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Metabolism of plasma lipids in partially hepatectomized rats
BIOCHI~ICAET BIOPHYSICAACTA
259
BBA 55668
METABOLISM
OF PLASMA
LIPIDS
IN PARTIALLY
HEPATECTOMIZED
RATS
T. OLIVECROT?IAAND G. FEX Department
of Chemistry, Se&m
90187 Umed(Sweden) (Received September gth,
on Physiological
Chemistry,
Universityof Urned,
S
rg6g)
SUMMARY To study the metabolism of plasma lipids in partially hepatectomized rats, various labeled lipids or lipid precursors were injected intravenously into laparotomized or partially hepatectomized rats, and their disappearance from plasma and incorporation into liver lipids was measured. The following observations were made. I. The disappearance from the circulation of fatty acid labeled chylomicra was retarded after partial hepatectomy in the rat. 2. The uptake into the liver of radioactivity from fatty acid labeled chylomicra was decreased after partial hepatectomy. However, the decrease was not proportional to the liver weight, and at 4 h after the operation the uptake of radioactivity per gram liver was about twice that in laparotomized controls. Similar results were obtained in experiments with labeled oleic acid bound to albumin. It is suggested that the relative increase in uptake of the labeled lipids per gram liver was caused by hemodynamic alterations known to occur after partial hepatectomy. 3. Incorporation of labeled glycerol into liver lipids was increased at 4 h after partial hepatectomy. 4. When chylomicra labeled in vi&o with cholesteryl palmitate were injected, about 80% of the radioactivity was in the liver 60 min after the injection in both partially hepatectomized and laparotomized rats.
INTRODUCTION
The liver plays a central role in the metabolism of plasma lipids. About 1/3 of the plasma free fatty acid flux is probably taken up by the liverI. Many of the other plasma lipids are both secreted into and taken up from the plasma by the liverZ-6. Previouslye!’ we reported that the incorporation of labeled plasma free fatty acids into liver triglycerides is enhanced after partial hepatectomy, and we speculated that this was due to the hemodynamic alterations that occur**s after partial liver resection. Furthermore, we have shown that the secretion of triglyceride into the plasma is not decreased during the first 24 h after partial hepatectomyl”. To obtain Biochiwa. Biophys.
Acta, 202 (1970) 259-268
260
T. OLIVECRONA,G. FEX
further information on the changes in the metabolism of plasma lipids after partial hepatectomy, we have studied the fate of various labeled lipids or lipid precursors after intravenous injection into partially hepatectomized or control rats. MATERIALSAND METHODS Isotopes
[g,ro-3H,]01eic acid (The Radiochemical Centre, Amersham, England) was purified according to previously described methodsa. Glyceryl [2-3H]trioleate, [I-l%]oleic acid, [7cQH]cholesteryl palmitate and [z3H]glycerol (The Radiochemical Centre, Amersham, England) were not further purified before use. The radiochemical purity of these compounds as shown by the manufacturer’s analysis was in all cases better than 95%. Preparation
of injection solutions
[3H]Oleic acid was complexed to 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO., U.S.A.) as described by BORGSTR~~MAND OLIVECRONA~.Chylomicra labeled with fatty acid or with both fatty acid and glycerol were prepared in the following manner: About I mC of [g,Io-3H,]oleic acid or, when doubly labeled chylomicra were to be produced, about 0.5 mC [I-14C]oleic acid and 1.0 mC glyceryl [z-3H]trioleate, were evaporated to dryness with nitrogen. 50 mg of sodium taurocholate, 50 mg of unlabeled oleic acid, 50 mg of monolein and 2 ml of 0.1 M phosphate TABLE I DISTRIBUTION OF RADIOACTIVITY IN LABELEDCHYLOMICRON LIPIDS Expt. I refers to chylomicra labeled in vivo with [I-W]oleic acid and [3H]glycerol. refers to chylomicra labeled in vitro with percent of recovered radioactivity.
[3H]cholesteryl
Expt.
Cholesteryl ester Triglyceride Free fatty acid Cholesterol + diglyceride Monoglyceride + phospholipid
Expt.
I aHI’%
1%
0.9 90.7 3.6 3.8 I.0
palmitate.
I .oo I.12
3H
97.7
2.3
I.20
TABLE II FATTYACIDCOMPOSITION OF LABELEDCHYLDMICRON LIPIDS See Table I for identification of experiments. Percentages by weight. Fatty acid carbon numbw
Exfit.
