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Plasmodium lophurae: Quantitative in vitro incorporation of 14C-1-acetate into lipids
EXPERIMENTAL
PARASITOLOGY
37, 193-204
( 1975)
Plasmodium lophurae:
Quantitative in Vitro of 14C-l-Acetate into Lipids
Incorporation
CHEIWL L. SMITH HARDY,~ L. T. HART,~ G. T. DIMOPOULLOS,~ AND E. N. LAMBFIEMONT 4 Department Louisiana
of Veterinary
Science and Nuclear Science Center, State University, Baton Rouge, Louisiana 70803
(Submitted
for publication
November
13, 1973)
HARDY, C. L. S., HART, L. T., DIMOPOULLOS, G. T., AND LAMBREMONT, E. N. 1975. Plasmodium Zophurae: Quantitative in uitro incorporation of W-l-acetate into lipids. Experimental Parasitology 37, 193-204. Blood from ducks parasitized with Plasmodium Zoph,urae and normal duck blood were incubated with sodium Y-l-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and. plasma and lipid classes separated by thin-layer chromatography. S#pecific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated %-labeled lipid precursors. Experiments employing sodium W-l-acetate in two concentrations, 50 PCi “C in 0.91 rmole sodium acetate/50 ml blood and 500 pCi “C in 9.1 kmole sodium acetate/50 ml blood ( 1.82 X lo-’ M and 1.82 X lo-” M ), showed higher l’C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At 1.82 X lo-’ M W-l-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration ( 1.82 X lo-” M ), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. Zophurae. Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols. At 24 hr of incubation, the lipids of partially purified P. Zophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides. In plasma from parasitized blood incubated 24 hr with W-l-acetate, the highest specific activity and greatest percent of “C incorporation was found in free fatty acids. Plasmodium Zophurae; Lipids; “C-acetate incorporation; INDEX DESCRIPTORS: Metabolism; Malaria; Chromatography, thin-layer; Ducks; Scintillation, liquid.
1 Present address: Clinical Laboratories, University of Mississippi Medical Center, Jackson, Mississippi 39216. 2 Department of Veterinary Science, Louisiana State University, Baton Rouge, Louisiana 70803. Author to whom reprint requests should be addressed. 3 Nuclear Science Center, Louisiana State University, Baton Rouge, Louisiana 70803.
The
of
integrity
of
(van
Deenen
and
malarial been shown
parasite well that
lipids
the
struc-
(RBC) de Gier 1964) and the erythrocyte
(von
Brand
documented. the
in
the
erythrocytic
It
1966)
has stages
also
has been
of some
Plusmodium species can take up fatty acids in vitro and incorporate them into cellular 193
Copyright c 1975 by Academic Press, Inc. All rights o4 reproduction in any form reserved.
importance
tural
194
HARDY
lipid (Gutierrez 1966; Cenedella et al. 1969). It is conceivable that the parasites may depend upon the RBC and plasma of the host for fatty acids instead of synthesizing their own (Cenedella 1968). Studies on the incorporation of ‘C-lacetate into the blood lipids of normal du,cks and ducks infected with P. Eophume were reported by Brundage et aZ. ( 1969). Although quantitative analyses were not made, it was demonstrated that a greater incorporation of 14C-l-acetate occurred in the lipids of the parasitized RBC. No attempt was made to separate the parasites from the RBC in their studies; therefore, the level of incorporation into the cellular components of the parasite could not be determined. In the present study, our objective was to measure quantitatively the radioactivity incorporated into the lipids of the parasite when whole blood from infected ducks was incubated with 14C-l-acetate. Supplementary data concerning the radioactivity found in plasma from infeacted ducks and data on normal blood were also collected. MATERIALS
AKD METHODS
Blood Samples The erythrocytic stages of P. lophurae were maintained in 4-6-week-old Pekin ducklings by intravenous passage of blood from infected ducks having greater than 80% of their RBC parasitized. When the desired degree of parasitemia was attained in the host (above 90% ), blood was collected by cardiac puncture in heparin so.dium (25-30 U.S.P. units/ml of blood). Uninfected ducks provided normal blood samples. Incubation
with l”C-l-Acetate
Normal blood and blood samples from infected ducks were dispensed into sterile 250-ml flasks in 50-ml portions. Normal blood was inscluded to establish the level of background activity of 14C incorporation
_,a. AlA. *-
I51
into the lipid classes that could be attributed to host RBC debris in the parasite preparations. An incubation mixture of penicillin (1000 I.U./ml blood), streptomycin (1290 I.U./ml blood) and sodium l”C-l-acetate (50 &i l”C in 0.91 pmole sodium acetate/50 ml blood, 1.82 x 10 5 hl; or 500 &i 14C in 9.1 pmole sodium acetate/50 ml blood, 1.82 x 10m4M) was added, and the flasks kept at 39 C for either 5 or 24 hr in an oscillating water bath. Recovery of Parasites After incubation, the blood samples were centrifuged and the plasma recovered for further study. The RBC were washed with buffer (Sherman and Hull 1960) and the buffy coat discarded at ea’ch washing. P. Zophurae cells were freed from RBC and purified according to the method described by Sherman and Hull (1960). The RBC were lysed with saponin and after centrifugal washing the debris was treated with DNAase. The resulting product was washed by centrifugation and the sediment recovered for lipid analysis. Yields of 8-10 ml of packed partially purified parasites per 50 ml of blood from infected ducks were recovered as compared to about 0.1 ml of packed erythrocytic debris per 50 ml of normal blood. Lipid Extraction The lipid extraction and recovery procedure described by Folch et al. (1957) was adopted for the extraction of normal plasma, plasma from infected ducks, partially purified parasites, and normal erythrocytic debris. Samples of plasma were added directly to 20 vol of chloroform; methanol (2:1, v/v) and mixed for 16-20 hr at 21 C on a magnetic stirrer. Erythrocytic debris and parasite preparations were first added directly to methanol and mixed for about 5 min. This step was found necessary to minimize clumping of
METABOLISM
OF
14C-ACETATE
the cellular material. Twice the volume of chloroform was then added to give a final extraction mixture of chloroform : methanol (2: 1, v/v) in a 20-fold excess of the volume of the preparation being extracted. The total lipid extracts were washed three times with distilled water to remove nonlipid material. The extraction proceeded as indicated for the plasma samples. After filtration and concentration, the total lipid solution was dried over anhydrous Na2S04 in a known volume of chloroform for the subsequent analyses and lipid class separations. Preparatizje Thin-Layer WC)
Chromatography
The techniques employed here for TLC have been described by Randerath (1966). Dupli’cate silica gel G plates were prepared in thicknesses of 250 pm and activated at 110 C for 30 min. One plate was used to assay the radioactivity in each lipid class and the other for the gravimetric analyses of lipids. A known weight of total lipid extract, not exceeding 15 mg, was applied to each plate. Lipid class standard, Hormel Institute II-A, containing a mixture of hydrogenated lecithin, oleic acid, triolein, cholesterol oleate and cholesterol was also spotted on each plate to aid in the identification of the separated fractions. After development of the TLC plates in a solvent system of petroleum ether, diethyl ether and glacial acetic acid (84: 15: 1, v/v/v), lipids for radioassay were made visible by exposing the plate to iodine vapor. Areas of the plate corresponding to separated standards [(free fatty acids (FFA), triglycerides (TG), sterol esters ( SE)] were removed and transferred to liquid scintillation vials. All of the remaining adsorbent on the plate which contained the more polar lipids was recovered and transferred quantitatively to a glass column plugged with defatted cotton. Lipid contained in this fraction was then eluted
IN
PLASMODIUM
19.5
with methanol: chloroform (2: 1, v/v). The eluate was con,centrated under a stream of nitrogen and quantitatively applied to a second silica gel G plate prepared as described above. Standards of monopalmitin and dipalmitin and as before, lipid class standard, Hormel Institute II-A, were spotted on separated lanes. The plates were developed in a solvent system of benzene, diethyl ether, absolute ethanol and glacial acetic acid (50:40:2:0.2, v/v/v/v) to resolve phospholipids ( PL), sterols ( ST), monoglycerides ( MG ), and diglycerides (DG) (Freeman and West 1966). The lipids were detected, identified and subsequently transferred to scintillation vials as previously described. The duplicate TLC plate employed for the gravimetric analyses of lipid classes was identically prepared and developed as des’cribed for the plates used for radioassays. The lanes containing only the H-A standard were sprayed with a 0.2% ethanolic solution of 2, 7’-dichlorofluorescein and the plates observed under ultraviolet light. Each lipid corresponding to a fraction assayed for radioactivity was eluted from the unsprayed lanes of adsorbent. Polar lipids were eluted with methanol: chloroform (2: 1, v/v) and neutral lipids with petroleum ether. After recovery and removal of solvent in uacuo, each lipid class was stored in a known volume of chloroform until the gravimetric analyses were made. Zonul Analysis of Lipids Total lipids from plasma of infected ducks and partially purified parasites were chromatographed on silica gel G TLC plates (5 x 20 cm). After visualization of the separated fractions, zones 2 mm in width were scraped directly into scintillation vials with the aid of a TLC zonal scraper (Snyder and Kimble 1965). The polar lipid fraction from several initial TLC separations was pooled and developed on a second plate as described earlier. Polar
196
HARDY PL.MG
-24 FIG. 1. Zonal analysis of 14C activity of total lipid from partially purified Plasmodium Zophurae. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84:15:1, v/v/v). Conditions: 500 &i %-acetate/50 ml of blood, incubation time 5 hr, 2 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), diglycerides (DG), free fatty acids (FFA), triglycerides ( TG).
