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Labeling of liver and serum cholesterol esters after the injection of cholesterol-4-C14 and cholesterol-4-C14 esters
ARCHIVES
OF
Labeling
BIOCHEMISTRY
of Liver
AKD
and
BIOPHYSICS
Serum
Cholesterol-4-C14 LEON Veterans
Administration
li‘ospital
113, 143-149
(1966)
Cholesterol and SWELL
after
Cholesterol-4-C’4 AiVD
July
the
Injection
of
Esters’
M. D. LAW
of Biochemistry, Virginia
and Department Richmond, Received
Esters
Medical
College
of Virginia,
9, 1965
Fasting rats were injected with trace amount of cholesterol-4-V and cholesterol4-W esters (palmitate oleate, linoleate and arachidonate) dipersed in rat serum. It was necessary to add an emulsifying agent (Tween 20) to disperse the 04 esters since they do not exchange with lipoprotein cholesterol esters. The serum C”-esters, 1 hour after the injection of cholesterol-4-V, consisted of 83y0 polyunsaturated esters of which 60y0 was arachidonate. At the same time (1 hour) the liver Cl4-esters consisted of approximately 50% polyunsaturated ester and 50’% saturated and monounsaturated esters. The specific activity of the polyunsaturated esters in liver and serum exceeded the specific activity of the saturated and monounsaturated esters; the liver arachidonate ester had the highest specific activity. The specific activities of the different liver esters were higher than the corresponding serum esters. After the injection of the CWesters, the labeling of serum and liver esters indicated that linoleate and arachidonate were metabolized more rapidly than the palmitate and oleate esters. There was a preferential conversion of the linoleate ester to the arachidonate ester. It is proposed that (a) a large portion of the serum cholesterol esters arise from the liver by selective processes, (b) that the major esters released into the blood of the postabsorptive rat by the liver are polyunsaturated, and (c) that a portion of the cholesterol arachidonate in the liver is synthesized by a transesterification process involving a reaction between a cholesterol ester (such as cholesterol linoleate) and an arachidonic acid donor.
Several tissues have been suggested as the major sources of the blood and tissue cholesterol esters. The intestine, through absorption and synthesis, furnishes considerable amounts of cholesterol esters to the body ester pool (1, 2). After the intestinal chylomicron cholesterol esters enter the circulation, they are removed to a large extent by the liver (3, 4). A portion of the cholesterol esters are hydrolyzed in the liver. The fate of the remaining cholesterol esters is not clear; they may either be incorporated into the different liver cholesterol ester pools or undergo transesterification reactions with fatty acid donors to form other cholesterol esters (5). The liver has also been shown to 1 Supported and HE-09040.
in part
by USPHS
grants
contain enzymic systems for synthesizing cholesterol esters from free cholesterol in the presence of ATP and CoA (6, 7). This enzymic system favors the formation of monounsaturated and saturated esters of cholesterol, which reflects the liver cholesterol ester fatty acid composition but not the serum cholesterol ester pattern. If the liver is a prime source of the blood cholesterol esters, then the transfer of the esters from liver to the blood must be highly selective since the blood cholesterol esters contain a high proportion of polyunsaturated fatty acids. Evidence in support of the liver as the chief site of formation of the blood cholesterol esters has been obtained in rats injected with C14-mevalonate and also in studies with perfused rat liver (8, 9). The individual liver
HE-09039 143
144
SWELL
AND
esters after the administration of mevalonate-Cl4 had a higher specific act,ivity than the pIasma esters. On the ot,her hand, it has also recently been shown (10) that, after the injection of cholesterol-4-C*4 lipoproteins into rats, the specific activit’y of the plasma polyunsat’urated esters exceeded the specific activity of the same esters in t,he liver during t.he first several hours. These findings suggest that the blood cholesterol kansferase enzyme (11) may synthesize part of the blood cholesterol esters. The purpose of the present study was to obtain additional informat’ion on the metabolism of liver and serum cholesterol esters wit*h particular emphasis on the turnover and interconversions of the individual cholesterol est,ers. Comparative data were obtained on the handling of individual injected cholesterol esters and free cholesterol. MATERIALS
AND
amount (1.5 1C) of cholesterol-4-Cl4 or choles tero1-4-Cl4 ester in 0.25 1111 rat serum. Tween 20 (15 mg/0.25 ml serum) was added to the serum before sonication to aid in the dispersal of the Cl&-esters. Cholesterol-MY* could be evenly dispersed in serum without the addition of an emulsifying agent. The Gl”-sterols were found to be uniformly dispersed in the sera by this procedure, and no chemical changes in the labeled compounds were noted when the labeled sera were subsequently extracted and analyzed by thin-layer chromatography. Fasting male rats (Wistar strain), maintained on Purina pellet chow and weighing 250-285 gm, were injected in the saphenous vein with 0.25 1111 of the Cl*-sterol labeled serum. The animals remained fasting after the inject.ion unt.il the time of killing. Groups of 5 animals were killed at 1, 4, and 24 hours after the injection of each Cl*-sterol labeled serum. Blood was obtained from the abdominal aorta, aud the liver was removed. The liver was quick-frozen with dry ice, pulverized, and extracted with 2:l chloroform-methanol. The blood was allowed to clot (5-10 minutes), and the serum was separated and immediately extracted with 2:l chloroformmethanol to prevent in, vitro cholesterol transesterificatiou (11). Uethods. The serum and liver lipids were separated bp silicic acid colunm chromatography (15). Cholesterol esters were elut.ed with diethyl etherpetroleum ether (1:99, v/v) and free cholesterol with diethyl ether-petroleum ether (25:i5, v/v). Free and esterified cholesterol concentrations were determined by the method of Sperry and Webb (IG). The C14-activity of the free and ester fractions were determined in a liquid scintillation system as described earlier (7). An aliquot of the cholesterol ester fraction was analyzed for fatty acid composition by gas-liquid chromatography (13). Another portion of the cholesterol ester fraction was separated into the four major cholesterol ester classes (saturated, monounsatllrated, linoleate, and arachidonate) by thirl-layer chro-
METHODS
Cholesterol-Q-C14 esters. Cholesterol-4-C14, cholesterol-4-V palmitate, cholesterol-4J.Y oleate, and cholesterol-4-V linoleate were obtained from Nuclear-Chicago Corp. The esters were assayed for radiochemical purity (97-99rr/c) by thin-layer chromatography on glass plates coated wit,h AgN08-impregnated Silica Gel G (12), and for chemical purity by gas-liquid chromatography (13). Cholest,erol-4-U4 arachidonate was synthesized from cholesterol-4-04 and 97”iL arachidonic acid (Hormel Foundation) enzymically (14). The ester was isolated by silicic acid column chromatography (15) and purified by thin-layer chromatography on AgNOa-impregnated Silica Gel G (12); the solvent system was 6: 1 (v/v) petroleum et,her-diethyl ether. Treatment 0s animals. The solrltion for injection was prepared by dispersing (by ultrasonic disintegration for 2 minutes at 5°C) a tracer
J,IS~~RIBI-TION
TABLE I IN SEI~GM AND LIVER
OF 0“-ACTIVITY THE
LAW
INJECTION
OF
‘:. Total Time
Liver
(hours) Saturated
1 4 24 Q Each
18.7 18.4 15.4 value
represent.s
Honounsaturated
33.0 31.2 32.0 analysis
CHOLESTEROL
E:STERS
C”-esters
C”-2sters“
Srrum
Linoleate
19.6 25.2 26.3 of a pool
AFTER
CHOI,ESTEROL--~-CY~
Arachidonate
28.7 25.2 26.3 of livers
Saturated
Monounsaturated
9.0 11.6 9.8 or serum
8.2 8.6 10.3 from
5 animals.