1‘+:0
2.3 13.5
16:o 16:1
Expt.
12.9
4.9
18:o
3.0 26.2 56.0 0.7 0.7
4.1
56.0 ‘4.4 2.5 2.5
18:1 18:2 18:3 20:4
Biochim.
I
Biophys.
Acta,
202
(1970)
259-258
II
Expt. Values are expressed
II
IJ as
PLASMALIPIDMETABOLISM
261
buffer (pH 7.4) were added and the mixture shaken. Dispersion was completed by repeated aspirations through a fine needle. No visible fat droplets were present in the final dispersion. The mixture was administered through a gastric fistula to a thoracic duct cannulated rat. Chylomicra were collected overnight according to ONTKOAND ZILVERSMIT~~. The lymph was filtered to remove fibrin clots and the chylomicra were isolated by centrifugation through o.go/o NaCl. The chylomicra were used within 24 h of their collection. The final chylomicron suspension contained 40-80 mg lipid per ml. 2-4 mg lipid were administered to each rat. Another batch of chylomicra isolated in a similar way, was labeled ilz vitro according to QCARFORDT AND GOODMAN'~ Typical analyses of these preparations are shown in Tables I and II. Animal procedures
Male Sprague-Dawley rats (AB Anticimex, Stockholm, Sweden) weighing about 200 g were used in all experiments. Partial hepatectomy was performed 13. Control rats were subjected to laparotomy according to HIGGINSAND ANDERSON and handling of the liver. Experiments were performed at a number of times 5 min51 h after operation. In the experiments which were done within 24 h after the respective operations, the preoperative fasting period was adjusted so that total fasting periods of about 24 h were obtained in all these groups. The 51-h postoperative groups were fasted from the operation to the time of killing. design The disappearance from the circulating blood of intravenously injected labeled chylomicra was studied in separate experiments. In other experiments the tissue distribution of the various isotopically labeled substances was determined. Tissue distribution of labeled oleic acid was studied 2 min after intravenous injection, distribution of [3H]oleic acid labeled or [W]oleic acid [3H]glycerol labeled chylomicron lipids 20 min after intravenous injection, distribution of chylomicra labeled with [3H]cholesteryl palmitate 60 min after intravenous injection, and distribution of [3H]glycerol 30 min after intravenous injection.
Experimental
Sampling of tissues and extraction of lipids
At appropriate times the animals ‘were anaesthetized with ether and the labeled material injected into the femoral vein. The disappearance of labeled chylomicra from the circulating blood was studied by withdrawing several small blood samples from an exposed neck vein at various intervals up to 20 min after injection. The blood samples were immediately transferred to preweighed tubes containing 0.5 ml of 0.1 M sodium oxalate and the tubes weighed again. Usually about 5 samples were taken from each rat. The total amount of blood withdrawn was less than I ml. The blood lipids were extracted with IO ml chloroform-methanol, 2:1 (v/v). 0.4 vol. of 2% KH,PO, was added and the tubes were shaken. After separation of the phases overnight, the lower, chloroform phase was collected. In the study of the disappearance of [3H]cholesteryl palmitate labeled chylomicra, these chloroform extracts were filtered directly into counting vials. In the experiment with fatty acid labeled chylomicra, the blood lipids were separated by thin-layer chromatography. Tissue distribution of various isotopically labeled lipids was studied in the following way: At appropriate times after injection the rats were killed by exsanguinaBiochim.
Biophys.
Acta,
202 (1970) 259-268
T. OLIVECRONA,
262
tion through
the abdominal
aorta under ether anaesthesia.
G. FEX
The livers were quickly
excised, rinsed in water, blotted dry, weighed, and the lipids extracted by homogenization in 20 vol. of chloroform-methanol, 2 : I (v/v). In some experiments 2 ml of whole blood were also taken
and extracted
in the same manner
as described
for the liver.