lipids were pooled in order to provide sufficient sample for qualitative clarification of lipid classes present. Zonal profile analyses were made on these plates by scraping zones, 5 mm in width, manually with a razor blade. Assay of ‘“C
Aliquots (10 ~1) of the samples were dispensed into the cups and the solvent evaporated in a vacuum oven at 40 C for 2 hr. After reaching ambient temperature in a desiccator, the cups were reweighed and placed in liquid scintillation vials for radioassay ( Lambremont and Graves 1969). All samples were analyzed in triplicate. The maximum amount of lipid weighed was 1.5 mg with a weighing accuracy of ~40 pg. Triplicates varying more than a40 yg were discarded and new weighings were made. RESULTS
Fiue-hour Incubations Parasites. A zonal profile analysis of total lipids from partially purified P. Zophurne is presented in Fig. 1. The results of a zonal analysis of the polar lipids from this initial TLC plate which were subsequently separated and developed in another solvent system are given in Fig. 2. The fraction exhibiting migration as PL possessed the highest level of radioactivity *.,0001
DL
,.375-
,500-
Analysis of 14C activity was conducted using a Beckman Liquid Scintillation Spectrometer, Model No. LS-250. The s’cintillation solution was prepared as described by Snyder (1968) for direct counting of silica gel G scrapings without elution. All sampIes were appropriateIy corrected for background radioactivity. Quench correction was calculated by the external standard method (Wang and Willis 1965). Gravimetric
ET AL.
Analysis of Lipids
Samples of total lipid and those fractionated as classes by TLC were dissolved in a known volume of chloroform and analyzed gravimetri8cally. Aluminum foil weighing cups for the Cahn Gram Electrobalance were tared.
5 ,200. F L" G 900.
300 75 7 600 i
FIG. 2. Thin-layer chromatography (TLC) of polar lipids from partially purified Plasmodium Zophurae from Fig. 1 by zonal analysis of ‘“C activity. Thin-layer chromatography on silica gel G in a solvent system of benzene:diethyI ether: ethanol:glacial acetic acid (50:40:2:0.2, v/v/v/v). Conditions: 500 &i %-acetate/50 ml blood, incubation time 5 hr, 5 mm zones. Abbreviations: phospholipids (PL), monogIycerides (MC), sterols (ST), lipid alcohol (LA), 1,2-diglycerides ( 1,2-DG), 1,3-diglycerides (1,3-DC).
METABOLISM
OF
14C-ACETATE
TABLE Distribution
Lipid
IN
I
of W among the Lipid Classes of Partially PuriJied Plasmodium lophurae and Debris from Normal RBC, Conditions: 60 pCi V-Acetate/SO ml Blood, Incubation !l’ime 5 hr Plasmodium
classes
Specific
RBC debris
bphurae
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters
%by
% by weight
10,525 25,266 -”
66.1 5.8
78.4 2.9 -
32 106
17.1 10.4 -
67.4 2.1 -
l.i6,412 20,923 1,623 10,788 738
18.1 6.6 0.5 23 0.4
1.4 3.9 3.9 2.9 6.5
560 198 760 525 63
9.2 6.5 12.4 37.8 6.6
2.0 4.1 2.1 9.1 13.2
(dpm/w)
Specific activity (dpm/w)
5% of
7;) of “C
activity
a Activity
197
PLASMODIUM
“C
weight
in lipid class not detected.
(Fig. 1); however, when the two most polar lipid fractions were rechromatographed MG was resolved from PL and it contributed significantly to the activity as shown in Fig. 2. In addition, DG was resolved from ST by this solvent system. The radioactivity previously observed in the fraction migrating as ST, DG in the initial TLC separation (Fig. 1) was due primarily to DG which chromatographitally overlapped ST (Fig. 2). Furthermore, considerable activity was present in the fractions identified as 1,2-diglyceride (1,2-DG) and 1,3-diglpceride (1,3-DG) (Fig. 2). In order to measure the 14C activity in each lipid class, gravimetric determinations were combined with radiochemical assay. The uptake of 50 &i 14C in 0.91 pmole sodium acetate/50 ml blood was investigated initially. The data given in Table I show that after accounting for the possible contribution of residual radioactivity by debris prepared from normal RBC, the lipid alcohol (LA) fraction of P. Zophurue was found to possess the highest specific activity. The greatest percent of label was found in PL, the largest frection by weight. No activity was detected in the area of the TLC plate corresponding to ST and 1,2-DG.
Five-hour incubations were also conducted with the amount of 14C acetate increased lo-fold (Table II). The percent incorporation of 14C into lipid was similar at both substrate concentrations, being 0.21% and 1.59% for normal blood and blood from infected ducks, respectively with 50 &i/50 ml blood, and 0.10 and 1.14%, respectively, at 500 &i/50 ml blood. As before, PL showed the highest percent in‘corporation of l”C whereas MG, 1,3-DG, and TG individually exceeded the specific activity found associated with PL. Plasma. Zonal analyses of total lipid extracted from the plasma of infected ducks are shown in Figs. 3 and 4. Essentially all of the radioactivity was observed in the FFA fraction and a small amount of activity was found in the most polar fraction (Fig. 3). From the profile formed as a result of the resolution of the most polar fractions (Fig. 4), all of the radioactivity from this area on the TLC plate as shown on Fig. 3 was found to remain there. The levels of activity in MG, 1,2-DG, 1,3-DG and the possible LA were negligible; however, the ST fraction displayed a great amount of activity ( Fig. 4). After incubation with the lower substrate concentration, the greatest specific
HARDY
I10
130
-Go
3. Zonal analysis of “C activity of total lipid from plasma which served as suspending medium for Plasmodium lo~jhurae-infected RHC. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84:15: 1, v/v/v). Conditions: 500 &i Y-acetate/50 ml blood, incubation time 5 hr, 2 mm zones. Abbreviations: phospholipids (PI,), sterols (ST), free fatty acids (FFA). FIG.
activity was found in the FFA fraction of normal plasma and plasma from infcctcd ducks, with the latter exhibiting 2.5 times greater specific activity than normal (Table III). The FFA contained greater than 80% of the total 14C and small percent of the total weight. In plasma from infected ducks, the ST fraction possessed a high specific activity but no comparable class
ET AL.
mm FK. 4. Thin-layer chromatography (TLC) of polar lipids from plasma which served as suspending medium for Pla.smodium lophurue-infected RBC by zonal analysis of “C activity. These lipids were the unresolved components from the TLC analysis in Fig. 3. TLC on silica gel G in a solvent system of benzene: cliethyl ether: ethanol: glacial acetic acid (50:40:2:0.2, v/v/v/v). Conditions: 500 pCi W-acetate/50 ml blood, incubation time 5 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), lipid alcohol (LA), l$diglycerides ( 1,2-DC), 1,3-diglycerides ( 1,3-DC ).
was observed in the plasma from normal ducks (Table IV). This component was also observed in P. lqwhurae lipid preparations (Table II). Specific activities of other classes did not appear to be significantly greater than those values for the lipid classes of normal plasma.
TABTX Distribution
Lipid
classes
Phospholipids Monoglycerides Sterols Lipid alcohol Diglycerides Free fatty acids Triglycerides Sterol esters a Activity
II
of ‘“C’ among the Lipid Classrs of Patfiull~/ P~cr(/ietl Plasmotliwa lophurae untl Debris SW/IL lVorma1 KBC. Conrlitions: 500 pCi W-Acetale,/ ml Blood, Inocbalion lime 5 hr F’lasmotliron
lophlrrcle
1tBC debris
Specific ac?ivil.,v (4m/md
“; of ‘T
47,410 1,270,700 4G,OOO
72.4 4.0 0.3
74.5 0.2 0.::
81,386 440,830
1.8 5.;i
1.1 0.6
32,468 69,203
9630
in lipid class not detected.
12.1 3. 4 O..i
Specific activity (dpm/mg)
18.2 2.4 2.7
4m fiOS -,‘
‘,; of 1%
7; kY weight,
2::.2 2.2
.wsi 3.8 -
31 3
12.3
1,481
5.8
4,048 4,458 492 _____~
13.2 18.2 6.0
4.0 3.4 4.2 12.7
2,642
~.