P-esters” Linoleate
22.3 15.5 19.7
Arachidonate
60.5 64.3 GO.2
LABELING
OF
CHOLESTEROL
total C14-esters. The serum (Fester patterns at 1, 4, and 24 hours were also similar. The major serum C14-ester was arachidonate, which accounted for 60-64X of the total C14-es&s. Comparison of the liver and serum (Festers indicates that the serum U4-esters cont,ained a much greater proportion of polyunsaturated C14-esters than the liver U4-esters at both early and later time periods. Table II shows the cholesterol ester fatty acid composit,ion of the serum and liver. Comparison of t.hese values with t’he C14ester patterns indicate t,hat the liver C14est’ers had a greater proportion of arachidonate-Cl4 and a lower percentage of C14saturated esters at the various time periods than the corresponding percentage of each ester as determined by fatty acid analysis. Table III shows the specific activity data. The free cholesterol of the serum and liver approached the same specific activity at 1 hour, indicating there was a very rapid equilibration of t,he blood and liver free cholesterol. In the early time periods (1 and 4 hours) the specific activity of the serum and liver polyunsaturated est’ers exceeded the specific act.ivit’y of the saturat,ed and monounsaturated est.crs. The liver arachidonate ester had a specific a&ivity considerably great’er than that of the other esters. At 24 hours the serum monounsaturated, linoleate, and arachidonat,e esters had the same specific activity, but. the saturated est,ers had a lower specific activity. At 24 hours, the liver arachidonate had a, sljecific activity much greater than the other esters; the saturat.ed est.ers had the lowest, specific activity. Comparison of the serum and liver esters indicates t,hat at no time did the spe-
mstography on Silica Gel G impregnated with ether-diethyl ether (6:1, AgN03 ; petroleum v/v) was used as ascending solvent. The esters were visualized by spraying the plates with 2,7dichlorofluoresceine and examining them under ultraviolet light. The respective cholesterol ester zones were scraped from the thin-layer plates directly into liqllid scintillation vials, methanol was added to elute the esters, and the C14-activity of the ester fractions was determined. The thinlayer chromatography procedure was checked with known pure colesterol-4-C14 esters (14). The recovery of CY4-activity was determined for each ester when pure stanards were analyzed singly and in mixtures. The recovery of each ester (pslmitate, oleate, linoleate, and arachidonate) in these test mixtures ranged from 93 to 99%. The specific activity of each cholesterol ester was calculated from the mass of each ester as determined by cholesterol analysis and fatty acid composition, and the C14-activity of each ester fraction. RESULTS
Injection of cholesterol-4-C14. Table I shows the distribution of C14-activity in the four ester classes of the serum and liver. The liver C14-ester patt’erns at 1, 4, and 24 hours were very similar. The polyunsaturated C14-esters accounted for approximately one half of the TABLE CHOLESTEROL Fatty
ESTEK
II
FATTY
ACID
5% Total
acids
Liver
Saturated Monounsaturated Linoleate Arachidonate
27.4 36.2 23.9 12.5
COMPOSITION fatty
acids
estersa
Serum
f dz f rt
16.4 11.0 17.8 54.8
2.5 4.1 3.8 2.G
esters-
f f f f
2.0 1.8 3.1 5.0
Q Values represent the average f SD of pools of liver or serum; each pool represents animals.
15 5
TABLE SPECIFIC
III
LIVER 0~
FREE AND CHOLESTEROL CHO~STE~~~L-4-Cl4 Serums
Time (hours)
Free
1 4 24
973 467 213
u Each
ACTIVITIES OF SERUM AND .PFTER THE INJECTION
145
ESTERS
value
195 164 121 represents
234 210 195 analysis
261 256 243 of a pool
65G 490 461 of livers
Free
Saturated
818 352 189
95 134 124
or serum
from
(cpm x 102)
~~~~~~t$
5 animals.
ESTERS
128 148 196
Linoleate
Arachidonate
215 216 232
190 224 230
146
SWELL TABLE
RECOVERY
Time
% Injected C%ictivity recovered
5%C’4-esteri-
palmitate
26.0 83.0 43.4 64.7 15.0 57.0 Cholesterol-4-U4
1 4 24
82.8 74.2 66.6 oleate
13.5 58.9 45.1 31.5 17.8 44.9 Cholesterol-4-Cl4
1 4 24
15.9 48.1 10.1 Cholesterol-4-P
1 4 24
20.2 25.2 11.3
INJECTION
7” C14-esteritied
fied
Cholest,erol-4-Cl4 1 4 24
THE
95.5 80.4 75.3 linoleate
48.7 26.8 23.5
97.7 79.8 78.4 arachidonate
51.3 22.6 15.2
97.0 89.4 83.5
a Each value represents analysis of a pool of livers or serum from 5 animals; recovery values calculated per whole liver. TABLE DISTRIBUTION
OF C14-A~~~~~~~ AFTER Liver
Time
(hours)
Saturated
P-esters0
Monounsaturated
IN
THE
(% total
LAW
cific activity of the serum esters exceed the specific activity of the liver esters. Injection of ckolesterol-4-C14 esters. Due t’o the inability of the various cholesterol esters in the lipoproteins to exchange with hhe added CWesters, it was only possible to administer cholest.erol-C14-esters emulsified (with Tween 20) in serum. The results obtained particularly at the earliest time (1 hour) would probably be indicative of t’he handling of an artifical emulsion of cholesterol esters. Table IV shows the recovery of CWactivity in the liver following the injection of C14-esters. After 24 hours, the oleate and palmitate groups had a greater percentage of the injected C14-activity in the liver than the linoleate and arachidonat,e groups. The percentage of recovered CWactivity in the esterfied form in the liver varied with the injected ester and the time period. The liver of the arachidonate-injected group had only 15% of the total CWactivity as C14ester after 24 hours; this percentage of ester is similar to that normally found in the liver. At 24 hours, the percentage of cholesterol-C14 in the esterified form in the serum ranged from 67 to 84 %.