The extracts were filtered into separatory funnels. 0.4 vol. of 2% KH,PO, and the funnels were shaken. After separation of the phases, the chloroform
was added phase was
collected. The remains of the rats were transferred to 1-1 erlenmeyer flasks containing 30 ml 30% ethanolic KOH. The flasks were warmed at 65” overnight, the volume made up to IOOO ml with 95% ethanol, and the fatty were extracted1 and assayed for radioactivity.
acids in aliquots
of the digest
Liver lipids were separated into two fractions on silicic acid columns14. Chloroform was used as eluent for the neutral lipids and methanol for the phospholipids. Blood
and chylomicron
lipids were separated
the spots were visualized
by spraying
z,5-dichlorofluorescein and eluted with ether.
in ethanol
0.2%
by thin-layer
chromatography’s,
with 1% iodine in methanol (w/v). Appropriate
spots
and
(w/v) or with
were scraped
off
To prepare methyl esters of chylomicron lipids, aliquots of the lipid extracts were evaporated to dryness under a gentle stream of nitrogen. 2 ml of 2% H,SO, in dry methanol were added, the tubes were flushed with nitrogen, stoppered, and warmed at 65” overnight. The methyl esters were extracted with light petroleum and purified on silicic acid columns as previously described’. The fatty acid methyl esters were separated by gas-liquid chromatography on a Perkin-Elmer model 81 gas chromatograph, as described in an accompanying paper’. The radioactivity of the various lipid extracts was measured model
2002
liquid scintillation
for by the channels
spectrometer.
Quenching,
in a Packard
when present, was corrected
ratio method.
RESULTS
Fig. I shows the disappearance acid labeled chylomicron triglyceride
3
6
9
3 12 Time after
6 9 12 iqectlon (mlnl
from blood of intravenously injected [3H]oleic at 4, 20 and 49 h after partial hepatectomy
3
6
9
12
Fig. I. Disappearance from blood of intravenously injected [3H]oleic acid labeled chylomicra at 4, 20 and 4g h after laparotomy or partial hepatectomy. Each curve represents the mean of values from 5 rats. The injected dose contained 3.5.106 counts/min. The half-lives & S.D. for the injected label were 4.75 + 1.06 and 2.79 & 0.79 min (I’ < 0.01); 6.64 + 1.45 and 4.37 + 0.96 min (P < 0.01) ; and 6.28 + 1.34 and 3.00 + 0.49 min (P < 0.01) for partially hepatectomized and laparotomized rats at 4, 20 and 49 h after operation. O-O, laparotomized rats; O---O, partially hepatectomized rats. Riochim.
Biophys.
Ada,
202 (1970) 259-268
PLASMA LIPID METABOLISM
01)
0
20
Time
after
40
operation
263
60
(h)
Fig. 2. Radioactivity in total liver lipids zo min after intravenous injection of [sH]- or [r4C]oleic acid labeled chylomicra and z min after intravenous injection of [sH]oleic acid complexed to albumin. Each point is the mean of values from 3 rats. The labeled chylomicra contained 3.7.106 complex contained counts/min sH or 0.6.106 counts/min 1% per dose; the fatty acid-albumin (% of injected [SH]oleic acid radioactivity in liver 29.4’ 106 counts/min per dose. O-O, lipids of partially hepatectomized rats/% of injected [3H]oleic acid radioactivity in liver lipids of laparotomized rats) x roe; /---I, (% of injected chylomicron radioactivity in liver lipids of partially hepatectomized rats/% of injected [sH]oleic acid radioactivity in liver lipids of laparotomized rats) x 100; O---O, liver wet weight after partial hepatectomy as 74 of preoperative liver weight.
or laparotomy. At each of the times studied the disappearance rate was slower in the partially hepatectomized rats. In the laparotomized control rats the half-lives for the injected intact
animal+.
label were 3-5 min, which is about the same as is usually This means that the disappearance
of chylomicron
found in
triglyceride
was
retarded by partial hepatectomy. The uptake of the labeled lipids by the liver is shown in Fig. 2. Liver radioactivity was measured 2 min after the injection in the experiments with [3H]oleic acid and 20 min after the injection in the experiments with [3H]oleic acid labeled chyle. These times were chosen so that most of the radioactivity had disappeared from the blood and were kept as short as possible to minimize redistribution. 2 min after injection of labeled fatty acid less than 4% of the radioactivity remained in the blood of laparotomized rats and less than 10% tectomized rats. 20 min after injection of labeled
in the blood of partially hepachylomicra, the corresponding
figures were less than 4% in all laparotomized groups and less than 6, 16 and 13% in the partially hepatectomized groups at 4,20 and 49 h after operation, respectively. In Fig. 2 the radioactivity in the liver of the partially hepatectomized rats is expressed as a percent of that in the liver of control rats. Thus, if the uptake of radioactivity by the liver is strictly proportional to the liver weight, this curve should coincide with that for liver weight as a percent of its preoperative value. The uptake of radioactivity by the residual liver was less in the partially hepatectomized rats than in the control rats, i.e. the values in Fig. 2 are less than IOO, but the radioactivity was not decreased in proportion to the liver weight during the first 18 h. Actually, at 4 h after the operation more than twice as much label was present per g liver in the partially hepatectomized rats than in the corresponding laparotomized group, regardless of which labeled precursor had been injected. During the second postoperative day, labeling of the liver lipids in the partially hepatectomized rats became proportional to the liver weight. Table III shows the distribution of label between liver lipid fractions 20 min after the intravenous injection of doubly labeled chylomicra at three separate times Biochim.
