENT
Plasmodium berghei
OF
TABLE
183
I
Comparison of Total Lipid Weight, Cholesterol and Lipid-P Content of Normal, Phenylhydrazine-Treated and Parasitized Eythrocyteti mg State of animal Normal Normal Normal
<5 <5 <5
Phenylhydrazine treated Phenylhydrazine treated Phenylhydrazine treated Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized a bers b that
% Parasitemia
% Reticulocytes
Total lipid wt
100 RBC’s Cholesterol
lipid-Pa
-
4.8 4.8 4.5
1.4 1.4 -
1.1 1.0 -
23
-
5.7
1.6
1.4
24
-
5.3
1.5
1.5
28 -
12 12 16 18 23 26 29 32 33 42
5.4 6.3 7.4 6.7 10.3 12.0 9.3 13.1 14.6 14.0 13.0
1.5 1.6 1.5 2.1 2.3 2.0 2.6 2.5 2.4
1.2 1.7 1.8 2.0 3.0 2.7 2.6 3.7 3.9 3.5 3.9
Each result was obtained from aliquots of blood pools which contained approximately equal numof red blood cells from at least ten rats. Values for mg of phospholipid may be calculated from lipid phosphorus data on the assumption in pure phosphohpid the phosphorus content is 4%.
TABLE
II
Lipid Content of Free Parasite@ B A
Experiment number I II
% Parasitemia 28 32
Total lipid of free parasites liberated from 100 RBC’s
Calculated total lipid of free parasites from 100 infected RBC’s (A/% parasitemia)
7.9 X 10-S mg 9.0 X 10-s mg
28.2 X 10-S mg 28.1 X 10-s mg
C Total lipid of free parasites from 100 cells (28 or 32% parasitized) + total lipid from 100 uninfected RBC’s 7.9 + 5.5b = 13.4 X 10-s 9.0 + 5.5b = 14.5 X 10-s
mg mg
a Parasites were freed from host red blood cells by saponin lysis according to Wallace et al. ( 1965). b From Table I, average total lipid content/100 uninfected RBC’s (Ave. 25% reticulocytes).
about 28 X lOWa mg of lipid as compared with about 5.5 X lop8 mg of lipid obtained from 100 uninfected red blood cells (average of the three values for phenylhydrazinetreated animals in Table I), Furthermore, for a given parasitemia, the total lipid content is shown to be a summation of the
lipid of the host cell plus that of the free parasites (Table II). A comparison of the relative changes in neutral and phospholipids was then carried out on the intact infected cells rather than on the isolated parasites since we believe that the quantitation would be
184
LAWRENCE
AND
more valid when expressed on the basis of intact cells with a given percentage parasitemia. Since most of the neutral lipid is considered to be cholesterol (Lewert, 1952), this parameter along with total lipid-P was determined. The results shown in Table I suggested that the increased lipid content of parasitized cells mainly reflects an increase in the phospholipids since the total amount of lipid-P increased from 1.0 X 10-Q mg/lOO RBC’s for the normal animal as compared with values of 1.7 X lo-” for the low parasitemia and increased progressively to 3.9 X 10eg through the parasitemias studied. Values obtained for cholesterol content of parasitized erythrocytes also increased from those obtained for normal erythrocytes, but the increase seen in this instance was not so marked as that seen with the phospholipid fraction. Results obtained for phenylhydrazine-treated animals were slightly higher than normal, but lower than those of the lowest parasitemia studied. Since the amount of lipid-P was greatly increased with increasing parasitemias, an attempt was made to determine whether
Relative
CENEDELLA
this increase was due to an increase in one or more specific phospholipids or whether it was a result of a general increase in all major phospholipid classes. The results are shown in Table III. No appreciable difference between the percentages of the phospholipids between the normal and phenylhydrazine-treated animals was seen. Thus, for the purpose of statistical analysis, the results were grouped together as unparasitized cells. There were some small although interesting changes in the relative composition of the phospholipid from the parasitized blood cells as compared with the uninfected cells which warrant comment. Phosphatidylethanolamine comprised about 30% of the total phospholipid in parasitized cells but only about 25% in unparasitized cells. This difference is highly statistically significant (p(t) < 0.001). Also, the fraction of the total phospholipid which was sphingomyelins plus lysolecithins was statistically significantly smaller in the highly parasitized as compared with unparasitized cells. The lecithins plus phosphatidlyserines of both parasitized and unparasitized cells were virtually identical
TABLE III Percentages of Major Phospholipid Classes Obtained from Normal, Phenylhydrazine-Treated and Parasitized Erythrocytesa Percentage
State of animal Normal Normal Phenylhydrazinetreated (23% Phenylhydrazinetreated (28% I2 % Parasitized 23 % Parasitized 26% Parasitized 29 % Parasitized 33 % Parasitized 42 % Parasitized
of total lipid-P in each lipid class”
A
B
C
D
E
1.2 1.3
24.8 25.8
58.0 59.9
16.0 13.0
0 0
Retie.)