IV
OF CWACTIVITY AFTER OF V-ESTERS
(hours)
AND
SERUM
INJECTION
V AND
LIVER
CL’ esters)
Linoleate
CHOLESTEROL
Serum
Arachidonate
Cholesterol-404
ESTERS
OF C14-Es~~~s
Saturated
WesterP Monounsaturated
(% total
Westers)
Linoleate
Arachidonate
palmitate
1 4 24
96.7 96.3 89.6
1.0 1.2 3.7
1.0 1.3 1.2 1.3 2.9 3.8 Cholesterol-,-Cl4
91.5 75.1 22.9 oleate
0.6 2.1 7.0
2.3 6.0 11.6
5.6 16.8 58.5
1 4 24
1.0 1.5 2.5
95.1 93.0 87.0
2.0 1.9 3.0 2.5 4.2 6.3 Cholesterol-.@74
0.7 2.9 7.6
94.0 72.2 33.1
1.8 6.6 10.4
3.5 18.3 48.9
1 4 24
0.4 1.9 4.5
1.1 3.7 8.5
91.1 7.4 0.3 85.5 8.9 2.2 59.6 27.4 5.0 Cholesterol-&Cl4 arachidonate
0.6 1.8 3.6
63.0 55.3 25.4
36.1 40.7 66.0
1 4 24
2.0 7.3 13.2
2.8 9.6 18.2
1.1 1.8 3.4
2.0 3.6 8.6
96.0 92.1 82.9
represents
analysis
n Each
value
3.2 7.7 17.9 of a pool
linoleate
92.0 75.4 50.7 of livers
0.9 2.5 5.1 or serum
from
5 animals.
LABELING
OF
CHOLESTEROL
Table V shows the distribution of C14-activity in the serum and liver esters after the injection of C14-esters. The liver (Fester pattern at the early time periods showed the presence of a high percentage of the ester corresponding to the CY4-ester injected. With time (at 24 hours) there was a marked decrease in the percentages of linoleate-Cl4 and arachidonate-Cl4 in the liver cholesterol esters of the groups injected with those polyunsaturated esters. At 24 hours, the liver U4-esters of the palmitate and oleate groups still consisted of 87-90 % monounsaturated and saturated esters. The serum CY4-esters showed a rapid shift toward the U4-ester pattern observed with injected cholesterol4-Cl4 (Table I). The serum (Pesters of the linoleate-injected group, at 1 hour, had appreciable amounts (36 %) of arachidonateC14; there was a rapid conversion of injected linoleate to arachidonate. Table VI shows the specific activity of the liver free and cholesterol esters. The specific TABLE SPECIFIC
Time (hours)
ACTIVITIES CHOLESTEROL INJECTION Free cholesterol
VI
OF LIVER ESTERS AFTER OF C14-Es~~~s Cholesterol
Saturated
esters’
~~~~~~
Cholesterol-4-Cl4 1 4 24
78 252 128
1 4 24
73 480 163
1 4 24
164 579 143
1 4 24
151 276 176
FREE THE
(cpm/mg
Linoleate
78 115 126
2335 86 4055 85 2226 Cholesterol-4.04
74 204 163 linoleate
-b
-b
81 120 52 76 Cholesterol-4-Cl4 96 189 117
122 189 122
X 102)
:zzi
palmitate
6602 -b 8088 76 3394 106 Cholesterol-4-Cl4 -b
AND
194 239 315 oleate 135 316 467
5895 915 4182 832 850 747 arachidonate 177 229 182
9712 4288 986
Li Values represent analysis of a pool of livers or serum from 5 animals; all animals received same amount of C14-activity (1.5 PC). b U4-activity too low for reliable specific activity.
147
ESTERS TABLE
SPECIFIC
Time (hours)
ACTIVITIES CHOLESTEROL INJECTION Free cholesterol
1
35
4 24
120 103
1 4 24
63 182 133
1 4 24
53 211 127
1 4 24
100 226 161
VII
OF SERUM ESTERS AFTER OF C14-Es~~~s Cholesterol
Saturated
ester+
~~~~~
FREE THE
(cpm/mg
AND
x
Linoleate
102)
tE$;
Cholesterol-4-C”
palmitate
1554
-2
-b
52 111
56 182
-b
841 35 237 108 Cholesterol-4-04 4645 68 2515 57 378 Cholesterol-4-Cl4 -b
-b
-b
46 56 54 58 Cholesterol-4.C*$ m-b 180 75
d 194 75
oleate d 142 82 linoleate
-b 128 111
4197 781 1062 253 254 214 arachidonate _6 239 117
2865 1985 366
a Each value represents analysis of a pool of liver or serum from 5 animals. b U4-activity too low for reliable specific activity.
activity relationships of the liver cholesterol esters showed that there was a more rapid appearance and disappearance of the linoleate and arachidonate esters than the palmitate and oleate esters. There was also a more rapid rate of appearance of CY4-activity in the arachidonate ester of the palmitate, oleate, and linoleate groups than the remaining esters. Of particular note is the handling of the injected linoleate ester which was more rapidly converted to arachidonate than the other injected cholesterol esters. Also, the specific activity of the arachidonate ester of that group exceeded the liver free cholesterol at all time periods. The specific activities of the serum cholesterol esters (Table VII) at 1 and 4 hours reflected the cholesterol ester injected; the ester injected had t,he highest specific activity. The serum arachidonate ester showed the greatest increase in specific activity with time in the palmitate-, oleate-, and linoleateinjected groups. The rate of appearance of
143
SWELL
AND
Cl4-act,ivit’y in the arachidonate est’er was highest for the linoleatc-injected ester. Also, the strum arachidonate ester of that group exceeded t#he specific activit,y of the fret cholesterol by almost 15 times at one hour.