METABOLISM
OF
%-ACETATE
TABLE Distribution
Lipid
IN
199
PLASMODIUM
III
of l4C among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Infected RBC and Debris from h’ormal RBC. Conditions 50 &‘i W-Acetate/50 ml Blood, Incubation Time 5 hr classes
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free Fatty acids Triglycerides Sterol esters
Plasma from infected
ducks
Specific activity (dpm/w)
% of 1%
% by weight
303 4537 7242 2790 BL* 63,422 577 283
5.4 2.4 3.2 2.1
69.1 2.0 1.8 2.9 3.8 5.2 7.6 7.6
85.2 1.1 0.6
Normal
plasma
Yoof
Specific activity (dpm/md
1%
% by weight
114 527:5 -a
3.8 1.4 -
51.2 0.4 -
1239 3750 24,800 216 32
4.6 1.0 88.0 0.5 0.7
5.6 0.4 5.4 3.3 33.7
a Activity in lipid class not detected. b Below limits of quantitative analysis
The specific activities in some of the lipid classes were higher in both normal plasma and plasma from infected ducks when the amount of labeled substrate was increased lo-fold (Table IV). The FFA fraction showed the highest specific activity, i.e., at least four-fold greater in infected ducks and lo-fold greater in normal plasma than that of any other single lipid fraction. Furthermore, the FFA fraction possessed the highest percent of 14C and TABLE Distribution
Lipid
IV
of W among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Infected and Debris from NoTma RBC. Conditions: 600 &i 14CAcetate/50 ml Blood, Incubation Time 5 hr classes
Plasma from infected Specific activity (dpmhd
Phospholipids Monoglycerides Sterols Lipid Alcohols Diglycerides Free fatty acids Triglycerides Sterol esters 0 Act,ivity
a low percent of total lipid by weight in both normal plasma and plasma from infected ducks. The SE fraction accounted for a significant portion of the total lipid by weight in normal plasma, whereas PL accounted for the largest portion by weight in normal plasma and plasma from infected ducks. As was observed in plasma from infected ducks, when whole blood was incubated with the lower con’centrations of substrate,
584 19,281 89,283 44,425 850 432,704 3925 1919
in lipid class not detected.
ducks
y. of 14C
% by weight
1.5 1.3 2.2 0.7 0.1 92.9 0.6 0.6
74.0 1.8 0.7 0.5 3.9 6.0 4.6 8.3
Normal Specific activity @pdmd 489 10,814 -0 8303 9021 111,190 1835 360
RBC
plasma % of W
% by weight
3.8 2.8 5.8 3.9 81.0 2.0 0.7
58.5 1.9 5.2 6.7 5.4 8.1 14.1
200
HARDY
ET AL.
of 14C (Table III) the SI fraction was not detected in normal plasma. A component designated at 1,2-DG which was not observed in the incubations with the lower substrate concentrations was found only in the lipids of normal plasma of blood incubated with the higher concentrations of 14C (Table IV). This also occurred with the normal blood samples incubated for 24 hr. Tu/>enty-Four-hour FIG. 5. Zonal analysis of 14C activity of total lipid from partially purified Plasmodiwm lophurae. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84: 15:1, v/v/v). Conditions: 500 pCi %-acetate/50 ml blood, incubation time 24 hr, 2 mm zones. Abbreviations: phospholipids (PL), sterols (ST), diglycerides (DG), free fatty acids (FFA), triglycerides (TG),
ST contained significant radioactivity. The LA and MG fractions had significantly higher specific activity than the remaining lipid classes of plasma from infected ducks with the exception of FFA ( Table III ) . In comparing the above data with results obtained on the lipid classes of blood inmcubated with the lesser concentrations TABLE Distribution
of Y.7 among the Lipid
RBC. Conditions: Lipid
classes
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters
Incubations
Parasites. Data presented in Figs. 5 and 6 were obtained by zonal analyses of total lipid extracted from partially-purified parasites, The activity in PL greatly exceed that found in the other fractions. Appearance of two peaks migrating ahead of PL was indicative of unresolved DG when this solvent system was employed (Fig. 5). By changing the system (Fig. 6) the contribution of unresolved glycerides to the previous profile was found. PL still exhibited a high level of incorporation. DG accounted for most of the radioactivity in the components that migrated ahead of PL in Fig. 5. By comparing data from Tables II and V, it appeared that the longer incubation V
Classes of Partially Purified Plasmodzkm lophurae and Debris from :Yormul 500 pCi X”-Acetate/SO ml Blood, Incubation Time 24 hr Plasmodium
lophurae
RBC debris
Specific act,ivit,y (dpm/mn)
yJ of “C
75 by weight
Specific act,ivity (dpm/w)
7; of W
56,2&i 470,333 -cl
32.8 3.8 -
253 207
BP 627,288 779,294 39,132 6353
0.3 21.8 37.4 3.8 0.3
71.3 1.0 .O 4.2 5.9 11.7 5 9.
21.4 13.4 5.1 10.1 27.8 16.0 6.2
395 482 2262 1302 461 __I_.
a Activity in lipid class not detected. b Below limits of quantitative analysis.
7,; by weight,
38.2 29.4 5.8 9.4 5 .5 5.5 6.1
METABOLISM
OF
14C-ACETATE
TABLE Distribution
Lipid
201
PLASMODIUM
VI
of 146 among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Znjected and Debris from Normal RBC. Conditions: 600 aCi 14C-acetate/50 ml Blood, Incubation Time 24 hr classes
Plasma from infected Specific activity @pm/md
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters a Activity
IN
ducks
Normal
RBC
plasma
% of 14C
% by weight
Specific activity (dpm/md
% of 1%
% by weight
2974 1186
1.6 0.3 -
30.7 14.4 -
866 11,088 -
10.2 10.0 -
64.1 4.9 -
2704 921 1,390,064 21,019 1881
0.6 0.4 94.8 2.1 0.2
13.4 24.8 3.9 5.7 7.1
5,555 4,555 84,267 1,675 1,105
5.0 2.9 68.4 1.5 2.0
4.8 7.1 4.4 5.0 9.8
-a
in class not det,ected.
period induced some changes in the distribution of 14C activity. The highest percent of 14C no longer resided in the PL fraction at 24 hr, although it still accounted for most of the extracted lipid by weight. The DG and FFA fra’ctions, which comprised a minor percentage of the total lipid by weight increased in percent of 14C incorporated and in specific activity. Shifts were found in the percent of l’C in the normal RBC debris after incubation for 24 hr; however, the specific activities were decreased in all lipid classes (Figs. 2 and 5). Pi’usmu. By zonal analysis, the highest activity was found in the unresolved PL fraction and in the FFA fraction of plasma from infected duscks (Fig. 7). By employing a more polar solvent system (Fig. 8) and resolving the polar fractions, the data indicated that the LA, ST, and DG fractions were significantly labeled. Quantitative analyses of lipid classes of plasma from blood of infected ducks when incubated for 24 hr with 500 &i/50 ml of blood, showed that an increase in the specific activities occurred in the FFA and TG fractions (Tables IV and VI). Samples of blood from infected ducks incubated for 24 hr exhibited some hemoly-
sis; however, normal blood did not appear to be affected. The increased osmotic fragility of infected RBC has been reported ( Fogel et al. 1966). DISCUSSION
It was the purpose of this study to define more clearly by quantitative means the distribution of radioactivity among the lipid classes of P. lophurae after incubation of blood from infected ducks with W-lacetate. Brundage et al. (1969) studied the uptake of this compound into the lipid classes of infected and normal blood cells and plasma and demonstrated that the lipids of parasitized blood cells possessed a greater level of activity of 14C than did components from normal blood. Only relative values were determined and no attempts were made to purify the parasites. The first important implication of the data presented in the present study was that the preparation of partially purified P. lophurae exhibited a specific pattern of incorporation of 14C different from that observed for the normal RBC debris. The debris from parasitized blood was similarly prepared as the partially purified parasite.
--.1.---
ki*nIJ
Y
--I
hl
.-
AL.
DG LA
FIG. 6. Thin-layer chromatography (TLC) of polar lipids from PIasmoclium Zophttrae by zonal analysis of “C activity. These lipids were the unresolved components from the TLC analysis in Fig. 5. Thin-layer chromatography on silica gel G in a solvent system of benzene :diethyl acid (50:40:2:0.2, ether: ethanol: glacial acetic v/v/v/v). Conditions: 500 pCi ‘%-acetate/50 ml blood, incubation time 24 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MC), sterols (ST), lipid alcohol (LA), diglycerides (DG).
Whether any parasite is able to bring about de novo fatty acid synthesis by a mechanism comparable to that in mammalian cells has not been established (von FFA
i I .E 24001 ~ 4c1600 i’ 1! 3200
FIG. 7. Zonal analysis of ‘“C activity of total lipid from plasma which served as suspending medium for Plasmodium lophurae-infected RBC. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether:formic acid 500 &i “C-ace(84: 15: 1, v/v/v). Conditions: tate/50 ml blood, incubation time 24 hr, 2 mm zones. Abbreviations: phospholipids (PL), sterols (ST), free fatty acids (FFA), triglycerides (TG).