Biophys.
Acta,
202 (1970)
259-268
T. OLIVECRONA, G. FEX
264 TABLE
III
R*~~ci*cr~vrTv IN r_rvRR mmn cL*ssRs 20 min AFTER INTRAVRNOU~rNJRcT*ON OF cnvLOivircR* LABELED WITH [~K]OLEIC ACID AND [3H]~~~~~~~~ INTO PARTIALLY HEPATECTOMIZEDOR LAPAROTOMIZEDRATS 4, 20 AND 4g h AFTER OPERATION Values are mean f S.D. of 5 rats in each group. The injected dose contained 1% and 3.4.106 counts/min 3H. Treatment
Time after operation
Radioactivity Neutral
( yO of injected
dose pev g liver)
lipids
Phospholipids
1%
(hJ -
0.6.106 counts/min
3H
w
3H
4
Laparotomy Partial hepatectomy
0.1
I.2
5.9 f
0.6
3.9 + 0.3
0.40 0.39
& 0.06 + 0.13
0.39 & 0.12
20
Laparotomy Partial hepatectomy
I.5 + 0.2 1.8 i 0.3
0.9 4 0.2 I.1 + 0.2
0.35 _c 0.11 0.18 * 0.05
0.24 f 0.04 0.20 & 0.07
49
Laparotomy Partial hepatectomy
I.4 5 0.2 I.4 + 0.3
0.8 & 0.3 1.0 f 0.3
0.29 h 0.06 0.23 + 0.07
0.26 & 0.03 0.29 * 0.11
after operation. activity
2.0
*
At 4 h after the operation
of the liver neutral
*
0.1
0.34
both fatty acid 14C and glycerol
lipids were significantly
higher
*
0.04
3H radio-
in the partially
hepa-
tectomized rats than in the controls. At 20 and 4g h after the operation, there was no significant difference in the labeling of the neutral lipids. Radioactivity in the liver phospholipids
was similar in partially
hepatectomized
and laparotomized
rats
at all three times studied, with regard to both glycerol and fatty acid label. Table IV shows the incorporation of [3H]glycerol into liver lipids 30 min after intravenous injection at two times after the operation. The radioactivity in liver neutral lipids, expressed per g tissue, was much higher in the partially hepatectomized rats than in the corresponding controls at 4 h after the operation. At zg h the values were less than at 4 h after the operation, but there was still a significant difference between the two groups. Labeling of liver phospholipids was also higher in the partially
hepatectomized
than in the laparotomized
rats at 4 h but not at 29 h after
the operation. TABLE
1V
RADIOACTIVITY
Is
GLYCEROL INTO
LIVER LIPID FRACTIONS 30 min AFTER INTRAVENOUS INJECTION OF [3H]HEPATECTOMIZED OR LAPAROTOMIZED RATS AT 4 AND 29 h AFTER
PARTIALLY
OPERATIOX
Data from individual Time
aftev
operation
rats. The injected dose contained Treatment
(h)
8.7’106
counts/min.
Radioactivity liver) Neutral
lipids
( yO of injected
Phospholipids
4
Partial hepatectomy
I .8 2.4 2.3
0.42 0.35 0.44
4
Laparotomy
0.15 0.13
0.21 0.25
29
Partial hepatectomy
0.22 0.26 0.36
0.13 0.09 0.14
29
Laparotomy
0.09 0.07 0.06
0.17 0.16 0.17
Biochim.
Biophys.