1.9
24.3
55.3
18.5
0
Retie. )
2.1 2.5 4.5 3.0 1.6 3.1 1.8
23.3 25.8 32.0 29.9 32.0 30.9 32.5
58.1 56.4 57.5 56.1 57.0 58.8 57.9
16.5 15.3 6.0 11.2 9.4 7.2 7.8
0 0 0 0 0 0 0
a Each result was obtained from aliquots of blood pools which contained the blood rats. b A, polyglycerol phosphatides, phosphatidic acid, neutral lipids; B, phosphatidyl C, phosphatidyl serines, lecithins; D, sphingomyelins and lysolecithins; E, origin.
of at least ten ethanolamines;
LIPID CONTENT OF Plasmodium
and accounted for between 55 and 60% of the total phospholipids with both cell types. Thus, although there was an approximate four-fold increase in the total phospholipid content of the highly parasitized cells as compared with unparasitized blood cells the relative composition of the phospholipid from the two cell types were very similar with only minor exception. Therefore, the increased phospholipid content of Plasmodium berghei parasitized blood cells largely represents a proportionate increase of all phospholipids studied rather than a selective increase of particular ones. DISCXJSSION
The lipid extracts of rat red blood cells parasitized with P. berghei showed increased amounts of total lipid when compared to unparasitized mature and immature cells. This increase in lipid content depended on the percentage of parasitized cells extracted; i.e., at low parasitemias ( 12%) the increase was small, while at high parasitemias (3242%) the increase amounted to approximately 2.5- to S-fold. Thus, at these higher parasitemias the extracted lipids should closely reflect the actual lipid composition of Plasmodium berghei. Morrison and Jeskey (1947) demonstrated that the lipids of erythrocytes of infected monkeys increased 550% over normal preparations. An investigation into the nature of the increase seen in the parasitized cells revealed that both neutral and phospholipid levels were raised. However, cholesterol, the major neutral lipid (Lewert, 1952) increased to only about two-fold while the lipid-P increased to more than four-fold over the range of parasitemias studied. Again, the increase in both these parameters was dependent upon the level of the parasitemia. In studies with the malarial parasite P. knowlesi, Ball et al. ( 1948)have shown that the parasitized cell content of
berghei
185
lipid-P was two-. to four-fold that of the normal red blood cell. Further, the magnitude of the increases that occurred were shown to be dependent on both the size and age of the parasites as well as on the total number of parasites present. Lewert ( 1952) has shown an increase in lipid-P in cells infected with P. galliwceum. Although the relative percent distribution of individual phospholipids between parasitized and unparasitized cells was similar, the phospholipids extracted from parasitized blood cells did contain a small but statistically significantly greater percentage of phosphatidyl ethanolamines and a significantly lower percentage of sphingomyelins plus lysolecithins than that of unparasitized cells. In vitro studies by Cenedella ( 1968) indicated that glucose carbon is incorporated into lipids by the erythrocytic form of Plasmodium berghei for the construction of phospholipid molecules to a greater extent th an in normal cells and that the glucose carbon enters the phospholipid primarily by way of a-glycerol phosphate. He also showed that the most highly labeled phospholipid was cephalin. Gutierrez ( 1966) recently reported studies of phospholipid synthesis by the erythrocytic form of P. fallax. When the parasitized erythrocytes were incubated in vitro with 14C-oleic, palmitic, or stearic acids, he observed that the parasitized cells readily incorporated these fatty acids into phospholipids, and that the greatest incorporation always occurred in the cephalin class. Since phospholipids are found almost exclusively associated with membranes, the pattern of phospholipids presented for intraerythrocytic Plasmodium berghei most probably reflects the phospholipid composition of the parasite membranes. In the parasitized rat erythrocyte, lecithins have been shown to be the most abundant phospholipid and thus of all the parasite phospholipids the net synthesis of lecithins must be greatest. This conclusion is sup-
