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Incorporation of labeled precursors into lipids of tumors induced by rous sarcoma virus
428
LIPIDS INDUCED
PAUL
H.
De$artment
FIGARD
AND
BY
ROUS SARCOMA VIRUS
ALVIN
of Micwbiology,
Indiana
(Received March Iqth, 1966) (Revised manuscript received
OF
June
S. LEVINE
University myth,
School of Medicine,
Indialzapolis,
Ind. (U.S.A.)
1966)
SUMMARY I. A comparison was made of the lipids in the chorioallantoic membranes of chicken eggs and in tumors induced in the membranes by Rous sarcoma virus. 2. Thin-layer chromatography of the lipids from the two tissues revealed only minor differences in their compositions. 3. Measurements were made of the incorporation of several labeled precursors into the lipids of the two tissues. From a buffered salt medium containing glucose, the greatest incorporation of radioactivity was found in the triglycerides. This was true for both tissues and for either glucose or acetate as the radioactive precursor. The tumor slices incorporated about six times as much radioactivity as did the normal membranes. Much of the increased incorporation took place in the triglycerides, but there appeared to be an increased incorporation by the tumors in almost all of the lipid fractions tested. There was considerable incorporation into the glycerol portion of the triglycerides from glucose and even from pyruvate. 4. The difference in incorporation by the two tissues appeared to be largely a matter of amount rather than of distribution.
INTRODUCTION
Tumors induced by Rous sarcoma virus utilized more glucose than the normal chorioallantoic membrane from which they were produced’. In addition, it was shown that the Rous tumors incorporated more amino acids into proteins than did the tissue of origin”. The principal products of the glucose utilization were lactate and carbon dioxide. There was also some conversion of glucose to fatty acids, and this occurred to a considerably greater extent in the tumors than in the normal membrane*. The present investigation extends the study into the lipid metabolism of these tissues. There have been some reports in the literatures@ that the distribution of the lipids among the various classes was different in certain tumors than in their normal tissues of origin. So it was of interest to find out if there were any striking differences in the lipid patterns of the Rous sarcoma tumors and of the membranes. Biochim.
Biophys.
Acta,
125 (1966) 428-434
Experimeuts
were x&soccmducted
to survey
the
ineorpraticm ofsome increased In-
corporation of the tumors was taking place,
tumors by ~~o~~~ationof Bryan ~~~~~t~te~strain of Rous sarcoma virus onto the dropped &~~~~~a~~~~~~~~ membrme af Ix-&q ~~~~~~~t~ &i&en eggs. They WXP, alfmved to grow fc~f5 days2 and then the tumors were dissected from the uninfected portions of the membrane and rinsed in cold sahne. S&es were prepared with a Stadie-Riggs microtome. For normal tissue the ~hor~oal~aI~~oi~ membranes from uninfected eggs of the same age were used. Other details of the method were as reported previously&. The tissues were @&racted with ~~l~~~~~~m-rnethano~(2:f,v/V) by the method: of ROUX & @LB. 30-g samples of tissue were used for the initial survey of the lipids, From the incubatiorrs described later all of the 600 mg of tisstxe was extracted. The tissue was filtered from the extract, and the extract was Iv&shed with a large volume of cold ~r%ter6. After the extract was removed from the water, the solvent was evaporated under nitrogen to give a sample of total lipids. These samples in q-ml centrifuge tubes were then treated with 8s ~1 of acetone fcx each rng of hpld. The tubes were ~~#~~~~~~~m&ml with a vortex mixer and centrifuged, The sohrtion of the acetone-soluble lipids was pipetted oE with a capillary pipette from the residue of the acetone-insoluble lipids, and the acetone was evaporated under nitrogen.