Fm. 8. Thin-layer chromatography (TLC) of poIar lipids from plasma which served as suspending medium for Plasmodium Zophurae-infected RBC by zonal analysis of ‘“C activity. These lipids were the unresolved components from the TLC analysis in Fig. 7. Thin-layer chromatography on silica gel G in a solvent system of benzene:diethyl acid, (50:40:2:0.2, ether:ethanol: glacial acetic v/v/v/v). Conditions: 500 &i ‘C-acetate/50 ml blood, incubation time 24 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), lipid alcohol (LA), 1,2-diglycerides ( 1,2-DG), 1,3-diglycerides ( 1,3-DC).
Brand 1966). Indeed, little is known as to what extent the malarial parasite is dependent upon its host for lipids or lipid precursors. The introduction of I’C-acetate in vitro to RBC results in its incorporation into a variety of ,compounds other than lipids. In addition to fatty acid elongation, “Clabeled lipids of P. Zophurue could be derived from turnover and recycling of other metabolites during incubation. The quantity of acetate utilized by blood cells increased with the length of the incubation period, and the lipids exhibited concomitant increases in specific activity indicating that net lipid synthesis was occurring. It was not possible to discern whether the purified parasites under in vitro conditions of incubation in the RBC actively exchanged labeled compounds with plasma. Lipid inter,change between the RBC and plasma has been well established, specifically cholesterol between RBC and plasma
METABOLISM
OF
14C-ACETATE
(Hagerman and Gould 1951; London and Schwarz 1953). Mulder et al. (1963) and Mulder and van Deenen (1965) noted the dynamic state of PL in membranes of mature RBC, as evidenced by renewal and transfer of fatty acids via the plasma. The data presented in Tables III and IV indicate that PL accounted for the majority of plasma lipids by weight; however, they were not involved in incorporation of the label to any great extent at 5 hr of incubation. This observation was expected because at 5 hr the partially purified parasites did not show significant interchange of PL (Tables I, II, V). The percent by weight of PL of the parasites remained relatively constant throughout, suggesting that it serves in a structural role with relatively little turnover. The data in Table II on the lipids of the parasite indicate a large increase in the specific activities of all glycerides when the substrate concentration was increased lQfold as compared to the results given in Table I. This may suggest the formation of lipid storage products by the parasites. Rudzinska and Trager (1957) demonstrated the presence of large defined areas of lipids in avian malarial parasites by electron microscopy. The data in Tables II and V suggest that lipolytic activity from the parasite could account for the decrease in the specific activity in TG of samples incubated for 24 hr because of the concomitant increase in the specific activities of FFA and DG and the percentage of W. Cenedella et al. (1969a) reported that P. berghei infected rat erythrocytes release large amounts of FFA when incubated in vitro and that the free parasite possesses phospholipase A enzyme activity. von Brand (1966) discussed the possibility that P. lophurae used either lipid or protein for energy in the absence of an exogenous energy supply since the parasite stores few polysaccharides. After incubating infected blood for 24 hr, there was evidence of hemolysis as ob-
IN
PLASMODIUM
203
served in the plasma after centrifugation of the blood cells. Obvious hemolysis was not observed in normal blood similarly treated. The high percentage of 14C in the FFA fraction (Fig. 7) suggested their release into the plasma by parasites and other blood cells from infected ducks. This observation is significant because the parasite did not appear greatly affected in its ability to retain l%Xabeled compounds (Table V) when hemolysis was taking place. Of interest in this respect is the fact that parasitized blood cells have been shown to exhibit an increase in osmotic fragility ( Fogel et al. 1966). In RBC debris recovered from blood of normal ducks incubated for 24 hr, the specific activity in each class was significantly decreased. This suggests that at 21 hr of incubation normal cells became less active in their ability to incorporate precursors and synthesize lipid, whereas the parasite was equally as active as at 5 hr and especially active in the synthesis of DG, FFA and TG. The relative increase in incorporation as indicated by zonal analysis when the incubation time was extended from 5 to 21 hr was in agreement with the data reported by Brundage et al. ( 1969). It is recognized that incubation of normal whole blood does not duplicate the experimental conditions present in the blood from infected ducks. The hematologic differences present in the diseased and healthy states are due not only to the presence of the parasite in the RBC, but also to the many other effects that infection exerts on the host. In any event, in this study debris from normal RBC established an adequate level of background activity of 14C incorporation into the lipid classes. It should also be recognized that the levels of label found in the lipids of plasma from both infected and normal ducks represent the composite synthesizing activities of the entire cellular population, i.e., leukocytes, platelets, and reticulocytes
HARDY ET AL,
204
(Marks et al. 1960) and the mature avian RBC (Webb et aE. 1960). REFERENCES BRUNUAGE, W. G., HYLAND, SH. MARIE CHHISTOPHER, AND DIMOPOULLOS, G. T. 1969. In vitro biosynthesis of lipids in blood from ducks infected with Plusmodium lophurae. American Journal of Tropical Medicine and Hygiene 18, 657-661. CENEDELLA, R. J. 19G8. Lipid synthesis from glucose carbon by Plasmodium berghei, in vitro. American Journal of Tropical Medicine and Hygiene 17, 680-684. CENEDELLA, R. J., JAHHELL, J. J., AND SAXE, L. H. 1969. Lipid synthesis in vioo from l-“C-oleic acid and 6-“H-glucose by intraerythrocytic Plasmodium berghei. Military Medicine 134, ( Suppl), 1045-1055. CENEDELLA, R. J., JARRELL, J. J., AND SAXE, L. H. 1969a. Plasmodium berghei: Production in vitro of free fatty acids. Experimental Parasitology 24, 130-136. FOGEL, B. J., SHIELDS, C. E., AND VON DOENHOFF, A. E., JR. 1966. The osmotic fragility of erythrocytes in experimental malaria. American Journal Tropical Medicine and Hygiene 15,269-276. FOLCH, J., LEES, M., AND SLOANE-STANLEY, G. II. 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497509. FREEMAN, C. P., AND WEST, D. 1966. Complete separation of lipid classes on a single thinlayer plate. Journal of Lipid Research 7, 324327. GUTIERAEZ, J. 1966. Effect of the antimalarial chloroquine on the phospholipid metabolism of avian malaria and heart tissue. American Journal of Tropicul Medicine und Hygiene 15,818-822. HAGERMAN, J. A. S., AND GOULD, R. G. 1951. The in vitro interchange of cholesterol between plasma and red cells. Proceedings of the Biology and MediSociety for Experimental cine 78, 329-332. LAMBREMONT, E. N., AND GRAVES, J. B. 1969. Incorporation of acetate-l-l% into neutral lipids and phospholipids during late development
stages of He&o&is zea. Comparative Biochemistry and Physiology 30, 347-357. LONDON, J. M., AND SCHWARZ, H. 1953. Erythrocyte metabolism. The metabolic behavior of the cholesterol of human erythrocytes. Journal of Clinical Investigation 32, 124881252. MARKS, P. A,, GILHORN, A., AND KIDSON, C. 1960. Lipid synthesis in human leukocytes, platelets, and erythrocytes. Journal of Biological Chemistry 235, 2579-2583. MULDER, E., DE GIER, J., AND VAX DEENEN, L. L. M. 1963. Selective incorporation of fatty acids into phospholipids of mature red cells. Biochimica et Biophysics Acta 70, 94-96. MULDER, E., AND VAN DEENEN, L. L. M. 1965. Metabolism of red cell lipids. I. Incorporation in t&o of fatty acids into phospholipids from mature erythrocytes. Biochimica et Biophysica Acta 106, 106-117. RANDERATH, K. 1966. “Thin-Layer Chromatography,” Academic Press, New York. RUDZINSKA, M. A., AND TRACER, W. 1957. Intracellu!ar phagotrophy by malaria parasites: an electron microscope study of Plasmodium lophurae. Journal of Protozoology 4, 190-199. SHERMAN, J. W., AND HULL, R. W. 1960. The pigment (Hemozoin) and proteins of the avian malaria parasite, Ptasmodi,um lophurue. Journal of Protozoology 7, 409416. SNYDER, F. 1968. Thin-layer chromatography radioassay: A review. In (S. Rothchild, Ed.), “Advances in Tracer Methodology,” vol. 4, pp. 81-104, Academic Press, New York. SNYDER, F., AND KIMBLE, H. 1965. An automatic zonal scraper and sample collector for radioassay of thin-layer chromatograms. Anulytical Biochemistry 11, 510-518. VAN DEENEN, L. L. M., AND DE GIER, J. 1964. Chemical composition and metabolism of lipids in red cells of various animal species. In (C. Bishop and D. M. Surgenor, Eds.), “The Red Blood Cell,” pp. 243-307, Academic Press, New York. VON BRAND, T. 1966. “Biochemistry of Parasites,” pp. 191-230, Academic Press, New York. WANG, C. H., AND WILLIS, D. L. 1965. “Radiotracer Methodology in Biological Sciences,” Prentice-Hall, Englewood Cliffs, New Jersey. WEBB, J. P. W., ALLISON, A. C., AND JAAIES, A. T. 1960. fn vitro lipid synthesis in fowl blood. Biochimica et Biophysics Acta 43, 89-94.