186
LAWRENCE
ArND CENEDELLA
ported by recent studies of Cenedella et al. (1969) measuring lipid synthesis in viva by intraerythrocytic Plasmodium berg&i. However, other data presented by Cendella (1968) and Gutierrez (1966) and by the present study indicate that the parasite cephalin, most probably phosphatidy1 ethanolamine, is an extremely active lipid fraction with a turnover rate apparently greater than that of the other phospholipids. In conclusion, the slight alterations in the relative composition of Plasmodium berghei-parasitized cell phospholipids as compared with unparasitized cells could reflect the phospholipid composition of the membranes characteristic of this malarial parasite. ACKNOWLEDGMENTS This work was carried out under terms of Contract No. DA-49-193-MD-2767 between the U.S. Army Medical Research and Development Command and the Board of Governors of West Virginia University and is Contribution No. 519 from the Army Research Program on Malaria. REFERENCES BALL, E. G., MCKEE, R. W., ANFINSEN, C. B., Cmz, W. O., AND GEIMAN, Q. M. 1948. Studies on malarial parasites, IX. Chemical and metabolic changes during growth and multiplication in vivo and in vitro. Journal of Biological Chemistry 175, 547-571. BISHOP, C., AND SURGENOR, D. M. 1964. The Red Blood Cell, 1st ed. Academic Press, New York, New York, p. 281-290. CENEDELLA, R. J, 1968. Lipid synthesis from glucose carbon by Plasmodium berghei in vitro. American Journal of Tropical Medicine and Hygiene 17, 680-684. CENEDELLA, R. J., JARRELL, J. J., AND SAXE, L. H.
1969. Lipid synthesis in vivo from l-14Coleic acid and 6-sH-Glucose by intraerythrocytic Plasmodium berghei. Military Medicine (in press). GIER, J. DE AND DEENEN, L. L. M., VAN. 1961. Some lipid characteristics of red cell membranes of various animal species. Biochimica and Biophysics Acta 49, 28&296. GUTIERREZ, J. 1966. Effect of the antimalarial chloroquine on the phospholipid metabolism of avian malaria and heart tissue. American Journal of Tropical Medicine and Hygiene 15, 818-822. 1952. Nucleic acids in plasLEWERT, R. M. modia and the phosphorus partition of cells infected with Plasmodium gallinaceum. Journal of Infectious Disease 91, 125-144. MORRISON, D. B., AND JESKEY, H. A. 1947. The pigment, lipids and proteins of the malaria parasite (P. knowlesi). Federation Proceedings 6, 279. ROUSER, G., SIAKOTOS, A. N., AND FLFXSCHER, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M. 1962. Separation of phosphatidyl ethanolamine, phosphatidyl serine, and other phospholipids by thin-layer chromatography. Journal of Lipid Research 3, 467-470. WALLACE, W. R., FINERTY, J. F., AND DIMOPOULLOS, G. T. 1965. Studies on the lipid of Plasmodium lophurae and Plasmodium berghei. American Journal of Tropical Medicine and Hygiene 14, 715-718. WAYS, P., AND HANAHAN, D. J, 1962. Characterization and quantification of red cell lipids in normal man. JournaE of Lipid Research, 5, 318-328. YOUNGBURG, G. E., AND YOUNGBURG, M. V. 1930. Phosphorus metabolism I. A system of blood phosphorus analysis. Journal of Laboratory and Clinical Medicine 16, 158-166. ZLATKIS, A., ZAK, B., AND BONE, A. J. 1953. A new method for the direct determination 01 serum cholesterol. Iournal of Laboratory and Clinical Medicine 41, 486492.