The two frictions of (2 : I, v/v] and spotted on silica gel G 0.25 mm thick, survey of the tissue lipids.
the lipids were dissolved in 30 ~1 of ~h~orofo~-methanol thin-layer ~h~~matogra~hy plates coated with a layer of 300-s quantities af the tipids were spotted for the initial Larger samplles were used from the inc&ated tissues, but in & caseS they were spotted on the plates in ~uant~ties of less than T mg per lane. For the acetone-soluble lipid sampies, the plates were developed with hexane-ethyl. ether-acetic acid (75 : ~5 : I, v/v).The acetone-insoluble lipids were separated by USE+ of chloroform-rn~t~a~ol-water (65 : zs : 4, v/v). The fractious were made visible on the plates by staining them with iodine, which could later be ~~~~orated, or by spray ing with 50% H&XI, and heating to chitr the lipids, When it was desired to recover the lipids from the p&es, guide lanes were prepared which could be stained to b&e the fractions. C~rres~~~i~ sections were then taken from the unstdned lanes.
For the i~co~~o~at~onstudies 600 mg of tumor slices or normal membrane were incubated in 5 ml of medium in q-ml ~~lenmeyer flasks at 3~~ far 3 h in a shaking water bath in air. The medium contained the following anlo~~t of salts in moles/l: Q&1,, 1.25; KCl, $3; MgSO,, 0.8; NaCt, x40. It had been butiered to pH 7.0 with a phosphate buffer giving a concentration of 25 m&T phosphate. It also contained
430
P.H.FIGARL),
A.S.LEVINE
2.5 mM n-glucose. This was the lowest concentration of glucose used in the previous study on the conversion of glucose to fatty acids I. Radioactive precursors were added as n-[14C]glucose (uniformly labeled) or sodium [I-‘*C]acetate in amounts of about 0.4 pmole and activity of approx. 10~ counts/min for each incubation. Blank incubations were also carried out with boiled tissue samples. After the incubations were
completed, described
the lipids were extracted
from the tissues and separated
by the procedures
above.
Measurement of radioactivity The determinations of radioactivity of the initial precursors and of the lipid fractions separated from the incubated tissues were done by liquid scintillation counting. One-tenth aliquots of the lipids were counted before the main portions of the samples were separated by thin-layer chromatography. For fractions from the chromatography plates the silica gel sections were transferred to counting vials for counting. The method followed was that of SNYDER AND STEPHENS'. The counting efficiency of these samples was measured
by addition
of internal
standards.
The silica gel caused
no significant quenching. The distribution of radioactivity within the triglyceride molecules was determined for some samples by eluting the lipid from the appropriate section of silica gel and hydrolyzing 3 h as suggested
it in 5 ml of 0.5 M KOH in 95% ethanol at reflux temperature for by HANAHAN~. The solutions were diluted with 5 ml of water, and
the ethanol was evaporated. The solutions were acidified with dilute HCl, and the fatty acids were extracted with hexane. Counts were made on both the fatty acid and glycerol
fractions.
Calculations The counts
found in the fractions
from the boiled tissues were subtracted
as
blanks from the counts measured for the corresponding fractions from the tissues which had not been heated, The blanks amounted to 5-10~/~ of the counts in the total lipid. After the fractionations they were found mainly near the origin of the phosphatide chromatograms. Corrections were also made for the incompleteness of the acetone separation. There was always some neutral lipid near the solvent front of the chromatograms of the acetone-insoluble lipids, and a little phosphatide was found at the origin of the chromatograms of the acetone-soluble lipids. The amount of incorporation for each lipid class was calculated from the total counts found in the samples before separation by thin-layer chromatography and the distribution of the counts found in the fractions from the plates. The amounts of incorporation occurring in the various classes of lipids were compared as the percentages of the initial radioactivity that was incorporated into each fraction and as specific activities. RESULTS
Lipid composition of chorioallantoic membrane and tumors The lipids were extracted from the normal chorioallantoic membranes and from the tumor tissues. These extractions and the separation of the lipid samples were carried out as described under METHODS. Biochim.
Biophys.