PARASITOLOGY
37, 193-204
( 1975)
Plasmodium lophurae:
Quantitative in Vitro of 14C-l-Acetate into Lipids
Incorporation
CHEIWL L. SMITH HARDY,~ L. T. HART,~ G. T. DIMOPOULLOS,~ AND E. N. LAMBFIEMONT 4 Department Louisiana
of Veterinary
Science and Nuclear Science Center, State University, Baton Rouge, Louisiana 70803
(Submitted
for publication
November
13, 1973)
HARDY, C. L. S., HART, L. T., DIMOPOULLOS, G. T., AND LAMBREMONT, E. N. 1975. Plasmodium Zophurae: Quantitative in uitro incorporation of W-l-acetate into lipids. Experimental Parasitology 37, 193-204. Blood from ducks parasitized with Plasmodium Zoph,urae and normal duck blood were incubated with sodium Y-l-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and. plasma and lipid classes separated by thin-layer chromatography. S#pecific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated %-labeled lipid precursors. Experiments employing sodium W-l-acetate in two concentrations, 50 PCi “C in 0.91 rmole sodium acetate/50 ml blood and 500 pCi “C in 9.1 kmole sodium acetate/50 ml blood ( 1.82 X lo-’ M and 1.82 X lo-” M ), showed higher l’C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At 1.82 X lo-’ M W-l-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration ( 1.82 X lo-” M ), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. Zophurae. Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols. At 24 hr of incubation, the lipids of partially purified P. Zophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides. In plasma from parasitized blood incubated 24 hr with W-l-acetate, the highest specific activity and greatest percent of “C incorporation was found in free fatty acids. Plasmodium Zophurae; Lipids; “C-acetate incorporation; INDEX DESCRIPTORS: Metabolism; Malaria; Chromatography, thin-layer; Ducks; Scintillation, liquid.
1 Present address: Clinical Laboratories, University of Mississippi Medical Center, Jackson, Mississippi 39216. 2 Department of Veterinary Science, Louisiana State University, Baton Rouge, Louisiana 70803. Author to whom reprint requests should be addressed. 3 Nuclear Science Center, Louisiana State University, Baton Rouge, Louisiana 70803.
The
of
integrity
of
(van
Deenen
and
malarial been shown
parasite well that
lipids
the
struc-
(RBC) de Gier 1964) and the erythrocyte
(von
Brand
documented. the
in
the
erythrocytic
It
1966)
has stages
also
has been
of some
Plusmodium species can take up fatty acids in vitro and incorporate them into cellular 193
Copyright c 1975 by Academic Press, Inc. All rights o4 reproduction in any form reserved.
importance
tural
194
HARDY
lipid (Gutierrez 1966; Cenedella et al. 1969). It is conceivable that the parasites may depend upon the RBC and plasma of the host for fatty acids instead of synthesizing their own (Cenedella 1968). Studies on the incorporation of ‘C-lacetate into the blood lipids of normal du,cks and ducks infected with P. Eophume were reported by Brundage et aZ. ( 1969). Although quantitative analyses were not made, it was demonstrated that a greater incorporation of 14C-l-acetate occurred in the lipids of the parasitized RBC. No attempt was made to separate the parasites from the RBC in their studies; therefore, the level of incorporation into the cellular components of the parasite could not be determined. In the present study, our objective was to measure quantitatively the radioactivity incorporated into the lipids of the parasite when whole blood from infected ducks was incubated with 14C-l-acetate. Supplementary data concerning the radioactivity found in plasma from infeacted ducks and data on normal blood were also collected. MATERIALS
AKD METHODS
Blood Samples The erythrocytic stages of P. lophurae were maintained in 4-6-week-old Pekin ducklings by intravenous passage of blood from infected ducks having greater than 80% of their RBC parasitized. When the desired degree of parasitemia was attained in the host (above 90% ), blood was collected by cardiac puncture in heparin so.dium (25-30 U.S.P. units/ml of blood). Uninfected ducks provided normal blood samples. Incubation
with l”C-l-Acetate
Normal blood and blood samples from infected ducks were dispensed into sterile 250-ml flasks in 50-ml portions. Normal blood was inscluded to establish the level of background activity of 14C incorporation
_,a. AlA. *-
I51
into the lipid classes that could be attributed to host RBC debris in the parasite preparations. An incubation mixture of penicillin (1000 I.U./ml blood), streptomycin (1290 I.U./ml blood) and sodium l”C-l-acetate (50 &i l”C in 0.91 pmole sodium acetate/50 ml blood, 1.82 x 10 5 hl; or 500 &i 14C in 9.1 pmole sodium acetate/50 ml blood, 1.82 x 10m4M) was added, and the flasks kept at 39 C for either 5 or 24 hr in an oscillating water bath. Recovery of Parasites After incubation, the blood samples were centrifuged and the plasma recovered for further study. The RBC were washed with buffer (Sherman and Hull 1960) and the buffy coat discarded at ea’ch washing. P. Zophurae cells were freed from RBC and purified according to the method described by Sherman and Hull (1960). The RBC were lysed with saponin and after centrifugal washing the debris was treated with DNAase. The resulting product was washed by centrifugation and the sediment recovered for lipid analysis. Yields of 8-10 ml of packed partially purified parasites per 50 ml of blood from infected ducks were recovered as compared to about 0.1 ml of packed erythrocytic debris per 50 ml of normal blood. Lipid Extraction The lipid extraction and recovery procedure described by Folch et al. (1957) was adopted for the extraction of normal plasma, plasma from infected ducks, partially purified parasites, and normal erythrocytic debris. Samples of plasma were added directly to 20 vol of chloroform; methanol (2:1, v/v) and mixed for 16-20 hr at 21 C on a magnetic stirrer. Erythrocytic debris and parasite preparations were first added directly to methanol and mixed for about 5 min. This step was found necessary to minimize clumping of
METABOLISM
OF
14C-ACETATE
the cellular material. Twice the volume of chloroform was then added to give a final extraction mixture of chloroform : methanol (2: 1, v/v) in a 20-fold excess of the volume of the preparation being extracted. The total lipid extracts were washed three times with distilled water to remove nonlipid material. The extraction proceeded as indicated for the plasma samples. After filtration and concentration, the total lipid solution was dried over anhydrous Na2S04 in a known volume of chloroform for the subsequent analyses and lipid class separations. Preparatizje Thin-Layer WC)
Chromatography
The techniques employed here for TLC have been described by Randerath (1966). Dupli’cate silica gel G plates were prepared in thicknesses of 250 pm and activated at 110 C for 30 min. One plate was used to assay the radioactivity in each lipid class and the other for the gravimetric analyses of lipids. A known weight of total lipid extract, not exceeding 15 mg, was applied to each plate. Lipid class standard, Hormel Institute II-A, containing a mixture of hydrogenated lecithin, oleic acid, triolein, cholesterol oleate and cholesterol was also spotted on each plate to aid in the identification of the separated fractions. After development of the TLC plates in a solvent system of petroleum ether, diethyl ether and glacial acetic acid (84: 15: 1, v/v/v), lipids for radioassay were made visible by exposing the plate to iodine vapor. Areas of the plate corresponding to separated standards [(free fatty acids (FFA), triglycerides (TG), sterol esters ( SE)] were removed and transferred to liquid scintillation vials. All of the remaining adsorbent on the plate which contained the more polar lipids was recovered and transferred quantitatively to a glass column plugged with defatted cotton. Lipid contained in this fraction was then eluted
IN
PLASMODIUM
19.5
with methanol: chloroform (2: 1, v/v). The eluate was con,centrated under a stream of nitrogen and quantitatively applied to a second silica gel G plate prepared as described above. Standards of monopalmitin and dipalmitin and as before, lipid class standard, Hormel Institute II-A, were spotted on separated lanes. The plates were developed in a solvent system of benzene, diethyl ether, absolute ethanol and glacial acetic acid (50:40:2:0.2, v/v/v/v) to resolve phospholipids ( PL), sterols ( ST), monoglycerides ( MG ), and diglycerides (DG) (Freeman and West 1966). The lipids were detected, identified and subsequently transferred to scintillation vials as previously described. The duplicate TLC plate employed for the gravimetric analyses of lipid classes was identically prepared and developed as des’cribed for the plates used for radioassays. The lanes containing only the H-A standard were sprayed with a 0.2% ethanolic solution of 2, 7’-dichlorofluorescein and the plates observed under ultraviolet light. Each lipid corresponding to a fraction assayed for radioactivity was eluted from the unsprayed lanes of adsorbent. Polar lipids were eluted with methanol: chloroform (2: 1, v/v) and neutral lipids with petroleum ether. After recovery and removal of solvent in uacuo, each lipid class was stored in a known volume of chloroform until the gravimetric analyses were made. Zonul Analysis of Lipids Total lipids from plasma of infected ducks and partially purified parasites were chromatographed on silica gel G TLC plates (5 x 20 cm). After visualization of the separated fractions, zones 2 mm in width were scraped directly into scintillation vials with the aid of a TLC zonal scraper (Snyder and Kimble 1965). The polar lipid fraction from several initial TLC separations was pooled and developed on a second plate as described earlier. Polar
196
HARDY PL.MG
-24 FIG. 1. Zonal analysis of 14C activity of total lipid from partially purified Plasmodium Zophurae. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84:15:1, v/v/v). Conditions: 500 &i %-acetate/50 ml of blood, incubation time 5 hr, 2 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), diglycerides (DG), free fatty acids (FFA), triglycerides ( TG).