LAW
The above findings on the turnover and site of synthesis of serum and liver cholesterol esters are not, in complete agreement with those obtained by several other invest,igators (8, 10). Goodman and Shiratori found that t’hc monounsaturated esters t,urnover more DISCUSSION rapidly in rat, plasma and liver. Those auAt least four enzymic systems for the forbhors concluded that the liver synthesizes mation of cholesterol esters may exist in the bulk of the plasma cholesterol esters and blood and tissues (1, 5, 7, 11). These are (a) t’hat the esters are synthesized by the ATP, the cholest’erol esterase system of the panCoA-dependent, enzyme system which favors creas and intestine; (b) t,he ATE’ and CoAthe formation of monounsat,urat#ed (Aholesdependent system in liver microsomes, and terol est,ers. The findings of Sugano and adrenal gland; (c) the free cholesterol-leciPortman (10) suggest that the serum t,ransthin transferase sysOem in blood; and (d) the ferase enzyme synthesizes a major portion cholest#erol ester-fat’ty acid donor tranxferase of t’he blood cholest’erol esters; this enzyme system in liver. Each of these enzymic sysfavors the format,ion of polyunsaturat,ed t,ems is specific for the formation of certain cholesterol esters. After the injection of t’ypes of cholesterol esters. The relative con- cholesterol-4-C’* labeled lipoproteins, the tribution of each of these cholest’erol ester blood polyunsaturat,ed esters had a higher synt,hesizing systems to the blood and tissue specific activity than those estersin the liver. cholest,erol ester pools is not’ 1mow-n. How The differences bet.ween the results of the ever, the c*omposit,ionand concentrat,ion of several studies may be due in part to the cholesterol est,erx in the blood and tissue nut,ribional state and handling of the animals. would bc det.ern1inec-lby t.he contribut,ion of It has been shown (18) that fast.ing increases cholest.erol esters from t,he (a) intestine; (b) the proportion of arachidonic acid in the the upt,ake, hydrolysis, transesterifications, serum cholesterol cst,crs. Previous diet might. and synt)hesis of cholesterol esters by the also be a factor. Liver and plasma cholest.erol liver and perhaps ot,her t#issues;and (c) the ester metabolism in the rat have also been incorporat.ion of liver cholesterol est,ersinto shown to be highly sensitive to stress (19). lipoproteins. The results of the present study It’ has been observed in the rat that the have provided some addit,ional information fatty acid composition of t-he serum choleson someof t,he above factors involved in the terol esters is vast’ly different from that of metabolism of t’he different cholesterol esters. liver cholesterol esters (5). This raises the The data obtained after the injection of question of whet’her t,he cholesterol esters of cholesteroMC’* suggest that t’he polyunthose tissues might be formed by different saturat.ed cholesterol esters of t,he serum mechanisms. The differences in the distribuand liver t’urnover more rapidly than the tion of C%ct,iviby in Dhe serum and liver saturat.ed and monounsaturated cholesterol cholesterol esters after the inject,ion of choesters. Also, the specific activity relation- lesterol-4-C14 indicates that the serum and ships suggest that a major portion of the liver ester pools do not, come into rapid serum cholesterol esters are derived from the equilibrium. This and the other data reliver in the postabsorptive rat. Several pos- ported in the present study subst’antiate the sible explanat.ions can be given for the higher view that t,he polyunsaturat,ed esters are turnover rate of t’he polyunsaturated choles- selectively released from t,he liver into the terol est’ers.A major portion of the polyun- blood. Gidez, and Roheim and co-workers sat,urated fatty acids may be transport’ed (9,20) have also present,edevidence for selecafrom the blood t,o the t’issuesesterified with t,ive releaseof cholesberolesters by Dheliver. cholesterol. Second, a recent observation (17) The higher density lipoprotein cholesterol indicates that’ there is a pathway involving esters were found to have a greater proporthe l~eferential conversion of cholesterol tion of polyunsaturated esters t,han the liver arachidonat,e to highly polar lipids in liver. cholesterol esters. On the ot.her hand, the low
LABELING
OF
CHOLESTEROL
densit,y lipoprotein cholesterol esters are similar in fat.ty acid composition to the liver cholesterol esters. Since cholesterol est,ers of the various lipoproteins do not exchange it was not possible to prepare lipoproteins labeled with a particular cholesterol est.er. Therefore, the data obtained at. t,he early t’imes after the injection of the individual esters emulsified in serum would probably not be indicat.ive of cndogenous cholesterol ester metabolism. However, the dat,a are of significance from a comparative standpoint, and at later Cmes, when adequate mixing of the injected esters into the ester pools probably had occurred. The findings w&h the inject’ed U4-esters support t,he results obtained with injected free cholesterol-4-U4. The injected linoleate and arachidonnte esters disappeared more rapidly from the liver than the palmitate and oleate esters. One of the most interest.ing features of the data relates to the possible preferential conversion of cholesterol linoleate to cholesterol arachidonate. There was a much greater formation of arachidonat’e from linoleate than when t,he other est.ers mere inject.ed. Xso, t,hc finding of a much higher specific :&vity of the liver arachidonate est,cr than the free cholesterol aft’er the injection of the linolcate ester suggesk that some of t,he arachidonate ester did not arise directly from the free fraction. This has been previously observed (5) in a related study. A possible mechanism for this reaction might be the int,eraction of a liver cholesterol ester such as cholesterol linoleate with an arachidonic: acid donor to form cholesterol arachidonat,e. This would be analogous to the t,ransferase reaction in blood (11)) but. would involve a cholesterol est,er as t,he substrate instead of free cholest.erol.
149
ESTERS REFERENCES
1. S\VELL, FIELD,
L., T~wwr, H.,
JR.,
AND
E.