Acta,
IZ=J (1966) 428-434
LIPIDS
43
OF ROWS TWMQRS
Then ~3s not emmgh of t&e fractions avaifabk from the thidayer chmmtagram for a complete chemical ~~e~~~~~2~i~~, lx& tfie p&x&d 03nstitnents ~~~~~~~ to be those given in Table I. The sterds and sterol esters gave the characteristic purple color when the chromatograms were sprayed with H,SO, and heated moderately. The phosphatidylethanolamine spot gave an amine test with ninhydrin, and the
0.56 0.13 0.14 0.05 0.01* 0.22 <
o.oI*
0.48 0.x8 O.?.J 0.07
* Approximate
values estimated from trace
spots on chromatograms.
lecithin spot gave a choline test with the Dragendorf reagerrt@, The identifications were based on the color reactions and on a comparison of the RF values with those of authentic s~~d~~ on the same plates. The standards for the neutral Iipids were commercial samples of cholesteryl stearate, tristearin, palmitic acid and cholesterol, The standards for phosphatides were phosphatidylethanol~mi~~ isolated from ratliver lipids by repeated column chromatography on silicic acid, lecithin obtained from egg lipids and sphingomyelin provided by Dr. H. E. CARTER of the University of Illinois. The amounts of the fractions listed. in Table I were calculated from the weights of the neutral lipids and ~ho~hatid~ and the densities of the spots on H,SO,-charred ~hromato~ams. There appeared to be no differences between the chromatograms of the phosphatides from the membranes ;uld those from the tumor, and the differences between the patterns for the neutral lipids from the two tissues were not great. The most noticeable differences were that the tumors seemed to have a greater proportion of fatty acids and. a smaller proportion of sterols as compared with the membranes.
The invigoration percentages of the precursors into the nentral hpids and ~hos~hatides are shown in Tables II and III. The tumor tissue i~~o~rat~ considerably more of the precursors than the membranes did. The amount of incorporation agreed satisfactorily for duplicate tissues in a given experiment, but there was quite a little variation from one sample of tissue to another. The standard errors of the means for the items in the table are for three or four different experiments. The membrane samples gave as much variation as the tumors. Table II shows the incorporation of glucose into the various classes of lipids.
P. II. FIGARD, A. S. LEVINE
432 TABLE
II
INCORPORATION
OF GLUCOSE
INTO
LIPIDS
OF CHORIOALLANTOIC
MEMBRANE
AND
Rows
TUMORS
Values for total counts added given as mean 5 standard error of mean. Per cent of total counts added x 10~
Probable identity
Fraction
_I-.
Membrane
Tl.@iKW
Neutral lipids I Sterol esters 2 Triglycerides 3+4 Fatty acids -C-diglycerides 5 Sterols Phosphatides
146 r!: 15 9+ 2 125 & 10 .5$: 1 7:k 2 178 * 20
1238 * 47 -I1029 + 99 c 63+ 443 i
87 Lecithin Phosphatidylethanolamine 9 Sphingomeelin Number of observations _-
86 i 13 65rt 9 271?; 4 3
173 i+ 239 3=+ %
TABLE
Specific activity (c~nts~~~n .._
104 13 58 34 14 38 2.5 22 7
pw
mg)
~~e~nbra~e Tumor ._ 380 1600 110
250
1400
4600
II0
960
50 660
230 1400
660 680 600
1400 1500 430
_
III
INCORPORATION
OF
ACETATE
INTO LIPIDS OF CHORlOALLANTOICMEMBRANE AND ROus TUMORS
Values for total counts added given as mean & standard error of mean for three observations -_ -. Specific activity Fraction Pev cent of total counls Probable identity (cou~ts~~~~ per mg) added x IO* ~e~b~a~e Twnor Mewtbrane T,U~~ Neutral lipids 99 %. 36 765 i 289 260 960 1 Sterol esters 6s1 2 31 * 12 70 160 2 Triglycerides 61 & 24 457 i 179 660 2000 3 Fatty acids 3%: 1 14?~ 1 X0 160 3000* 1000* Diglycerides 14* 4 98i3 Sterols 5I-41 2 102 3 40 390 5” 6 Monoglycerides 1oir 633 *Z* 2000* 3 24 Phosphatides s7
Lecithin Phosphatidylethanolamine
9
40 -& I2 11zk 4 6 16f ‘3i. 2
290 f 107 120 95 ”3: 36 42 755 9
Sphingomyelin -* Approximate values estimated from trace spots on chromatograms.