lipids were pooled in order to provide sufficient sample for qualitative clarification of lipid classes present. Zonal profile analyses were made on these plates by scraping zones, 5 mm in width, manually with a razor blade. Assay of ‘“C
Aliquots (10 ~1) of the samples were dispensed into the cups and the solvent evaporated in a vacuum oven at 40 C for 2 hr. After reaching ambient temperature in a desiccator, the cups were reweighed and placed in liquid scintillation vials for radioassay ( Lambremont and Graves 1969). All samples were analyzed in triplicate. The maximum amount of lipid weighed was 1.5 mg with a weighing accuracy of ~40 pg. Triplicates varying more than a40 yg were discarded and new weighings were made. RESULTS
Fiue-hour Incubations Parasites. A zonal profile analysis of total lipids from partially purified P. Zophurne is presented in Fig. 1. The results of a zonal analysis of the polar lipids from this initial TLC plate which were subsequently separated and developed in another solvent system are given in Fig. 2. The fraction exhibiting migration as PL possessed the highest level of radioactivity *.,0001
DL
,.375-
,500-
Analysis of 14C activity was conducted using a Beckman Liquid Scintillation Spectrometer, Model No. LS-250. The s’cintillation solution was prepared as described by Snyder (1968) for direct counting of silica gel G scrapings without elution. All sampIes were appropriateIy corrected for background radioactivity. Quench correction was calculated by the external standard method (Wang and Willis 1965). Gravimetric
ET AL.
Analysis of Lipids
Samples of total lipid and those fractionated as classes by TLC were dissolved in a known volume of chloroform and analyzed gravimetri8cally. Aluminum foil weighing cups for the Cahn Gram Electrobalance were tared.
5 ,200. F L" G 900.
300 75 7 600 i
FIG. 2. Thin-layer chromatography (TLC) of polar lipids from partially purified Plasmodium Zophurae from Fig. 1 by zonal analysis of ‘“C activity. Thin-layer chromatography on silica gel G in a solvent system of benzene:diethyI ether: ethanol:glacial acetic acid (50:40:2:0.2, v/v/v/v). Conditions: 500 &i %-acetate/50 ml blood, incubation time 5 hr, 5 mm zones. Abbreviations: phospholipids (PL), monogIycerides (MC), sterols (ST), lipid alcohol (LA), 1,2-diglycerides ( 1,2-DG), 1,3-diglycerides (1,3-DC).
METABOLISM
OF
14C-ACETATE
TABLE Distribution
Lipid
IN
I
of W among the Lipid Classes of Partially PuriJied Plasmodium lophurae and Debris from Normal RBC, Conditions: 60 pCi V-Acetate/SO ml Blood, Incubation !l’ime 5 hr Plasmodium
classes
Specific
RBC debris
bphurae
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters
%by
% by weight
10,525 25,266 -”
66.1 5.8
78.4 2.9 -
32 106
17.1 10.4 -
67.4 2.1 -
l.i6,412 20,923 1,623 10,788 738
18.1 6.6 0.5 23 0.4
1.4 3.9 3.9 2.9 6.5
560 198 760 525 63
9.2 6.5 12.4 37.8 6.6
2.0 4.1 2.1 9.1 13.2
(dpm/w)
Specific activity (dpm/w)
5% of
7;) of “C
activity
a Activity
197
PLASMODIUM
“C
weight
in lipid class not detected.
(Fig. 1); however, when the two most polar lipid fractions were rechromatographed MG was resolved from PL and it contributed significantly to the activity as shown in Fig. 2. In addition, DG was resolved from ST by this solvent system. The radioactivity previously observed in the fraction migrating as ST, DG in the initial TLC separation (Fig. 1) was due primarily to DG which chromatographitally overlapped ST (Fig. 2). Furthermore, considerable activity was present in the fractions identified as 1,2-diglyceride (1,2-DG) and 1,3-diglpceride (1,3-DG) (Fig. 2). In order to measure the 14C activity in each lipid class, gravimetric determinations were combined with radiochemical assay. The uptake of 50 &i 14C in 0.91 pmole sodium acetate/50 ml blood was investigated initially. The data given in Table I show that after accounting for the possible contribution of residual radioactivity by debris prepared from normal RBC, the lipid alcohol (LA) fraction of P. Zophurue was found to possess the highest specific activity. The greatest percent of label was found in PL, the largest frection by weight. No activity was detected in the area of the TLC plate corresponding to ST and 1,2-DG.
Five-hour incubations were also conducted with the amount of 14C acetate increased lo-fold (Table II). The percent incorporation of 14C into lipid was similar at both substrate concentrations, being 0.21% and 1.59% for normal blood and blood from infected ducks, respectively with 50 &i/50 ml blood, and 0.10 and 1.14%, respectively, at 500 &i/50 ml blood. As before, PL showed the highest percent in‘corporation of l”C whereas MG, 1,3-DG, and TG individually exceeded the specific activity found associated with PL. Plasma. Zonal analyses of total lipid extracted from the plasma of infected ducks are shown in Figs. 3 and 4. Essentially all of the radioactivity was observed in the FFA fraction and a small amount of activity was found in the most polar fraction (Fig. 3). From the profile formed as a result of the resolution of the most polar fractions (Fig. 4), all of the radioactivity from this area on the TLC plate as shown on Fig. 3 was found to remain there. The levels of activity in MG, 1,2-DG, 1,3-DG and the possible LA were negligible; however, the ST fraction displayed a great amount of activity ( Fig. 4). After incubation with the lower substrate concentration, the greatest specific
HARDY
I10
130
-Go
3. Zonal analysis of “C activity of total lipid from plasma which served as suspending medium for Plasmodium lo~jhurae-infected RHC. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84:15: 1, v/v/v). Conditions: 500 &i Y-acetate/50 ml blood, incubation time 5 hr, 2 mm zones. Abbreviations: phospholipids (PI,), sterols (ST), free fatty acids (FFA). FIG.
activity was found in the FFA fraction of normal plasma and plasma from infcctcd ducks, with the latter exhibiting 2.5 times greater specific activity than normal (Table III). The FFA contained greater than 80% of the total 14C and small percent of the total weight. In plasma from infected ducks, the ST fraction possessed a high specific activity but no comparable class
ET AL.
mm FK. 4. Thin-layer chromatography (TLC) of polar lipids from plasma which served as suspending medium for Pla.smodium lophurue-infected RBC by zonal analysis of “C activity. These lipids were the unresolved components from the TLC analysis in Fig. 3. TLC on silica gel G in a solvent system of benzene: cliethyl ether: ethanol: glacial acetic acid (50:40:2:0.2, v/v/v/v). Conditions: 500 pCi W-acetate/50 ml blood, incubation time 5 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), lipid alcohol (LA), l$diglycerides ( 1,2-DC), 1,3-diglycerides ( 1,3-DC ).
was observed in the plasma from normal ducks (Table IV). This component was also observed in P. lqwhurae lipid preparations (Table II). Specific activities of other classes did not appear to be significantly greater than those values for the lipid classes of normal plasma.
TABTX Distribution
Lipid
classes
Phospholipids Monoglycerides Sterols Lipid alcohol Diglycerides Free fatty acids Triglycerides Sterol esters a Activity
II
of ‘“C’ among the Lipid Classrs of Patfiull~/ P~cr(/ietl Plasmotliwa lophurae untl Debris SW/IL lVorma1 KBC. Conrlitions: 500 pCi W-Acetale,/ ml Blood, Inocbalion lime 5 hr F’lasmotliron
lophlrrcle
1tBC debris
Specific ac?ivil.,v (4m/md
“; of ‘T
47,410 1,270,700 4G,OOO
72.4 4.0 0.3
74.5 0.2 0.::
81,386 440,830
1.8 5.;i
1.1 0.6
32,468 69,203
9630
in lipid class not detected.
12.1 3. 4 O..i
Specific activity (dpm/mg)
18.2 2.4 2.7
4m fiOS -,‘
‘,; of 1%
7; kY weight,
2::.2 2.2
.wsi 3.8 -
31 3
12.3
1,481
5.8
4,048 4,458 492 _____~
13.2 18.2 6.0
4.0 3.4 4.2 12.7
2,642
~.