Acta,
202
(1970)
259-268
dose per g
PLASMA
LIPID
TABLE
V
METABOLISM
IN
RADIOACTIVITY CHOLESTERYL ROTOMIZED
LIVER
PALMITATE
BLOOD
LABELED
LIPIDS
60 min
CHYLOMICRA
after opevation
rats. The injected dose contained (h)
Treatme?zt
INJECTION
HEPATECTOMIZED
OF
i3H]-
OR
LAPA-
z.6.10@
counts/min.
Radioactivity
(“/b
ofinjected dose) Ester Free
(per whole livev) Partial hepatectomy Laparotomy
4 Blood
INTRAVENOUS
PARTIALLY
Total cholesterol
-_Liver 4
AFTER
INTO
RATS
Data from individual Time
AND
2%
80 74 85 80
0.56 0.79 0.30 0.30
(per ml blood)
4
Partial hepatectomy
4
Laparotomy
0.39 0.3r
5.2 I.8
0.6 0.7
0.22
0.74
Timeafter injectionimin)
Fig. 3. Disappearance from blood of intravenously injected [3H]cholesteryl pahnitate labeled chylomicra 4 h after partial hepatectomy or laparotomy. Curves from individual rats are plotted. The injected dose contained 2.4.106 counts/mm O-O, laparotomized rats; a------0, partially hepatectomized rats.
The disappearance from the circulating blood of intravenously injected chylomicra labeled with [3HJcl~olesteryl palmitate 4 h after the operation is shown in Fig. 3. The disappearance curve consists of at least two exponentials, the first one much more rapid than the second. The slope of the second part of the curve is less for the partially hepatectomized rats. The uptake of labeled cholesteryl palmitate by the liver is shown in Table V. Total uptake of radioactivity was similar in the two groups and represented about 8074 of the injected radioactivity. However, in the partially hepatectomized rats a lower fraction of label was present as free cholesterol, indicating that the cholesteryl esters were hydrolyzed more slowly in these rats. Very little radioactivity was present in the blood 60 min after injection in either group (Table V). In the partially hepatectomized rats this radioactivity was mainly in the cholesteryl esters, whereas in the laparotomized rats most of it was in the free cholesterol fraction. DISCUSSION
A current view of the metabolism of chylomicra and other very low density lipoprotein triglycerides by the liver is that they are first “trapped” in the spaces of Disse311’. Here they are hydrolyzed by a lipase, and the liberated fatty acids Biochim. Biophys.
Acta,
202
(1970)
259-268
T. OLIVECRONA, G. FEX
266
are taken up into the liver cells where they are further metabolized”,l*,l9. At this stage their metabolism is probably indistinguishable from that of plasma free fatty acidsSO. However, FELTS AND MAYES~~ have suggested that chylomicron triglyceride fatty acids enter the liver only after hydrolysis of chylomicron triglycerides in extrahepatic tissues. During at least the first day after partial hepatectomy, the portal pressure is increased and blood flow per g liver is higher than in the normal liverapg. This means that the force that filters chylomicra
out into the spaces of Disse
increases,
in these
and an increased
“trapping”
of chylomicra
spaces
appears
a
logical consequence. The present data show that the radioactivity in the liver after injection of fatty acid labeled chylomicra is considerably higher than corresponds to the liver weight during the first 20 h after partial hepatectomy. becomes approximately proportional the above hypothesis on chylomicron
Later the uptake
to the liver weight. These results agree with metabolism. They also concur with the data
of MoRRW~, which show that the utilization of chylomicron lipids by the perfused rat liver increases with the perfusion rate. The liver is not essential for the clearing of chylomicra from the circulation, and probably a large proportion of chylomicron triglyceride is normally taken up that by extrahepatic tissues. NESTEL, HAVEL AND BEZMAN~~ have demonstrated chylomicron lipids are removed from the circulation in dogs with the liver circulation excluded and BEZMAN-TARCHER, OTWAY AND ROBINSON~~ have shown that injected labeled chylomicra disappear from the blood at similar rates in the isolated supradiaphragmatic portion of the rat and in the intact rat. Our data show that the disappearance was retarded
from the circulating in the partially
blood of the injected
hepatectomized
fatty acid labeled chylomicra
rats. This slower removal
rate can be
partially explained by the decreased total capacity of the liver to take up chylomicra. However, this may not be a sufficient explanation, and the extrahepatic uptake of chylomicron triglyceride from the circulation may also be retarded in the partially hepatectomized rats. Our data are in good agreement with those of NESTEL et aLz3 who showed that partial exclusion of the liver from the circulation leads to a retardation in theclearing
of labeled chylomicron lipids from the blood. In previous studies 3,17920various techniques have been used to evaluate the chylomicron triglyceride as presence in the liver of “unhydrolyzed” or “intact” distinguished from “hydrolyzed” chylomicron triglyceride, also called “newly synthesized”
triglyceride.