1.50 130 180 II0
960 I300 800 770
The diglycerides were removed with the fatty acids since there appeared to be very little of them present, The triglycerides contained much of the radioactivity. The next most active classes were lecithin and phosphatidylethanolamine. The incorporation of glucose by the tumor tissue appeared to be greater than that by the membranes in all classes of lipid. Only in the case of the sphingomyelin was there no significant difference. From preliminary experiments with acetate as the radioactive precursor, it appeared that the activities in the diglycerides and monoglycerides were of more importance here than in the incubations with radioactive glucose. Measurements on the radioactivity in the areas of the chromatograms where the diglycerides and monoglycerides occur were made, and the results were included in the data of Table IIf even though they were scarcely visible on the chromatograms. The amount of activity in these fractions was a significant part of the total in spite of the fact that they were present in very small quantities. Biochim. Biophys. Acta, ~25 (1966) 428-434
LIPIDS OF ROUS
433
TUMOURS
Several samples of triglycerides from incubations with various radioactive substrates were hydrolyzed to determine the dist~buiion of the activity between the glycerol and fatty acid portions of the molecules. From the glucose incubations 36 yO of the radioactivity was found in the glycerol moiety. Experiments with sodium [a-Xlpyruvate as the substrate yielded 13 7, of the label in the glycerol. With acetate as the precursor there was 3% of the activity in the glycerol portion of the triglycerides. DISCUSSION
The triglycerides appeared to be the class of lipids most actively incorporating the radioactivity. This was true for both of the precursors and for both tissues. They represented about half of the total incorporation even though the triglyceride content of the tissues was less than zoo/O of the total lipid. The relative importance of the fraction was also shown by a comparison of the specific activities. The tumor slices incorporated about 6 times as much radioactivity as did the normal membranes. Since so much of the incorporation appeared to be taking place in the triglyceride fraction, this fraction also accounted for much of the increased incorporation by the tumor. However, there appeared to be increased incorporation by the tumor in all lipid classes. The only possible exception was in the case of sphingomyelin with glucose as the precursor. The finding that 13% of the radioactivity of the triglycerides from pyruvate incorporation was in the glycerol portion of the molecules indicated that there was a considerable amount of the pyruvate being converted through oxaloacetate to phosphoglycerate. Acetate incorporation into diglycerides and monoglycerides was high, although the small amounts of the compounds present and their incomplete separation from the major components on the chromatograms permitted only estimations of their specific activities. In spite of their high specific activities, their contribution to the total lipid activity was not large, because they were present in such small amounts. Their high activity could be a result of incomplete triglyceride synthesis, or it could result from degradation of triglyceride formed earlier during the period of incubation. It should be possible to distin~ish between these alternatives by observations for periods of incubation of different duration. This was not done because the phenomenon appeared to occur in both tissues and this investigation was aimed primarily at looking for differences associated with the malignant state. The difference in incorporation by the two tissues appeared to be largely a matter of amount rather than of distribution.
Health
This investigation was supported by research grants from the U. S. Public Service (CA-04692-07) and from the Marion County Cancer Society, Inc. We wish to thank Dr. J. ASHMORE for helpful discussions.
Biachim. Biophys.
Acta,
125
(1966) 428-434
P. H. FIGARD,
434
A. S. LEVINE
REFERENCES I 2 3 4 5 6 7 8 g
A. S. LEVINE, R. UHL AND J. ASHMORE, J. Natl. Cancer Inst., 27 (1961) 597. S. R. WAGLE, J. ASHMORE AND A. S. LEVINE, Cancer Res., 23 (1963) 1808. F. L. HAVEN AND W. R. BLOOR, Aduan. Cancer Res., IV (1956) 237. H. 0. CHRISTENSEN Lou, J. CLAUSEN AND F. BIERRING, ,I. Neurochem., 12 (1965) 619. W. C. LOVE, A. S. LEVINE AND J. ASHMORE, J. Natl. Cancer Inst., 32 (1964) 731. J. FOLCH, I. ASCOLI, M. LEES, J. A. MEATH AND F. N. LEBARON, J. Biol. Chem., IgI (1951) 833. F. SNYDER AND N. STEPHENS, Anal. Biochem., 4 (1962) 128. D. J. HANAHAN, Lipide Chemistry, John Wiley and Sons, New York, 1960, p. 187. W. D. SKIDMORE AND C. ENTENMAN, J. Lipid Res., 3 (1962) 471.