C., JR., TREAD~ELL,
HOPPER,
J. R.,
C. R., Ann.
N. Y. Acad. sci. 72, 813 (1959). 2. LINDSEY, C. A., JR., AND WILSON, J. D., J. Lipid Res. 6, 173 (1965). 3. GOODMAN, D. S., J. Clin. Invest. 41, 1886 (1962). 4. Losso\v, W. J., BWT, N., AND CHAIKOFF, I. L., J. Lipid Res. 3, 207 (1962). L., L.lw, M. D., SND TREADM-ELL, C. 5. SLVELL, R., Arch. Hiochem. Biophys. 105, 541 (1964). D. S., DEYKIN, D., AND SHIR~TORI, 6. GOODMAN, T., J. Bid. Chem. 239, 1335 (1964). L., Law, M. D., BND TREAD\\-ELL, C. 7. SWELL, Il., Brch. Biochem. Biophys. 104, 128 (1964). 8. GOODMAN, 1). S., AND SHIRATORI, T., J. Lipid Res. 5, 578 (1964). 9. ROHEIM, P. S., HAFT, D. E., GIDEZ, L. I., WHITE, A., AND EDER, H., J. Clin. Invest. 42, 1277 (1963). 10. SUGANO, M., AND PORTMaN, 0. W., Arch. Biochem. Biophys. 109, 302 (1965). 11. GLOMSET, J. A., I’.~RKER, F., TJ‘IDEN, RI., AND WILLUMH, I<. H., Biochim. Biophys. dcta 58, 398 (1962). 12. MORRIS, L. J., J. Lipid Res. 4, 357 (1963). L., FIELL’, H., JR., SCHOOLS, P. E., 13. SWELL, JIM., 2i~~ TI
OF
Labeling
BIOCHEMISTRY
of Liver
AKD
and
BIOPHYSICS
Serum
Cholesterol-4-C14 LEON Veterans
Administration
li‘ospital
113, 143-149
(1966)
Cholesterol and SWELL
after
Cholesterol-4-C’4 AiVD
July
the
Injection
of
Esters’
M. D. LAW
of Biochemistry, Virginia
and Department Richmond, Received
Esters
Medical
College
of Virginia,
9, 1965
Fasting rats were injected with trace amount of cholesterol-4-V and cholesterol4-W esters (palmitate oleate, linoleate and arachidonate) dipersed in rat serum. It was necessary to add an emulsifying agent (Tween 20) to disperse the 04 esters since they do not exchange with lipoprotein cholesterol esters. The serum C”-esters, 1 hour after the injection of cholesterol-4-V, consisted of 83y0 polyunsaturated esters of which 60y0 was arachidonate. At the same time (1 hour) the liver Cl4-esters consisted of approximately 50% polyunsaturated ester and 50’% saturated and monounsaturated esters. The specific activity of the polyunsaturated esters in liver and serum exceeded the specific activity of the saturated and monounsaturated esters; the liver arachidonate ester had the highest specific activity. The specific activities of the different liver esters were higher than the corresponding serum esters. After the injection of the CWesters, the labeling of serum and liver esters indicated that linoleate and arachidonate were metabolized more rapidly than the palmitate and oleate esters. There was a preferential conversion of the linoleate ester to the arachidonate ester. It is proposed that (a) a large portion of the serum cholesterol esters arise from the liver by selective processes, (b) that the major esters released into the blood of the postabsorptive rat by the liver are polyunsaturated, and (c) that a portion of the cholesterol arachidonate in the liver is synthesized by a transesterification process involving a reaction between a cholesterol ester (such as cholesterol linoleate) and an arachidonic acid donor.
Several tissues have been suggested as the major sources of the blood and tissue cholesterol esters. The intestine, through absorption and synthesis, furnishes considerable amounts of cholesterol esters to the body ester pool (1, 2). After the intestinal chylomicron cholesterol esters enter the circulation, they are removed to a large extent by the liver (3, 4). A portion of the cholesterol esters are hydrolyzed in the liver. The fate of the remaining cholesterol esters is not clear; they may either be incorporated into the different liver cholesterol ester pools or undergo transesterification reactions with fatty acid donors to form other cholesterol esters (5). The liver has also been shown to 1 Supported and HE-09040.
in part
by USPHS
grants
contain enzymic systems for synthesizing cholesterol esters from free cholesterol in the presence of ATP and CoA (6, 7). This enzymic system favors the formation of monounsaturated and saturated esters of cholesterol, which reflects the liver cholesterol ester fatty acid composition but not the serum cholesterol ester pattern. If the liver is a prime source of the blood cholesterol esters, then the transfer of the esters from liver to the blood must be highly selective since the blood cholesterol esters contain a high proportion of polyunsaturated fatty acids. Evidence in support of the liver as the chief site of formation of the blood cholesterol esters has been obtained in rats injected with C14-mevalonate and also in studies with perfused rat liver (8, 9). The individual liver
HE-09039 143
144
SWELL
AND
esters after the administration of mevalonate-Cl4 had a higher specific act,ivity than the pIasma esters. On the ot,her hand, it has also recently been shown (10) that, after the injection of cholesterol-4-C*4 lipoproteins into rats, the specific activit’y of the plasma polyunsat’urated esters exceeded the specific activity of the same esters in t,he liver during t.he first several hours. These findings suggest that the blood cholesterol kansferase enzyme (11) may synthesize part of the blood cholesterol esters. The purpose of the present study was to obtain additional informat’ion on the metabolism of liver and serum cholesterol esters wit*h particular emphasis on the turnover and interconversions of the individual cholesterol est,ers. Comparative data were obtained on the handling of individual injected cholesterol esters and free cholesterol. MATERIALS
AND
amount (1.5 1C) of cholesterol-4-Cl4 or choles tero1-4-Cl4 ester in 0.25 1111 rat serum. Tween 20 (15 mg/0.25 ml serum) was added to the serum before sonication to aid in the dispersal of the Cl&-esters. Cholesterol-MY* could be evenly dispersed in serum without the addition of an emulsifying agent. The Gl”-sterols were found to be uniformly dispersed in the sera by this procedure, and no chemical changes in the labeled compounds were noted when the labeled sera were subsequently extracted and analyzed by thin-layer chromatography. Fasting male rats (Wistar strain), maintained on Purina pellet chow and weighing 250-285 gm, were injected in the saphenous vein with 0.25 1111 of the Cl*-sterol labeled serum. The animals remained fasting after the inject.ion unt.il the time of killing. Groups of 5 animals were killed at 1, 4, and 24 hours after the injection of each Cl*-sterol labeled serum. Blood was obtained from the abdominal aorta, aud the liver was removed. The liver was quick-frozen with dry ice, pulverized, and extracted with 2:l chloroform-methanol. The blood was allowed to clot (5-10 minutes), and the serum was separated and immediately extracted with 2:l chloroformmethanol to prevent in, vitro cholesterol transesterificatiou (11). Uethods. The serum and liver lipids were separated bp silicic acid colunm chromatography (15). Cholesterol esters were elut.ed with diethyl etherpetroleum ether (1:99, v/v) and free cholesterol with diethyl ether-petroleum ether (25:i5, v/v). Free and esterified cholesterol concentrations were determined by the method of Sperry and Webb (IG). The C14-activity of the free and ester fractions were determined in a liquid scintillation system as described earlier (7). An aliquot of the cholesterol ester fraction was analyzed for fatty acid composition by gas-liquid chromatography (13). Another portion of the cholesterol ester fraction was separated into the four major cholesterol ester classes (saturated, monounsatllrated, linoleate, and arachidonate) by thirl-layer chro-
METHODS
Cholesterol-Q-C14 esters. Cholesterol-4-C14, cholesterol-4-V palmitate, cholesterol-4J.Y oleate, and cholesterol-4-V linoleate were obtained from Nuclear-Chicago Corp. The esters were assayed for radiochemical purity (97-99rr/c) by thin-layer chromatography on glass plates coated wit,h AgN08-impregnated Silica Gel G (12), and for chemical purity by gas-liquid chromatography (13). Cholest,erol-4-U4 arachidonate was synthesized from cholesterol-4-04 and 97”iL arachidonic acid (Hormel Foundation) enzymically (14). The ester was isolated by silicic acid column chromatography (15) and purified by thin-layer chromatography on AgNOa-impregnated Silica Gel G (12); the solvent system was 6: 1 (v/v) petroleum et,her-diethyl ether. Treatment 0s animals. The solrltion for injection was prepared by dispersing (by ultrasonic disintegration for 2 minutes at 5°C) a tracer
J,IS~~RIBI-TION
TABLE I IN SEI~GM AND LIVER
OF 0“-ACTIVITY THE
LAW
INJECTION
OF
‘:. Total Time
Liver
(hours) Saturated
1 4 24 Q Each
18.7 18.4 15.4 value
represent.s
Honounsaturated
33.0 31.2 32.0 analysis
CHOLESTEROL
E:STERS
C”-esters
C”-2sters“
Srrum
Linoleate
19.6 25.2 26.3 of a pool
AFTER
CHOI,ESTEROL--~-CY~
Arachidonate
28.7 25.2 26.3 of livers
Saturated
Monounsaturated
9.0 11.6 9.8 or serum
8.2 8.6 10.3 from
5 animals.