Lipid
Content
(1969)
of Plasmodium Rat Red Blood
C. W.
Lawrence
and
Richard
berghei-Infected Cells J. Cenedella
Department of Pharmacology, West Virginia University Medical Center, Morgantown, West Virginia 26506 (Submitted
for publication
18 February
1969)
LAWFIENCE, C. W. AND CENEDELLA, R. J. 1969. Lipid content of Plasmodium berghei-infected rat red blood cells. Experimental ParasitoZogy 26, 181-186. The lipid extracts of rat red blood cells infected with the malarial parasite Plasmodium berghei contained increased amounts of total lipid when compared to extracts of cells from either normal or phenylhydrazine-treated rats. Total cholesterol increased approximately two-fold while the total lipid-P increased more than four-fold. The four-fold increase in phospholipid content of Plasmodium berghei parasitized over unparasitized blood cells largely represents a proportionate increase of all phospholipids studied rather than a selective increase of particular ones. However, the phospholipids of parasitized cells did contain a small but statistically significant higher percentage of phosphatidylethanolamine and also a significantly lower percentage of sphingomylins plus lysolecitbins than unparasitized cell phospholipids. These minor changes could reflect the phospholipid composition of the membranes characteristic of this malarial parasite. INDEX DESCRIPTORS: Plasmodium berghei, lipids; Red blood cells; Phosphohpids; Cholesterol; Phosphatidyl ethanolamine; Sphingomylein; Lysolecithin; Malaria; Metabolism; Phenylhydrazine.
Growth and multiplication of the malarial parasite in the mammalian red blood cells is accompanied by a marked increase in the lipid content of these cells (Ball et al., 1946; Lewert, 1952; Morrison and Jeskey, 1947). Since mammalian erythrocytes possess little ability to synthesize these lipids de nouo (Bishop and Surgenor, 1964), this increase in the lipid content of malarial-infected erythrocytes most reasonably reflects the lipid composition of the growing parasite. Although the increase in lipid content of malarial-infected blood cells is well documented, the identity of individual lipids producing this increase has been largely undetermined. The purpose of this investigation was to quantitate accurately the increase in lipid content of Plasmodium berghei infected rat red blood cells, to
determine the distribution of this lipid between neutral and phospholipid, and to identify and quantitate the major phospholipids. MATERIALS
AND METHODS
Blood from at least 10 male rats weighing 140-160 gm was collected by into 12 ml syringes cardiac puncture containing 1.2 ml of sodium citrate anticoagulant (3.69 gm tri-sodium citrate plus 1.13 gm citric acid/100 ml distilled water) and then pooled. The animals were of three types: ( 1) normal ( untreated) ; (2) Plasmodium berghei-parasitized and (3) phenylhydrazine-treated. PZusmodium berghei (strain KBG 173), obtained initially from Dr. William Trager of the Rockefeller University, was used for 181
182
LAWRENCE
AND CENEDELLA
these investigations. Parasites were maintained by blood passage from infected to healthy 6-week old male rats. In order to obtain uninfected rats with about the same reticulocytosis at the time of experimentation as the infected animals, normal rats were treated twice with phenylhydrazine ( 15 mg/kg body weight 5 and 3 days before being used). Total red cell counts were established with a hemocytometer; reticulocyte counts were made subsequent to staining with methylene blue N. Thin smears were made from infected blood pools, stained (Wright’s) and counted to determine the percentage of parasitized cells. Subsequently aliquots (26-40 ml) of the parasitized or unparasitized blood were centrifuged at about 1OOOg for 10 minutes at 5°C. After the plasma