Biochim.
Biophys. Acta, 125 (1966) 428-434
LIPIDS INDUCED
PAUL
H.
De$artment
FIGARD
AND
BY
ROUS SARCOMA VIRUS
ALVIN
of Micwbiology,
Indiana
(Received March Iqth, 1966) (Revised manuscript received
OF
June
S. LEVINE
University myth,
School of Medicine,
Indialzapolis,
Ind. (U.S.A.)
1966)
SUMMARY I. A comparison was made of the lipids in the chorioallantoic membranes of chicken eggs and in tumors induced in the membranes by Rous sarcoma virus. 2. Thin-layer chromatography of the lipids from the two tissues revealed only minor differences in their compositions. 3. Measurements were made of the incorporation of several labeled precursors into the lipids of the two tissues. From a buffered salt medium containing glucose, the greatest incorporation of radioactivity was found in the triglycerides. This was true for both tissues and for either glucose or acetate as the radioactive precursor. The tumor slices incorporated about six times as much radioactivity as did the normal membranes. Much of the increased incorporation took place in the triglycerides, but there appeared to be an increased incorporation by the tumors in almost all of the lipid fractions tested. There was considerable incorporation into the glycerol portion of the triglycerides from glucose and even from pyruvate. 4. The difference in incorporation by the two tissues appeared to be largely a matter of amount rather than of distribution.
INTRODUCTION
Tumors induced by Rous sarcoma virus utilized more glucose than the normal chorioallantoic membrane from which they were produced’. In addition, it was shown that the Rous tumors incorporated more amino acids into proteins than did the tissue of origin”. The principal products of the glucose utilization were lactate and carbon dioxide. There was also some conversion of glucose to fatty acids, and this occurred to a considerably greater extent in the tumors than in the normal membrane*. The present investigation extends the study into the lipid metabolism of these tissues. There have been some reports in the literatures@ that the distribution of the lipids among the various classes was different in certain tumors than in their normal tissues of origin. So it was of interest to find out if there were any striking differences in the lipid patterns of the Rous sarcoma tumors and of the membranes. Biochim.
Biophys.
Acta,
125 (1966) 428-434
Experimeuts
were x&soccmducted
to survey
the
ineorpraticm ofsome increased In-
corporation of the tumors was taking place,
tumors by ~~o~~~ationof Bryan ~~~~~t~te~strain of Rous sarcoma virus onto the dropped &~~~~~a~~~~~~~~ membrme af Ix-&q ~~~~~~~t~ &i&en eggs. They WXP, alfmved to grow fc~f5 days2 and then the tumors were dissected from the uninfected portions of the membrane and rinsed in cold sahne. S&es were prepared with a Stadie-Riggs microtome. For normal tissue the ~hor~oal~aI~~oi~ membranes from uninfected eggs of the same age were used. Other details of the method were as reported previously&. The tissues were @&racted with ~~l~~~~~~m-rnethano~(2:f,v/V) by the method: of ROUX & @LB. 30-g samples of tissue were used for the initial survey of the lipids, From the incubatiorrs described later all of the 600 mg of tisstxe was extracted. The tissue was filtered from the extract, and the extract was Iv&shed with a large volume of cold ~r%ter6. After the extract was removed from the water, the solvent was evaporated under nitrogen to give a sample of total lipids. These samples in q-ml centrifuge tubes were then treated with 8s ~1 of acetone fcx each rng of hpld. The tubes were ~~#~~~~~~~m&ml with a vortex mixer and centrifuged, The sohrtion of the acetone-soluble lipids was pipetted oE with a capillary pipette from the residue of the acetone-insoluble lipids, and the acetone was evaporated under nitrogen.