Provided
some assumptions
are made,
these
techniques
allow an approximate calculation of the amount of trapped chylomicron triglyceride present in the liver. It would be logical to apply some of these techniques to the present experimental situation. Our data from the experiment with chylomicra labeled in both the glycerol and fatty acid moiety suggest the presence of “trapped” chylomicra in the livers of the partially hepatectomized rats 4 h after operation. However, no rigorous interpretation of these data is possible, since other changes. in lipid metabolism occur after partial hepatectomy, which make such calculations. difficult. Thus, we find that the incorporation of free glycerol into liver triglyceride is increased several fold during the early stage of liver regeneration. AS previously demonstrated6,’ and extended in the present publication, the uptake of plasma free fatty acids per g liver is also increased, and the distribution of both labeled percursors. between liver lipid classes is different from that in the controls. Biochzm.
Biophys.
Acta,
202 (1970)
2.59-268
PLASMA
LIPID
METABOLISM
267
The uptake of plasma free fatty acids by the liver is considered to be governed mainly by the plasma concentration of free fatty acids and the rate of blood flow through the liverz4. Hormonal or nutritional manipulations do not influence the uptake of plasma free fatty acids by the liver a5. Blood flow per g liver tissue is increased during the early stages of liver regeneration*yg. Conditions for an increased uptake of plasma free fatty acids would thus be expected to prevail. The data suggest that this was the case during the first 18 h after operation. Actually, the relative uptakes of labeled plasma free fatty acids and of labeled chylomicron triglyceride were increased to a similar degree during the first 18 h after the partial liver resection (Fig. z), suggesting that the two changes may be caused by the same factors. Chylomicron cholesteryl ester is removed from the circulation almost exclusively by the liver4y26.The present data show that this is also true after partial hepatectomy. Thus, even when the liver parenchyma is reduced to about r/3 of its original weight, no other tissue can substitute to any great extent for the liver in the removal of chylomicron cholesteryl ester from the circulation. The exact mechanism by which the chylomicron cholesteryl esters are metabolized is not well-defined. We favor the following hypothesis which is consistent with the present data. Most of the chylomicron triglyceride is removed from the circulation by extrahepatic tissues, probably by the action of lipoprotein lipase. The residue of the lipoprotein then appears in the plasma as a higher density lipoprotein which is subsequently metabolized mainly by the liver. The first steep part of the disappearance curve would thus represent the removal of chylomicron triglyceride and the subsequent rapid removal of the lipoprotein residues by the liver, while the second slower part of the disappearance curve represents the metabolism of cholesteryl esters mixed into a liver plasma pool of cholesteryl esters. The present data show the main difference between laparotomized and partially hepatectomized rats to be a slower second part of the disappearance curve in the partially hepatectomized rats. No detailed determination was made of the first part of the slope. It is possible that the initial disappearance of radioactivity was retarded in the partially hepatectomized rats to the same degree as the disappearance of fatty acid labeled chylomicra, although this cannot be definitely stated from our data. ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Medical Research Council (B68-13x-7z7-o3A) and the Swedish Cancer Society (245-K69-01X). The skilful technical assistance of Miss K. Lindberg is gratefully acknowledged. REFERENCES
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G. G~RANSSON AND T. OLIVECRONA, Acta Physiol. Stand., 62 (1964) 224. B. BORGSTRGM AND T. OLIVECRONA, J. Lipid Res., 2 (1961) 263. M. C. SCHOTZ, B. ARNESJ~ AND T. OLIVECRONA, Biochim.Biophys. Acta, 125 (1966) 485. DEW. S. GOODMAN. I. C&n. Invest.. 41 (1962) 1886. R. W. MAHLEY, R:i. HAMILTON ANTI G.-S. REQUIRE, J. Lipid Res., IO (1969) 433. G. FEX AND T. OLIVECRONA, Biochim.Biophys. Acta. 152 (1968) 217. G. FEX, Biochim. Biophys. Acta, in the p&s. J. MENYHART AND L. SIMON, Acta Physiol. Acad. Sci. Hung., 30 (1966) 161. J. MENYHART AND L. SIMON, Acta Physiol. Acad. Sci. Hung., 30 (1966) 169. G. FEX AND T. OLIVECRONA, Biochim. Biophys. Acta, 164 (1968) 424. Biochim.