was removed and discarded, the cells were suspended to the original volume in Kreb’s phosphate buffer (pH 7.4, Ca2+ omitted) and centrifuged as above. This washing procedure was repeated twice. For preparation of free parasites, blood from parasitized rats was collected and the cells were centrifuged and washed as above. The washed erythrocytes were lysed by incubation at 37°C for 30 minutes in 40 volumes of a l:lO,OOO dilution of saponin in buffer according to Wallace et al. ( 1965). Lipid extraction of the red cells was carried out according to procedure III of Ways and Hanahan (1962). After extraction the lipid was transferred to tared vials, at weighed, and stored in chloroform -10°C. The phospholipid content of these total lipid extracts was measured by phosphorus analysis (Youngburg and Youngburg, 1930). Cholesterol content was determined by the method of Zlatkis, et al. ( 1953). Aliquots of each lipid sample were subjected to thin-layer chromatography on silica gel G with chloroform:methanol:acetic acid: water (65:25:8:4) (Skipski et al., 1962)
and phosphorus analyses on the individual phospholipid fractions recovered after chromatography were carried out after digestion of samples and blanks with 72% perchloric acid followed by treatment with ammonium molybdate solution and 10% ascorbic acid (Rouser et al., 1966). The plate was divided into five areas. Area A included the area from the solvent front down to the phosphatidyl ethanolamines. This area contains polyglycerol phosphatides, phosphatidic acid, and neutral lipids. Area B included the phosphatidyl ethanolamines down to the phosphatidyl serine area. Area C included the phosphatidyl serines as a minor component (Gier and VanDeenen, 1961) and lecithins down to the sphingomyelin area while area D included both sphingomyelin and lysolecithin (minor component according to Gier and van Deenen, 1961). Area E included material remaining at the origin. Values for total lipid-P applied to the plates was determined by taking the sum of these five areas since these included all the lipid originally applied. RESULTS
The results shown in Table I indicate that there was a marked increase in the total lipid weight (mg lipid/100 RBC’s) extracted from the parasitized erythrocytes when compared to the values obtained from either the normal or phenylhydrazinetreated animals. This increase progressed rather consistently from low parasitemias (12%) through the highest parasitemia studied (42%). In order to substantiate that the increase lipid content of the parasitized cells was attributable to the parasite and not to changes in the host cell, measurements were made of the total lipid content of free parasites isolated from infected cells. The data (Table II) indicate that the parasites freed from 100 infected cells would contain
LIPID
CONTENT
Plasmodium berghei
OF
TABLE
183
I
Comparison of Total Lipid Weight, Cholesterol and Lipid-P Content of Normal, Phenylhydrazine-Treated and Parasitized Eythrocyteti mg State of animal Normal Normal Normal
<5 <5 <5
Phenylhydrazine treated Phenylhydrazine treated Phenylhydrazine treated Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized Parasitized a bers b that
% Parasitemia
% Reticulocytes
Total lipid wt
100 RBC’s Cholesterol
lipid-Pa
-
4.8 4.8 4.5
1.4 1.4 -
1.1 1.0 -
23
-
5.7
1.6
1.4
24
-
5.3
1.5
1.5
28 -
12 12 16 18 23 26 29 32 33 42
5.4 6.3 7.4 6.7 10.3 12.0 9.3 13.1 14.6 14.0 13.0
1.5 1.6 1.5 2.1 2.3 2.0 2.6 2.5 2.4
1.2 1.7 1.8 2.0 3.0 2.7 2.6 3.7 3.9 3.5 3.9
Each result was obtained from aliquots of blood pools which contained approximately equal numof red blood cells from at least ten rats. Values for mg of phospholipid may be calculated from lipid phosphorus data on the assumption in pure phosphohpid the phosphorus content is 4%.