METABOLISM
OF
%-ACETATE
TABLE Distribution
Lipid
IN
199
PLASMODIUM
III
of l4C among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Infected RBC and Debris from h’ormal RBC. Conditions 50 &‘i W-Acetate/50 ml Blood, Incubation Time 5 hr classes
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free Fatty acids Triglycerides Sterol esters
Plasma from infected
ducks
Specific activity (dpm/w)
% of 1%
% by weight
303 4537 7242 2790 BL* 63,422 577 283
5.4 2.4 3.2 2.1
69.1 2.0 1.8 2.9 3.8 5.2 7.6 7.6
85.2 1.1 0.6
Normal
plasma
Yoof
Specific activity (dpm/md
1%
% by weight
114 527:5 -a
3.8 1.4 -
51.2 0.4 -
1239 3750 24,800 216 32
4.6 1.0 88.0 0.5 0.7
5.6 0.4 5.4 3.3 33.7
a Activity in lipid class not detected. b Below limits of quantitative analysis
The specific activities in some of the lipid classes were higher in both normal plasma and plasma from infected ducks when the amount of labeled substrate was increased lo-fold (Table IV). The FFA fraction showed the highest specific activity, i.e., at least four-fold greater in infected ducks and lo-fold greater in normal plasma than that of any other single lipid fraction. Furthermore, the FFA fraction possessed the highest percent of 14C and TABLE Distribution
Lipid
IV
of W among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Infected and Debris from NoTma RBC. Conditions: 600 &i 14CAcetate/50 ml Blood, Incubation Time 5 hr classes
Plasma from infected Specific activity (dpmhd
Phospholipids Monoglycerides Sterols Lipid Alcohols Diglycerides Free fatty acids Triglycerides Sterol esters 0 Act,ivity
a low percent of total lipid by weight in both normal plasma and plasma from infected ducks. The SE fraction accounted for a significant portion of the total lipid by weight in normal plasma, whereas PL accounted for the largest portion by weight in normal plasma and plasma from infected ducks. As was observed in plasma from infected ducks, when whole blood was incubated with the lower con’centrations of substrate,
584 19,281 89,283 44,425 850 432,704 3925 1919
in lipid class not detected.
ducks
y. of 14C
% by weight
1.5 1.3 2.2 0.7 0.1 92.9 0.6 0.6
74.0 1.8 0.7 0.5 3.9 6.0 4.6 8.3
Normal Specific activity @pdmd 489 10,814 -0 8303 9021 111,190 1835 360
RBC
plasma % of W
% by weight
3.8 2.8 5.8 3.9 81.0 2.0 0.7
58.5 1.9 5.2 6.7 5.4 8.1 14.1
200
HARDY
ET AL.
of 14C (Table III) the SI fraction was not detected in normal plasma. A component designated at 1,2-DG which was not observed in the incubations with the lower substrate concentrations was found only in the lipids of normal plasma of blood incubated with the higher concentrations of 14C (Table IV). This also occurred with the normal blood samples incubated for 24 hr. Tu/>enty-Four-hour FIG. 5. Zonal analysis of 14C activity of total lipid from partially purified Plasmodiwm lophurae. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether: formic acid (84: 15:1, v/v/v). Conditions: 500 pCi %-acetate/50 ml blood, incubation time 24 hr, 2 mm zones. Abbreviations: phospholipids (PL), sterols (ST), diglycerides (DG), free fatty acids (FFA), triglycerides (TG),
ST contained significant radioactivity. The LA and MG fractions had significantly higher specific activity than the remaining lipid classes of plasma from infected ducks with the exception of FFA ( Table III ) . In comparing the above data with results obtained on the lipid classes of blood inmcubated with the lesser concentrations TABLE Distribution
of Y.7 among the Lipid
RBC. Conditions: Lipid
classes
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters
Incubations
Parasites. Data presented in Figs. 5 and 6 were obtained by zonal analyses of total lipid extracted from partially-purified parasites, The activity in PL greatly exceed that found in the other fractions. Appearance of two peaks migrating ahead of PL was indicative of unresolved DG when this solvent system was employed (Fig. 5). By changing the system (Fig. 6) the contribution of unresolved glycerides to the previous profile was found. PL still exhibited a high level of incorporation. DG accounted for most of the radioactivity in the components that migrated ahead of PL in Fig. 5. By comparing data from Tables II and V, it appeared that the longer incubation V
Classes of Partially Purified Plasmodzkm lophurae and Debris from :Yormul 500 pCi X”-Acetate/SO ml Blood, Incubation Time 24 hr Plasmodium
lophurae
RBC debris
Specific act,ivit,y (dpm/mn)
yJ of “C
75 by weight
Specific act,ivity (dpm/w)
7; of W
56,2&i 470,333 -cl
32.8 3.8 -
253 207
BP 627,288 779,294 39,132 6353
0.3 21.8 37.4 3.8 0.3
71.3 1.0 .O 4.2 5.9 11.7 5 9.
21.4 13.4 5.1 10.1 27.8 16.0 6.2
395 482 2262 1302 461 __I_.
a Activity in lipid class not detected. b Below limits of quantitative analysis.
7,; by weight,
38.2 29.4 5.8 9.4 5 .5 5.5 6.1
METABOLISM
OF
14C-ACETATE
TABLE Distribution
Lipid
201
PLASMODIUM
VI
of 146 among the Lipid Classes of the Suspending Medium of Plasmodium lophurae-Znjected and Debris from Normal RBC. Conditions: 600 aCi 14C-acetate/50 ml Blood, Incubation Time 24 hr classes
Plasma from infected Specific activity @pm/md
Phospholipids Monoglycerides Sterols Lipid alcohols Diglycerides Free fatty acids Triglycerides Sterol esters a Activity
IN
ducks
Normal
RBC
plasma
% of 14C
% by weight
Specific activity (dpm/md
% of 1%
% by weight
2974 1186
1.6 0.3 -
30.7 14.4 -
866 11,088 -
10.2 10.0 -
64.1 4.9 -
2704 921 1,390,064 21,019 1881
0.6 0.4 94.8 2.1 0.2
13.4 24.8 3.9 5.7 7.1
5,555 4,555 84,267 1,675 1,105
5.0 2.9 68.4 1.5 2.0
4.8 7.1 4.4 5.0 9.8
-a
in class not det,ected.
period induced some changes in the distribution of 14C activity. The highest percent of 14C no longer resided in the PL fraction at 24 hr, although it still accounted for most of the extracted lipid by weight. The DG and FFA fra’ctions, which comprised a minor percentage of the total lipid by weight increased in percent of 14C incorporated and in specific activity. Shifts were found in the percent of l’C in the normal RBC debris after incubation for 24 hr; however, the specific activities were decreased in all lipid classes (Figs. 2 and 5). Pi’usmu. By zonal analysis, the highest activity was found in the unresolved PL fraction and in the FFA fraction of plasma from infected duscks (Fig. 7). By employing a more polar solvent system (Fig. 8) and resolving the polar fractions, the data indicated that the LA, ST, and DG fractions were significantly labeled. Quantitative analyses of lipid classes of plasma from blood of infected ducks when incubated for 24 hr with 500 &i/50 ml of blood, showed that an increase in the specific activities occurred in the FFA and TG fractions (Tables IV and VI). Samples of blood from infected ducks incubated for 24 hr exhibited some hemoly-
sis; however, normal blood did not appear to be affected. The increased osmotic fragility of infected RBC has been reported ( Fogel et al. 1966). DISCUSSION
It was the purpose of this study to define more clearly by quantitative means the distribution of radioactivity among the lipid classes of P. lophurae after incubation of blood from infected ducks with W-lacetate. Brundage et al. (1969) studied the uptake of this compound into the lipid classes of infected and normal blood cells and plasma and demonstrated that the lipids of parasitized blood cells possessed a greater level of activity of 14C than did components from normal blood. Only relative values were determined and no attempts were made to purify the parasites. The first important implication of the data presented in the present study was that the preparation of partially purified P. lophurae exhibited a specific pattern of incorporation of 14C different from that observed for the normal RBC debris. The debris from parasitized blood was similarly prepared as the partially purified parasite.
--.1.---
ki*nIJ
Y
--I
hl
.-
AL.
DG LA
FIG. 6. Thin-layer chromatography (TLC) of polar lipids from PIasmoclium Zophttrae by zonal analysis of “C activity. These lipids were the unresolved components from the TLC analysis in Fig. 5. Thin-layer chromatography on silica gel G in a solvent system of benzene :diethyl acid (50:40:2:0.2, ether: ethanol: glacial acetic v/v/v/v). Conditions: 500 pCi ‘%-acetate/50 ml blood, incubation time 24 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MC), sterols (ST), lipid alcohol (LA), diglycerides (DG).
Whether any parasite is able to bring about de novo fatty acid synthesis by a mechanism comparable to that in mammalian cells has not been established (von FFA
i I .E 24001 ~ 4c1600 i’ 1! 3200
FIG. 7. Zonal analysis of ‘“C activity of total lipid from plasma which served as suspending medium for Plasmodium lophurae-infected RBC. Thin-layer chromatography on silica gel G in a solvent system of petroleum ether:diethyl ether:formic acid 500 &i “C-ace(84: 15: 1, v/v/v). Conditions: tate/50 ml blood, incubation time 24 hr, 2 mm zones. Abbreviations: phospholipids (PL), sterols (ST), free fatty acids (FFA), triglycerides (TG).