P-esters” Linoleate
22.3 15.5 19.7
Arachidonate
60.5 64.3 GO.2
LABELING
OF
CHOLESTEROL
total C14-esters. The serum (Fester patterns at 1, 4, and 24 hours were also similar. The major serum C14-ester was arachidonate, which accounted for 60-64X of the total C14-es&s. Comparison of the liver and serum (Festers indicates that the serum U4-esters cont,ained a much greater proportion of polyunsaturated C14-esters than the liver U4-esters at both early and later time periods. Table II shows the cholesterol ester fatty acid composit,ion of the serum and liver. Comparison of t.hese values with t’he C14ester patterns indicate t,hat the liver C14est’ers had a greater proportion of arachidonate-Cl4 and a lower percentage of C14saturated esters at the various time periods than the corresponding percentage of each ester as determined by fatty acid analysis. Table III shows the specific activity data. The free cholesterol of the serum and liver approached the same specific activity at 1 hour, indicating there was a very rapid equilibration of t,he blood and liver free cholesterol. In the early time periods (1 and 4 hours) the specific activity of the serum and liver polyunsaturated est’ers exceeded the specific act.ivit’y of the saturat,ed and monounsaturated est.crs. The liver arachidonate ester had a specific a&ivity considerably great’er than that of the other esters. At 24 hours the serum monounsaturated, linoleate, and arachidonat,e esters had the same specific activity, but. the saturated est,ers had a lower specific activity. At 24 hours, the liver arachidonate had a, sljecific activity much greater than the other esters; the saturat.ed est.ers had the lowest, specific activity. Comparison of the serum and liver esters indicates t,hat at no time did the spe-
mstography on Silica Gel G impregnated with ether-diethyl ether (6:1, AgN03 ; petroleum v/v) was used as ascending solvent. The esters were visualized by spraying the plates with 2,7dichlorofluoresceine and examining them under ultraviolet light. The respective cholesterol ester zones were scraped from the thin-layer plates directly into liqllid scintillation vials, methanol was added to elute the esters, and the C14-activity of the ester fractions was determined. The thinlayer chromatography procedure was checked with known pure colesterol-4-C14 esters (14). The recovery of CY4-activity was determined for each ester when pure stanards were analyzed singly and in mixtures. The recovery of each ester (pslmitate, oleate, linoleate, and arachidonate) in these test mixtures ranged from 93 to 99%. The specific activity of each cholesterol ester was calculated from the mass of each ester as determined by cholesterol analysis and fatty acid composition, and the C14-activity of each ester fraction. RESULTS
Injection of cholesterol-4-C14. Table I shows the distribution of C14-activity in the four ester classes of the serum and liver. The liver C14-ester patt’erns at 1, 4, and 24 hours were very similar. The polyunsaturated C14-esters accounted for approximately one half of the TABLE CHOLESTEROL Fatty
ESTEK
II
FATTY
ACID
5% Total
acids
Liver
Saturated Monounsaturated Linoleate Arachidonate
27.4 36.2 23.9 12.5
COMPOSITION fatty
acids
estersa
Serum
f dz f rt
16.4 11.0 17.8 54.8
2.5 4.1 3.8 2.G
esters-
f f f f
2.0 1.8 3.1 5.0
Q Values represent the average f SD of pools of liver or serum; each pool represents animals.
15 5
TABLE SPECIFIC
III
LIVER 0~
FREE AND CHOLESTEROL CHO~STE~~~L-4-Cl4 Serums
Time (hours)
Free
1 4 24
973 467 213
u Each
ACTIVITIES OF SERUM AND .PFTER THE INJECTION
145
ESTERS
value
195 164 121 represents
234 210 195 analysis
261 256 243 of a pool
65G 490 461 of livers
Free
Saturated
818 352 189
95 134 124
or serum
from
(cpm x 102)
~~~~~~t$
5 animals.
ESTERS
128 148 196
Linoleate
Arachidonate
215 216 232
190 224 230
146
SWELL TABLE
RECOVERY
Time
% Injected C%ictivity recovered
5%C’4-esteri-
palmitate
26.0 83.0 43.4 64.7 15.0 57.0 Cholesterol-4-U4
1 4 24
82.8 74.2 66.6 oleate
13.5 58.9 45.1 31.5 17.8 44.9 Cholesterol-4-Cl4
1 4 24
15.9 48.1 10.1 Cholesterol-4-P
1 4 24
20.2 25.2 11.3
INJECTION
7” C14-esteritied
fied
Cholest,erol-4-Cl4 1 4 24
THE
95.5 80.4 75.3 linoleate
48.7 26.8 23.5
97.7 79.8 78.4 arachidonate
51.3 22.6 15.2
97.0 89.4 83.5
a Each value represents analysis of a pool of livers or serum from 5 animals; recovery values calculated per whole liver. TABLE DISTRIBUTION
OF C14-A~~~~~~~ AFTER Liver
Time
(hours)
Saturated
P-esters0
Monounsaturated
IN
THE
(% total
LAW
cific activity of the serum esters exceed the specific activity of the liver esters. Injection of ckolesterol-4-C14 esters. Due t’o the inability of the various cholesterol esters in the lipoproteins to exchange with hhe added CWesters, it was only possible to administer cholest.erol-C14-esters emulsified (with Tween 20) in serum. The results obtained particularly at the earliest time (1 hour) would probably be indicative of t’he handling of an artifical emulsion of cholesterol esters. Table IV shows the recovery of CWactivity in the liver following the injection of C14-esters. After 24 hours, the oleate and palmitate groups had a greater percentage of the injected C14-activity in the liver than the linoleate and arachidonat,e groups. The percentage of recovered CWactivity in the esterfied form in the liver varied with the injected ester and the time period. The liver of the arachidonate-injected group had only 15% of the total CWactivity as C14ester after 24 hours; this percentage of ester is similar to that normally found in the liver. At 24 hours, the percentage of cholesterol-C14 in the esterified form in the serum ranged from 67 to 84 %.