TABLE
II
Lipid Content of Free Parasite@ B A
Experiment number I II
% Parasitemia 28 32
Total lipid of free parasites liberated from 100 RBC’s
Calculated total lipid of free parasites from 100 infected RBC’s (A/% parasitemia)
7.9 X 10-S mg 9.0 X 10-s mg
28.2 X 10-S mg 28.1 X 10-s mg
C Total lipid of free parasites from 100 cells (28 or 32% parasitized) + total lipid from 100 uninfected RBC’s 7.9 + 5.5b = 13.4 X 10-s 9.0 + 5.5b = 14.5 X 10-s
mg mg
a Parasites were freed from host red blood cells by saponin lysis according to Wallace et al. ( 1965). b From Table I, average total lipid content/100 uninfected RBC’s (Ave. 25% reticulocytes).
about 28 X lOWa mg of lipid as compared with about 5.5 X lop8 mg of lipid obtained from 100 uninfected red blood cells (average of the three values for phenylhydrazinetreated animals in Table I), Furthermore, for a given parasitemia, the total lipid content is shown to be a summation of the
lipid of the host cell plus that of the free parasites (Table II). A comparison of the relative changes in neutral and phospholipids was then carried out on the intact infected cells rather than on the isolated parasites since we believe that the quantitation would be
184
LAWRENCE
AND
more valid when expressed on the basis of intact cells with a given percentage parasitemia. Since most of the neutral lipid is considered to be cholesterol (Lewert, 1952), this parameter along with total lipid-P was determined. The results shown in Table I suggested that the increased lipid content of parasitized cells mainly reflects an increase in the phospholipids since the total amount of lipid-P increased from 1.0 X 10-Q mg/lOO RBC’s for the normal animal as compared with values of 1.7 X lo-” for the low parasitemia and increased progressively to 3.9 X 10eg through the parasitemias studied. Values obtained for cholesterol content of parasitized erythrocytes also increased from those obtained for normal erythrocytes, but the increase seen in this instance was not so marked as that seen with the phospholipid fraction. Results obtained for phenylhydrazine-treated animals were slightly higher than normal, but lower than those of the lowest parasitemia studied. Since the amount of lipid-P was greatly increased with increasing parasitemias, an attempt was made to determine whether
Relative
CENEDELLA
this increase was due to an increase in one or more specific phospholipids or whether it was a result of a general increase in all major phospholipid classes. The results are shown in Table III. No appreciable difference between the percentages of the phospholipids between the normal and phenylhydrazine-treated animals was seen. Thus, for the purpose of statistical analysis, the results were grouped together as unparasitized cells. There were some small although interesting changes in the relative composition of the phospholipid from the parasitized blood cells as compared with the uninfected cells which warrant comment. Phosphatidylethanolamine comprised about 30% of the total phospholipid in parasitized cells but only about 25% in unparasitized cells. This difference is highly statistically significant (p(t) < 0.001). Also, the fraction of the total phospholipid which was sphingomyelins plus lysolecithins was statistically significantly smaller in the highly parasitized as compared with unparasitized cells. The lecithins plus phosphatidlyserines of both parasitized and unparasitized cells were virtually identical
TABLE III Percentages of Major Phospholipid Classes Obtained from Normal, Phenylhydrazine-Treated and Parasitized Erythrocytesa Percentage
State of animal Normal Normal Phenylhydrazinetreated (23% Phenylhydrazinetreated (28% I2 % Parasitized 23 % Parasitized 26% Parasitized 29 % Parasitized 33 % Parasitized 42 % Parasitized
of total lipid-P in each lipid class”
A
B
C
D
E
1.2 1.3
24.8 25.8
58.0 59.9
16.0 13.0
0 0
Retie.)