Fm. 8. Thin-layer chromatography (TLC) of poIar lipids from plasma which served as suspending medium for Plasmodium Zophurae-infected RBC by zonal analysis of ‘“C activity. These lipids were the unresolved components from the TLC analysis in Fig. 7. Thin-layer chromatography on silica gel G in a solvent system of benzene:diethyl acid, (50:40:2:0.2, ether:ethanol: glacial acetic v/v/v/v). Conditions: 500 &i ‘C-acetate/50 ml blood, incubation time 24 hr, 5 mm zones. Abbreviations: phospholipids (PL), monoglycerides (MG), sterols (ST), lipid alcohol (LA), 1,2-diglycerides ( 1,2-DG), 1,3-diglycerides ( 1,3-DC).
Brand 1966). Indeed, little is known as to what extent the malarial parasite is dependent upon its host for lipids or lipid precursors. The introduction of I’C-acetate in vitro to RBC results in its incorporation into a variety of ,compounds other than lipids. In addition to fatty acid elongation, “Clabeled lipids of P. Zophurue could be derived from turnover and recycling of other metabolites during incubation. The quantity of acetate utilized by blood cells increased with the length of the incubation period, and the lipids exhibited concomitant increases in specific activity indicating that net lipid synthesis was occurring. It was not possible to discern whether the purified parasites under in vitro conditions of incubation in the RBC actively exchanged labeled compounds with plasma. Lipid inter,change between the RBC and plasma has been well established, specifically cholesterol between RBC and plasma
METABOLISM
OF
14C-ACETATE
(Hagerman and Gould 1951; London and Schwarz 1953). Mulder et al. (1963) and Mulder and van Deenen (1965) noted the dynamic state of PL in membranes of mature RBC, as evidenced by renewal and transfer of fatty acids via the plasma. The data presented in Tables III and IV indicate that PL accounted for the majority of plasma lipids by weight; however, they were not involved in incorporation of the label to any great extent at 5 hr of incubation. This observation was expected because at 5 hr the partially purified parasites did not show significant interchange of PL (Tables I, II, V). The percent by weight of PL of the parasites remained relatively constant throughout, suggesting that it serves in a structural role with relatively little turnover. The data in Table II on the lipids of the parasite indicate a large increase in the specific activities of all glycerides when the substrate concentration was increased lQfold as compared to the results given in Table I. This may suggest the formation of lipid storage products by the parasites. Rudzinska and Trager (1957) demonstrated the presence of large defined areas of lipids in avian malarial parasites by electron microscopy. The data in Tables II and V suggest that lipolytic activity from the parasite could account for the decrease in the specific activity in TG of samples incubated for 24 hr because of the concomitant increase in the specific activities of FFA and DG and the percentage of W. Cenedella et al. (1969a) reported that P. berghei infected rat erythrocytes release large amounts of FFA when incubated in vitro and that the free parasite possesses phospholipase A enzyme activity. von Brand (1966) discussed the possibility that P. lophurae used either lipid or protein for energy in the absence of an exogenous energy supply since the parasite stores few polysaccharides. After incubating infected blood for 24 hr, there was evidence of hemolysis as ob-
IN
PLASMODIUM
203
served in the plasma after centrifugation of the blood cells. Obvious hemolysis was not observed in normal blood similarly treated. The high percentage of 14C in the FFA fraction (Fig. 7) suggested their release into the plasma by parasites and other blood cells from infected ducks. This observation is significant because the parasite did not appear greatly affected in its ability to retain l%Xabeled compounds (Table V) when hemolysis was taking place. Of interest in this respect is the fact that parasitized blood cells have been shown to exhibit an increase in osmotic fragility ( Fogel et al. 1966). In RBC debris recovered from blood of normal ducks incubated for 24 hr, the specific activity in each class was significantly decreased. This suggests that at 21 hr of incubation normal cells became less active in their ability to incorporate precursors and synthesize lipid, whereas the parasite was equally as active as at 5 hr and especially active in the synthesis of DG, FFA and TG. The relative increase in incorporation as indicated by zonal analysis when the incubation time was extended from 5 to 21 hr was in agreement with the data reported by Brundage et al. ( 1969). It is recognized that incubation of normal whole blood does not duplicate the experimental conditions present in the blood from infected ducks. The hematologic differences present in the diseased and healthy states are due not only to the presence of the parasite in the RBC, but also to the many other effects that infection exerts on the host. In any event, in this study debris from normal RBC established an adequate level of background activity of 14C incorporation into the lipid classes. It should also be recognized that the levels of label found in the lipids of plasma from both infected and normal ducks represent the composite synthesizing activities of the entire cellular population, i.e., leukocytes, platelets, and reticulocytes
HARDY ET AL,
204
(Marks et al. 1960) and the mature avian RBC (Webb et aE. 1960). REFERENCES BRUNUAGE, W. G., HYLAND, SH. MARIE CHHISTOPHER, AND DIMOPOULLOS, G. T. 1969. In vitro biosynthesis of lipids in blood from ducks infected with Plusmodium lophurae. American Journal of Tropical Medicine and Hygiene 18, 657-661. CENEDELLA, R. J. 19G8. Lipid synthesis from glucose carbon by Plasmodium berghei, in vitro. American Journal of Tropical Medicine and Hygiene 17, 680-684. CENEDELLA, R. J., JAHHELL, J. J., AND SAXE, L. H. 1969. Lipid synthesis in vioo from l-“C-oleic acid and 6-“H-glucose by intraerythrocytic Plasmodium berghei. Military Medicine 134, ( Suppl), 1045-1055. CENEDELLA, R. J., JARRELL, J. J., AND SAXE, L. H. 1969a. Plasmodium berghei: Production in vitro of free fatty acids. Experimental Parasitology 24, 130-136. FOGEL, B. J., SHIELDS, C. E., AND VON DOENHOFF, A. E., JR. 1966. The osmotic fragility of erythrocytes in experimental malaria. American Journal Tropical Medicine and Hygiene 15,269-276. FOLCH, J., LEES, M., AND SLOANE-STANLEY, G. II. 1957. A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497509. FREEMAN, C. P., AND WEST, D. 1966. Complete separation of lipid classes on a single thinlayer plate. Journal of Lipid Research 7, 324327. GUTIERAEZ, J. 1966. Effect of the antimalarial chloroquine on the phospholipid metabolism of avian malaria and heart tissue. American Journal of Tropicul Medicine und Hygiene 15,818-822. HAGERMAN, J. A. S., AND GOULD, R. G. 1951. The in vitro interchange of cholesterol between plasma and red cells. Proceedings of the Biology and MediSociety for Experimental cine 78, 329-332. LAMBREMONT, E. N., AND GRAVES, J. B. 1969. Incorporation of acetate-l-l% into neutral lipids and phospholipids during late development
stages of He&o&is zea. Comparative Biochemistry and Physiology 30, 347-357. LONDON, J. M., AND SCHWARZ, H. 1953. Erythrocyte metabolism. The metabolic behavior of the cholesterol of human erythrocytes. Journal of Clinical Investigation 32, 124881252. MARKS, P. A,, GILHORN, A., AND KIDSON, C. 1960. Lipid synthesis in human leukocytes, platelets, and erythrocytes. Journal of Biological Chemistry 235, 2579-2583. MULDER, E., DE GIER, J., AND VAX DEENEN, L. L. M. 1963. Selective incorporation of fatty acids into phospholipids of mature red cells. Biochimica et Biophysics Acta 70, 94-96. MULDER, E., AND VAN DEENEN, L. L. M. 1965. Metabolism of red cell lipids. I. Incorporation in t&o of fatty acids into phospholipids from mature erythrocytes. Biochimica et Biophysica Acta 106, 106-117. RANDERATH, K. 1966. “Thin-Layer Chromatography,” Academic Press, New York. RUDZINSKA, M. A., AND TRACER, W. 1957. Intracellu!ar phagotrophy by malaria parasites: an electron microscope study of Plasmodium lophurae. Journal of Protozoology 4, 190-199. SHERMAN, J. W., AND HULL, R. W. 1960. The pigment (Hemozoin) and proteins of the avian malaria parasite, Ptasmodi,um lophurue. Journal of Protozoology 7, 409416. SNYDER, F. 1968. Thin-layer chromatography radioassay: A review. In (S. Rothchild, Ed.), “Advances in Tracer Methodology,” vol. 4, pp. 81-104, Academic Press, New York. SNYDER, F., AND KIMBLE, H. 1965. An automatic zonal scraper and sample collector for radioassay of thin-layer chromatograms. Anulytical Biochemistry 11, 510-518. VAN DEENEN, L. L. M., AND DE GIER, J. 1964. Chemical composition and metabolism of lipids in red cells of various animal species. In (C. Bishop and D. M. Surgenor, Eds.), “The Red Blood Cell,” pp. 243-307, Academic Press, New York. VON BRAND, T. 1966. “Biochemistry of Parasites,” pp. 191-230, Academic Press, New York. WANG, C. H., AND WILLIS, D. L. 1965. “Radiotracer Methodology in Biological Sciences,” Prentice-Hall, Englewood Cliffs, New Jersey. WEBB, J. P. W., ALLISON, A. C., AND JAAIES, A. T. 1960. fn vitro lipid synthesis in fowl blood. Biochimica et Biophysics Acta 43, 89-94.