The two frictions of (2 : I, v/v] and spotted on silica gel G 0.25 mm thick, survey of the tissue lipids.
the lipids were dissolved in 30 ~1 of ~h~orofo~-methanol thin-layer ~h~~matogra~hy plates coated with a layer of 300-s quantities af the tipids were spotted for the initial Larger samplles were used from the inc&ated tissues, but in & caseS they were spotted on the plates in ~uant~ties of less than T mg per lane. For the acetone-soluble lipid sampies, the plates were developed with hexane-ethyl. ether-acetic acid (75 : ~5 : I, v/v).The acetone-insoluble lipids were separated by USE+ of chloroform-rn~t~a~ol-water (65 : zs : 4, v/v). The fractious were made visible on the plates by staining them with iodine, which could later be ~~~~orated, or by spray ing with 50% H&XI, and heating to chitr the lipids, When it was desired to recover the lipids from the p&es, guide lanes were prepared which could be stained to b&e the fractions. C~rres~~~i~ sections were then taken from the unstdned lanes.
For the i~co~~o~at~onstudies 600 mg of tumor slices or normal membrane were incubated in 5 ml of medium in q-ml ~~lenmeyer flasks at 3~~ far 3 h in a shaking water bath in air. The medium contained the following anlo~~t of salts in moles/l: Q&1,, 1.25; KCl, $3; MgSO,, 0.8; NaCt, x40. It had been butiered to pH 7.0 with a phosphate buffer giving a concentration of 25 m&T phosphate. It also contained
430
P.H.FIGARL),
A.S.LEVINE
2.5 mM n-glucose. This was the lowest concentration of glucose used in the previous study on the conversion of glucose to fatty acids I. Radioactive precursors were added as n-[14C]glucose (uniformly labeled) or sodium [I-‘*C]acetate in amounts of about 0.4 pmole and activity of approx. 10~ counts/min for each incubation. Blank incubations were also carried out with boiled tissue samples. After the incubations were
completed, described
the lipids were extracted
from the tissues and separated
by the procedures
above.
Measurement of radioactivity The determinations of radioactivity of the initial precursors and of the lipid fractions separated from the incubated tissues were done by liquid scintillation counting. One-tenth aliquots of the lipids were counted before the main portions of the samples were separated by thin-layer chromatography. For fractions from the chromatography plates the silica gel sections were transferred to counting vials for counting. The method followed was that of SNYDER AND STEPHENS'. The counting efficiency of these samples was measured
by addition
of internal
standards.
The silica gel caused
no significant quenching. The distribution of radioactivity within the triglyceride molecules was determined for some samples by eluting the lipid from the appropriate section of silica gel and hydrolyzing 3 h as suggested
it in 5 ml of 0.5 M KOH in 95% ethanol at reflux temperature for by HANAHAN~. The solutions were diluted with 5 ml of water, and
the ethanol was evaporated. The solutions were acidified with dilute HCl, and the fatty acids were extracted with hexane. Counts were made on both the fatty acid and glycerol
fractions.
Calculations The counts
found in the fractions
from the boiled tissues were subtracted
as
blanks from the counts measured for the corresponding fractions from the tissues which had not been heated, The blanks amounted to 5-10~/~ of the counts in the total lipid. After the fractionations they were found mainly near the origin of the phosphatide chromatograms. Corrections were also made for the incompleteness of the acetone separation. There was always some neutral lipid near the solvent front of the chromatograms of the acetone-insoluble lipids, and a little phosphatide was found at the origin of the chromatograms of the acetone-soluble lipids. The amount of incorporation for each lipid class was calculated from the total counts found in the samples before separation by thin-layer chromatography and the distribution of the counts found in the fractions from the plates. The amounts of incorporation occurring in the various classes of lipids were compared as the percentages of the initial radioactivity that was incorporated into each fraction and as specific activities. RESULTS
Lipid composition of chorioallantoic membrane and tumors The lipids were extracted from the normal chorioallantoic membranes and from the tumor tissues. These extractions and the separation of the lipid samples were carried out as described under METHODS. Biochim.