1.9
24.3
55.3
18.5
0
Retie. )
2.1 2.5 4.5 3.0 1.6 3.1 1.8
23.3 25.8 32.0 29.9 32.0 30.9 32.5
58.1 56.4 57.5 56.1 57.0 58.8 57.9
16.5 15.3 6.0 11.2 9.4 7.2 7.8
0 0 0 0 0 0 0
a Each result was obtained from aliquots of blood pools which contained the blood rats. b A, polyglycerol phosphatides, phosphatidic acid, neutral lipids; B, phosphatidyl C, phosphatidyl serines, lecithins; D, sphingomyelins and lysolecithins; E, origin.
of at least ten ethanolamines;
LIPID CONTENT OF Plasmodium
and accounted for between 55 and 60% of the total phospholipids with both cell types. Thus, although there was an approximate four-fold increase in the total phospholipid content of the highly parasitized cells as compared with unparasitized blood cells the relative composition of the phospholipid from the two cell types were very similar with only minor exception. Therefore, the increased phospholipid content of Plasmodium berghei parasitized blood cells largely represents a proportionate increase of all phospholipids studied rather than a selective increase of particular ones. DISCXJSSION
The lipid extracts of rat red blood cells parasitized with P. berghei showed increased amounts of total lipid when compared to unparasitized mature and immature cells. This increase in lipid content depended on the percentage of parasitized cells extracted; i.e., at low parasitemias ( 12%) the increase was small, while at high parasitemias (3242%) the increase amounted to approximately 2.5- to S-fold. Thus, at these higher parasitemias the extracted lipids should closely reflect the actual lipid composition of Plasmodium berghei. Morrison and Jeskey (1947) demonstrated that the lipids of erythrocytes of infected monkeys increased 550% over normal preparations. An investigation into the nature of the increase seen in the parasitized cells revealed that both neutral and phospholipid levels were raised. However, cholesterol, the major neutral lipid (Lewert, 1952) increased to only about two-fold while the lipid-P increased to more than four-fold over the range of parasitemias studied. Again, the increase in both these parameters was dependent upon the level of the parasitemia. In studies with the malarial parasite P. knowlesi, Ball et al. ( 1948)have shown that the parasitized cell content of
berghei
185
lipid-P was two-. to four-fold that of the normal red blood cell. Further, the magnitude of the increases that occurred were shown to be dependent on both the size and age of the parasites as well as on the total number of parasites present. Lewert ( 1952) has shown an increase in lipid-P in cells infected with P. galliwceum. Although the relative percent distribution of individual phospholipids between parasitized and unparasitized cells was similar, the phospholipids extracted from parasitized blood cells did contain a small but statistically significantly greater percentage of phosphatidyl ethanolamines and a significantly lower percentage of sphingomyelins plus lysolecithins than that of unparasitized cells. In vitro studies by Cenedella ( 1968) indicated that glucose carbon is incorporated into lipids by the erythrocytic form of Plasmodium berghei for the construction of phospholipid molecules to a greater extent th an in normal cells and that the glucose carbon enters the phospholipid primarily by way of a-glycerol phosphate. He also showed that the most highly labeled phospholipid was cephalin. Gutierrez ( 1966) recently reported studies of phospholipid synthesis by the erythrocytic form of P. fallax. When the parasitized erythrocytes were incubated in vitro with 14C-oleic, palmitic, or stearic acids, he observed that the parasitized cells readily incorporated these fatty acids into phospholipids, and that the greatest incorporation always occurred in the cephalin class. Since phospholipids are found almost exclusively associated with membranes, the pattern of phospholipids presented for intraerythrocytic Plasmodium berghei most probably reflects the phospholipid composition of the parasite membranes. In the parasitized rat erythrocyte, lecithins have been shown to be the most abundant phospholipid and thus of all the parasite phospholipids the net synthesis of lecithins must be greatest. This conclusion is sup-