IV
OF CWACTIVITY AFTER OF V-ESTERS
(hours)
AND
SERUM
INJECTION
V AND
LIVER
CL’ esters)
Linoleate
CHOLESTEROL
Serum
Arachidonate
Cholesterol-404
ESTERS
OF C14-Es~~~s
Saturated
WesterP Monounsaturated
(% total
Westers)
Linoleate
Arachidonate
palmitate
1 4 24
96.7 96.3 89.6
1.0 1.2 3.7
1.0 1.3 1.2 1.3 2.9 3.8 Cholesterol-,-Cl4
91.5 75.1 22.9 oleate
0.6 2.1 7.0
2.3 6.0 11.6
5.6 16.8 58.5
1 4 24
1.0 1.5 2.5
95.1 93.0 87.0
2.0 1.9 3.0 2.5 4.2 6.3 Cholesterol-.@74
0.7 2.9 7.6
94.0 72.2 33.1
1.8 6.6 10.4
3.5 18.3 48.9
1 4 24
0.4 1.9 4.5
1.1 3.7 8.5
91.1 7.4 0.3 85.5 8.9 2.2 59.6 27.4 5.0 Cholesterol-&Cl4 arachidonate
0.6 1.8 3.6
63.0 55.3 25.4
36.1 40.7 66.0
1 4 24
2.0 7.3 13.2
2.8 9.6 18.2
1.1 1.8 3.4
2.0 3.6 8.6
96.0 92.1 82.9
represents
analysis
n Each
value
3.2 7.7 17.9 of a pool
linoleate
92.0 75.4 50.7 of livers
0.9 2.5 5.1 or serum
from
5 animals.
LABELING
OF
CHOLESTEROL
Table V shows the distribution of C14-activity in the serum and liver esters after the injection of C14-esters. The liver (Fester pattern at the early time periods showed the presence of a high percentage of the ester corresponding to the CY4-ester injected. With time (at 24 hours) there was a marked decrease in the percentages of linoleate-Cl4 and arachidonate-Cl4 in the liver cholesterol esters of the groups injected with those polyunsaturated esters. At 24 hours, the liver U4-esters of the palmitate and oleate groups still consisted of 87-90 % monounsaturated and saturated esters. The serum CY4-esters showed a rapid shift toward the U4-ester pattern observed with injected cholesterol4-Cl4 (Table I). The serum (Pesters of the linoleate-injected group, at 1 hour, had appreciable amounts (36 %) of arachidonateC14; there was a rapid conversion of injected linoleate to arachidonate. Table VI shows the specific activity of the liver free and cholesterol esters. The specific TABLE SPECIFIC
Time (hours)
ACTIVITIES CHOLESTEROL INJECTION Free cholesterol
VI
OF LIVER ESTERS AFTER OF C14-Es~~~s Cholesterol
Saturated
esters’
~~~~~~
Cholesterol-4-Cl4 1 4 24
78 252 128
1 4 24
73 480 163
1 4 24
164 579 143
1 4 24
151 276 176
FREE THE
(cpm/mg
Linoleate
78 115 126
2335 86 4055 85 2226 Cholesterol-4.04
74 204 163 linoleate
-b
-b
81 120 52 76 Cholesterol-4-Cl4 96 189 117
122 189 122
X 102)
:zzi
palmitate
6602 -b 8088 76 3394 106 Cholesterol-4-Cl4 -b
AND
194 239 315 oleate 135 316 467
5895 915 4182 832 850 747 arachidonate 177 229 182
9712 4288 986
Li Values represent analysis of a pool of livers or serum from 5 animals; all animals received same amount of C14-activity (1.5 PC). b U4-activity too low for reliable specific activity.
147
ESTERS TABLE
SPECIFIC
Time (hours)
ACTIVITIES CHOLESTEROL INJECTION Free cholesterol
1
35
4 24
120 103
1 4 24
63 182 133
1 4 24
53 211 127
1 4 24
100 226 161
VII
OF SERUM ESTERS AFTER OF C14-Es~~~s Cholesterol
Saturated
ester+
~~~~~
FREE THE
(cpm/mg
AND
x
Linoleate
102)
tE$;
Cholesterol-4-C”
palmitate
1554
-2
-b
52 111
56 182
-b
841 35 237 108 Cholesterol-4-04 4645 68 2515 57 378 Cholesterol-4-Cl4 -b
-b
-b
46 56 54 58 Cholesterol-4.C*$ m-b 180 75
d 194 75
oleate d 142 82 linoleate
-b 128 111
4197 781 1062 253 254 214 arachidonate _6 239 117
2865 1985 366
a Each value represents analysis of a pool of liver or serum from 5 animals. b U4-activity too low for reliable specific activity.
activity relationships of the liver cholesterol esters showed that there was a more rapid appearance and disappearance of the linoleate and arachidonate esters than the palmitate and oleate esters. There was also a more rapid rate of appearance of CY4-activity in the arachidonate ester of the palmitate, oleate, and linoleate groups than the remaining esters. Of particular note is the handling of the injected linoleate ester which was more rapidly converted to arachidonate than the other injected cholesterol esters. Also, the specific activity of the arachidonate ester of that group exceeded the liver free cholesterol at all time periods. The specific activities of the serum cholesterol esters (Table VII) at 1 and 4 hours reflected the cholesterol ester injected; the ester injected had t,he highest specific activity. The serum arachidonate ester showed the greatest increase in specific activity with time in the palmitate-, oleate-, and linoleateinjected groups. The rate of appearance of
143
SWELL
AND
Cl4-act,ivit’y in the arachidonate est’er was highest for the linoleatc-injected ester. Also, the strum arachidonate ester of that group exceeded t#he specific activit,y of the fret cholesterol by almost 15 times at one hour.