Biophys.
Acta,
IZ=J (1966) 428-434
LIPIDS
43
OF ROWS TWMQRS
Then ~3s not emmgh of t&e fractions avaifabk from the thidayer chmmtagram for a complete chemical ~~e~~~~~2~i~~, lx& tfie p&x&d 03nstitnents ~~~~~~~ to be those given in Table I. The sterds and sterol esters gave the characteristic purple color when the chromatograms were sprayed with H,SO, and heated moderately. The phosphatidylethanolamine spot gave an amine test with ninhydrin, and the
0.56 0.13 0.14 0.05 0.01* 0.22 <
o.oI*
0.48 0.x8 O.?.J 0.07
* Approximate
values estimated from trace
spots on chromatograms.
lecithin spot gave a choline test with the Dragendorf reagerrt@, The identifications were based on the color reactions and on a comparison of the RF values with those of authentic s~~d~~ on the same plates. The standards for the neutral Iipids were commercial samples of cholesteryl stearate, tristearin, palmitic acid and cholesterol, The standards for phosphatides were phosphatidylethanol~mi~~ isolated from ratliver lipids by repeated column chromatography on silicic acid, lecithin obtained from egg lipids and sphingomyelin provided by Dr. H. E. CARTER of the University of Illinois. The amounts of the fractions listed. in Table I were calculated from the weights of the neutral lipids and ~ho~hatid~ and the densities of the spots on H,SO,-charred ~hromato~ams. There appeared to be no differences between the chromatograms of the phosphatides from the membranes ;uld those from the tumor, and the differences between the patterns for the neutral lipids from the two tissues were not great. The most noticeable differences were that the tumors seemed to have a greater proportion of fatty acids and. a smaller proportion of sterols as compared with the membranes.
The invigoration percentages of the precursors into the nentral hpids and ~hos~hatides are shown in Tables II and III. The tumor tissue i~~o~rat~ considerably more of the precursors than the membranes did. The amount of incorporation agreed satisfactorily for duplicate tissues in a given experiment, but there was quite a little variation from one sample of tissue to another. The standard errors of the means for the items in the table are for three or four different experiments. The membrane samples gave as much variation as the tumors. Table II shows the incorporation of glucose into the various classes of lipids.
P. II. FIGARD, A. S. LEVINE
432 TABLE
II
INCORPORATION
OF GLUCOSE
INTO
LIPIDS
OF CHORIOALLANTOIC
MEMBRANE
AND
Rows
TUMORS
Values for total counts added given as mean 5 standard error of mean. Per cent of total counts added x 10~
Probable identity
Fraction
_I-.
Membrane
Tl.@iKW
Neutral lipids I Sterol esters 2 Triglycerides 3+4 Fatty acids -C-diglycerides 5 Sterols Phosphatides
146 r!: 15 9+ 2 125 & 10 .5$: 1 7:k 2 178 * 20
1238 * 47 -I1029 + 99 c 63+ 443 i
87 Lecithin Phosphatidylethanolamine 9 Sphingomeelin Number of observations _-
86 i 13 65rt 9 271?; 4 3
173 i+ 239 3=+ %
TABLE
Specific activity (c~nts~~~n .._
104 13 58 34 14 38 2.5 22 7
pw
mg)
~~e~nbra~e Tumor ._ 380 1600 110
250
1400
4600
II0
960
50 660
230 1400
660 680 600
1400 1500 430
_
III
INCORPORATION
OF
ACETATE
INTO LIPIDS OF CHORlOALLANTOICMEMBRANE AND ROus TUMORS
Values for total counts added given as mean & standard error of mean for three observations -_ -. Specific activity Fraction Pev cent of total counls Probable identity (cou~ts~~~~ per mg) added x IO* ~e~b~a~e Twnor Mewtbrane T,U~~ Neutral lipids 99 %. 36 765 i 289 260 960 1 Sterol esters 6s1 2 31 * 12 70 160 2 Triglycerides 61 & 24 457 i 179 660 2000 3 Fatty acids 3%: 1 14?~ 1 X0 160 3000* 1000* Diglycerides 14* 4 98i3 Sterols 5I-41 2 102 3 40 390 5” 6 Monoglycerides 1oir 633 *Z* 2000* 3 24 Phosphatides s7
Lecithin Phosphatidylethanolamine
9
40 -& I2 11zk 4 6 16f ‘3i. 2
290 f 107 120 95 ”3: 36 42 755 9
Sphingomyelin -* Approximate values estimated from trace spots on chromatograms.