186
LAWRENCE
ArND CENEDELLA
ported by recent studies of Cenedella et al. (1969) measuring lipid synthesis in viva by intraerythrocytic Plasmodium berg&i. However, other data presented by Cendella (1968) and Gutierrez (1966) and by the present study indicate that the parasite cephalin, most probably phosphatidy1 ethanolamine, is an extremely active lipid fraction with a turnover rate apparently greater than that of the other phospholipids. In conclusion, the slight alterations in the relative composition of Plasmodium berghei-parasitized cell phospholipids as compared with unparasitized cells could reflect the phospholipid composition of the membranes characteristic of this malarial parasite. ACKNOWLEDGMENTS This work was carried out under terms of Contract No. DA-49-193-MD-2767 between the U.S. Army Medical Research and Development Command and the Board of Governors of West Virginia University and is Contribution No. 519 from the Army Research Program on Malaria. REFERENCES BALL, E. G., MCKEE, R. W., ANFINSEN, C. B., Cmz, W. O., AND GEIMAN, Q. M. 1948. Studies on malarial parasites, IX. Chemical and metabolic changes during growth and multiplication in vivo and in vitro. Journal of Biological Chemistry 175, 547-571. BISHOP, C., AND SURGENOR, D. M. 1964. The Red Blood Cell, 1st ed. Academic Press, New York, New York, p. 281-290. CENEDELLA, R. J, 1968. Lipid synthesis from glucose carbon by Plasmodium berghei in vitro. American Journal of Tropical Medicine and Hygiene 17, 680-684. CENEDELLA, R. J., JARRELL, J. J., AND SAXE, L. H.
1969. Lipid synthesis in vivo from l-14Coleic acid and 6-sH-Glucose by intraerythrocytic Plasmodium berghei. Military Medicine (in press). GIER, J. DE AND DEENEN, L. L. M., VAN. 1961. Some lipid characteristics of red cell membranes of various animal species. Biochimica and Biophysics Acta 49, 28&296. GUTIERREZ, J. 1966. Effect of the antimalarial chloroquine on the phospholipid metabolism of avian malaria and heart tissue. American Journal of Tropical Medicine and Hygiene 15, 818-822. 1952. Nucleic acids in plasLEWERT, R. M. modia and the phosphorus partition of cells infected with Plasmodium gallinaceum. Journal of Infectious Disease 91, 125-144. MORRISON, D. B., AND JESKEY, H. A. 1947. The pigment, lipids and proteins of the malaria parasite (P. knowlesi). Federation Proceedings 6, 279. ROUSER, G., SIAKOTOS, A. N., AND FLFXSCHER, S. 1966. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1, 85-86. SKIPSKI, V. P., PETERSON, R. F., AND BARCLAY, M. 1962. Separation of phosphatidyl ethanolamine, phosphatidyl serine, and other phospholipids by thin-layer chromatography. Journal of Lipid Research 3, 467-470. WALLACE, W. R., FINERTY, J. F., AND DIMOPOULLOS, G. T. 1965. Studies on the lipid of Plasmodium lophurae and Plasmodium berghei. American Journal of Tropical Medicine and Hygiene 14, 715-718. WAYS, P., AND HANAHAN, D. J, 1962. Characterization and quantification of red cell lipids in normal man. JournaE of Lipid Research, 5, 318-328. YOUNGBURG, G. E., AND YOUNGBURG, M. V. 1930. Phosphorus metabolism I. A system of blood phosphorus analysis. Journal of Laboratory and Clinical Medicine 16, 158-166. ZLATKIS, A., ZAK, B., AND BONE, A. J. 1953. A new method for the direct determination 01 serum cholesterol. Iournal of Laboratory and Clinical Medicine 41, 486492.