LAW
The above findings on the turnover and site of synthesis of serum and liver cholesterol esters are not, in complete agreement with those obtained by several other invest,igators (8, 10). Goodman and Shiratori found that t’hc monounsaturated esters t,urnover more DISCUSSION rapidly in rat, plasma and liver. Those auAt least four enzymic systems for the forbhors concluded that the liver synthesizes mation of cholesterol esters may exist in the bulk of the plasma cholesterol esters and blood and tissues (1, 5, 7, 11). These are (a) t’hat the esters are synthesized by the ATP, the cholest’erol esterase system of the panCoA-dependent, enzyme system which favors creas and intestine; (b) t,he ATE’ and CoAthe formation of monounsat,urat#ed (Aholesdependent system in liver microsomes, and terol est,ers. The findings of Sugano and adrenal gland; (c) the free cholesterol-leciPortman (10) suggest that the serum t,ransthin transferase sysOem in blood; and (d) the ferase enzyme synthesizes a major portion cholest#erol ester-fat’ty acid donor tranxferase of t’he blood cholest’erol esters; this enzyme system in liver. Each of these enzymic sysfavors the format,ion of polyunsaturat,ed t,ems is specific for the formation of certain cholesterol esters. After the injection of t’ypes of cholesterol esters. The relative con- cholesterol-4-C’* labeled lipoproteins, the tribution of each of these cholest’erol ester blood polyunsaturat,ed esters had a higher synt,hesizing systems to the blood and tissue specific activity than those estersin the liver. cholest,erol ester pools is not’ 1mow-n. How The differences bet.ween the results of the ever, the c*omposit,ionand concentrat,ion of several studies may be due in part to the cholesterol est,erx in the blood and tissue nut,ribional state and handling of the animals. would bc det.ern1inec-lby t.he contribut,ion of It has been shown (18) that fast.ing increases cholest.erol esters from t,he (a) intestine; (b) the proportion of arachidonic acid in the the upt,ake, hydrolysis, transesterifications, serum cholesterol cst,crs. Previous diet might. and synt)hesis of cholesterol esters by the also be a factor. Liver and plasma cholest.erol liver and perhaps ot,her t#issues;and (c) the ester metabolism in the rat have also been incorporat.ion of liver cholesterol est,ersinto shown to be highly sensitive to stress (19). lipoproteins. The results of the present study It’ has been observed in the rat that the have provided some addit,ional information fatty acid composition of t-he serum choleson someof t,he above factors involved in the terol esters is vast’ly different from that of metabolism of t’he different cholesterol esters. liver cholesterol esters (5). This raises the The data obtained after the injection of question of whet’her t,he cholesterol esters of cholesteroMC’* suggest that t’he polyunthose tissues might be formed by different saturat.ed cholesterol esters of t,he serum mechanisms. The differences in the distribuand liver t’urnover more rapidly than the tion of C%ct,iviby in Dhe serum and liver saturat.ed and monounsaturated cholesterol cholesterol esters after the inject,ion of choesters. Also, the specific activity relation- lesterol-4-C14 indicates that the serum and ships suggest that a major portion of the liver ester pools do not, come into rapid serum cholesterol esters are derived from the equilibrium. This and the other data reliver in the postabsorptive rat. Several pos- ported in the present study subst’antiate the sible explanat.ions can be given for the higher view that t,he polyunsaturat,ed esters are turnover rate of t’he polyunsaturated choles- selectively released from t,he liver into the terol est’ers.A major portion of the polyun- blood. Gidez, and Roheim and co-workers sat,urated fatty acids may be transport’ed (9,20) have also present,edevidence for selecafrom the blood t,o the t’issuesesterified with t,ive releaseof cholesberolesters by Dheliver. cholesterol. Second, a recent observation (17) The higher density lipoprotein cholesterol indicates that’ there is a pathway involving esters were found to have a greater proporthe l~eferential conversion of cholesterol tion of polyunsaturated esters t,han the liver arachidonat,e to highly polar lipids in liver. cholesterol esters. On the ot.her hand, the low
LABELING
OF
CHOLESTEROL
densit,y lipoprotein cholesterol esters are similar in fat.ty acid composition to the liver cholesterol esters. Since cholesterol est,ers of the various lipoproteins do not exchange it was not possible to prepare lipoproteins labeled with a particular cholesterol est.er. Therefore, the data obtained at. t,he early t’imes after the injection of the individual esters emulsified in serum would probably not be indicat.ive of cndogenous cholesterol ester metabolism. However, the dat,a are of significance from a comparative standpoint, and at later Cmes, when adequate mixing of the injected esters into the ester pools probably had occurred. The findings w&h the inject’ed U4-esters support t,he results obtained with injected free cholesterol-4-U4. The injected linoleate and arachidonnte esters disappeared more rapidly from the liver than the palmitate and oleate esters. One of the most interest.ing features of the data relates to the possible preferential conversion of cholesterol linoleate to cholesterol arachidonate. There was a much greater formation of arachidonat’e from linoleate than when t,he other est.ers mere inject.ed. Xso, t,hc finding of a much higher specific :&vity of the liver arachidonate est,cr than the free cholesterol aft’er the injection of the linolcate ester suggesk that some of t,he arachidonate ester did not arise directly from the free fraction. This has been previously observed (5) in a related study. A possible mechanism for this reaction might be the int,eraction of a liver cholesterol ester such as cholesterol linoleate with an arachidonic: acid donor to form cholesterol arachidonat,e. This would be analogous to the t,ransferase reaction in blood (11)) but. would involve a cholesterol est,er as t,he substrate instead of free cholest.erol.
149
ESTERS REFERENCES
1. S\VELL, FIELD,
L., T~wwr, H.,
JR.,
AND
E.
C., JR., TREAD~ELL,
HOPPER,
J. R.,
C. R., Ann.
N. Y. Acad. sci. 72, 813 (1959). 2. LINDSEY, C. A., JR., AND WILSON, J. D., J. Lipid Res. 6, 173 (1965). 3. GOODMAN, D. S., J. Clin. Invest. 41, 1886 (1962). 4. Losso\v, W. J., BWT, N., AND CHAIKOFF, I. L., J. Lipid Res. 3, 207 (1962). L., L.lw, M. D., SND TREADM-ELL, C. 5. SLVELL, R., Arch. Hiochem. Biophys. 105, 541 (1964). D. S., DEYKIN, D., AND SHIR~TORI, 6. GOODMAN, T., J. Bid. Chem. 239, 1335 (1964). L., Law, M. D., BND TREAD\\-ELL, C. 7. SWELL, Il., Brch. Biochem. Biophys. 104, 128 (1964). 8. GOODMAN, 1). S., AND SHIRATORI, T., J. Lipid Res. 5, 578 (1964). 9. ROHEIM, P. S., HAFT, D. E., GIDEZ, L. I., WHITE, A., AND EDER, H., J. Clin. Invest. 42, 1277 (1963). 10. SUGANO, M., AND PORTMaN, 0. W., Arch. Biochem. Biophys. 109, 302 (1965). 11. GLOMSET, J. A., I’.~RKER, F., TJ‘IDEN, RI., AND WILLUMH, I<. H., Biochim. Biophys. dcta 58, 398 (1962). 12. MORRIS, L. J., J. Lipid Res. 4, 357 (1963). L., FIELL’, H., JR., SCHOOLS, P. E., 13. SWELL, JIM., 2i~~ TI