1.50 130 180 II0
960 I300 800 770
The diglycerides were removed with the fatty acids since there appeared to be very little of them present, The triglycerides contained much of the radioactivity. The next most active classes were lecithin and phosphatidylethanolamine. The incorporation of glucose by the tumor tissue appeared to be greater than that by the membranes in all classes of lipid. Only in the case of the sphingomyelin was there no significant difference. From preliminary experiments with acetate as the radioactive precursor, it appeared that the activities in the diglycerides and monoglycerides were of more importance here than in the incubations with radioactive glucose. Measurements on the radioactivity in the areas of the chromatograms where the diglycerides and monoglycerides occur were made, and the results were included in the data of Table IIf even though they were scarcely visible on the chromatograms. The amount of activity in these fractions was a significant part of the total in spite of the fact that they were present in very small quantities. Biochim. Biophys. Acta, ~25 (1966) 428-434
LIPIDS OF ROUS
433
TUMOURS
Several samples of triglycerides from incubations with various radioactive substrates were hydrolyzed to determine the dist~buiion of the activity between the glycerol and fatty acid portions of the molecules. From the glucose incubations 36 yO of the radioactivity was found in the glycerol moiety. Experiments with sodium [a-Xlpyruvate as the substrate yielded 13 7, of the label in the glycerol. With acetate as the precursor there was 3% of the activity in the glycerol portion of the triglycerides. DISCUSSION
The triglycerides appeared to be the class of lipids most actively incorporating the radioactivity. This was true for both of the precursors and for both tissues. They represented about half of the total incorporation even though the triglyceride content of the tissues was less than zoo/O of the total lipid. The relative importance of the fraction was also shown by a comparison of the specific activities. The tumor slices incorporated about 6 times as much radioactivity as did the normal membranes. Since so much of the incorporation appeared to be taking place in the triglyceride fraction, this fraction also accounted for much of the increased incorporation by the tumor. However, there appeared to be increased incorporation by the tumor in all lipid classes. The only possible exception was in the case of sphingomyelin with glucose as the precursor. The finding that 13% of the radioactivity of the triglycerides from pyruvate incorporation was in the glycerol portion of the molecules indicated that there was a considerable amount of the pyruvate being converted through oxaloacetate to phosphoglycerate. Acetate incorporation into diglycerides and monoglycerides was high, although the small amounts of the compounds present and their incomplete separation from the major components on the chromatograms permitted only estimations of their specific activities. In spite of their high specific activities, their contribution to the total lipid activity was not large, because they were present in such small amounts. Their high activity could be a result of incomplete triglyceride synthesis, or it could result from degradation of triglyceride formed earlier during the period of incubation. It should be possible to distin~ish between these alternatives by observations for periods of incubation of different duration. This was not done because the phenomenon appeared to occur in both tissues and this investigation was aimed primarily at looking for differences associated with the malignant state. The difference in incorporation by the two tissues appeared to be largely a matter of amount rather than of distribution.
Health
This investigation was supported by research grants from the U. S. Public Service (CA-04692-07) and from the Marion County Cancer Society, Inc. We wish to thank Dr. J. ASHMORE for helpful discussions.
Biachim. Biophys.
Acta,
125
(1966) 428-434
P. H. FIGARD,
434
A. S. LEVINE
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Biochim.
Biophys. Acta, 125 (1966) 428-434