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(1965) 411. 25 M. B. FINE AND R. H. WILLIAMS, Am. J. Physiol., Igg (1960) 403. 26 S. H. QUARFORDT AKD D. S. GOODMAN, J. Lipid Res., 8 (1967) 264. B&him.
Biophys. Acta, 202 (1970) 259-268
259
BBA 55668
METABOLISM
OF PLASMA
LIPIDS
IN PARTIALLY
HEPATECTOMIZED
RATS
T. OLIVECROT?IAAND G. FEX Department
of Chemistry, Se&m
90187 Umed(Sweden) (Received September gth,
on Physiological
Chemistry,
Universityof Urned,
S
rg6g)
SUMMARY To study the metabolism of plasma lipids in partially hepatectomized rats, various labeled lipids or lipid precursors were injected intravenously into laparotomized or partially hepatectomized rats, and their disappearance from plasma and incorporation into liver lipids was measured. The following observations were made. I. The disappearance from the circulation of fatty acid labeled chylomicra was retarded after partial hepatectomy in the rat. 2. The uptake into the liver of radioactivity from fatty acid labeled chylomicra was decreased after partial hepatectomy. However, the decrease was not proportional to the liver weight, and at 4 h after the operation the uptake of radioactivity per gram liver was about twice that in laparotomized controls. Similar results were obtained in experiments with labeled oleic acid bound to albumin. It is suggested that the relative increase in uptake of the labeled lipids per gram liver was caused by hemodynamic alterations known to occur after partial hepatectomy. 3. Incorporation of labeled glycerol into liver lipids was increased at 4 h after partial hepatectomy. 4. When chylomicra labeled in vi&o with cholesteryl palmitate were injected, about 80% of the radioactivity was in the liver 60 min after the injection in both partially hepatectomized and laparotomized rats.
INTRODUCTION
The liver plays a central role in the metabolism of plasma lipids. About 1/3 of the plasma free fatty acid flux is probably taken up by the liverI. Many of the other plasma lipids are both secreted into and taken up from the plasma by the liverZ-6. Previouslye!’ we reported that the incorporation of labeled plasma free fatty acids into liver triglycerides is enhanced after partial hepatectomy, and we speculated that this was due to the hemodynamic alterations that occur**s after partial liver resection. Furthermore, we have shown that the secretion of triglyceride into the plasma is not decreased during the first 24 h after partial hepatectomyl”. To obtain Biochiwa. Biophys.
Acta, 202 (1970) 259-268
260
T. OLIVECRONA,G. FEX
further information on the changes in the metabolism of plasma lipids after partial hepatectomy, we have studied the fate of various labeled lipids or lipid precursors after intravenous injection into partially hepatectomized or control rats. MATERIALSAND METHODS Isotopes
[g,ro-3H,]01eic acid (The Radiochemical Centre, Amersham, England) was purified according to previously described methodsa. Glyceryl [2-3H]trioleate, [I-l%]oleic acid, [7cQH]cholesteryl palmitate and [z3H]glycerol (The Radiochemical Centre, Amersham, England) were not further purified before use. The radiochemical purity of these compounds as shown by the manufacturer’s analysis was in all cases better than 95%. Preparation
of injection solutions
[3H]Oleic acid was complexed to 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO., U.S.A.) as described by BORGSTR~~MAND OLIVECRONA~.Chylomicra labeled with fatty acid or with both fatty acid and glycerol were prepared in the following manner: About I mC of [g,Io-3H,]oleic acid or, when doubly labeled chylomicra were to be produced, about 0.5 mC [I-14C]oleic acid and 1.0 mC glyceryl [z-3H]trioleate, were evaporated to dryness with nitrogen. 50 mg of sodium taurocholate, 50 mg of unlabeled oleic acid, 50 mg of monolein and 2 ml of 0.1 M phosphate TABLE I DISTRIBUTION OF RADIOACTIVITY IN LABELEDCHYLOMICRON LIPIDS Expt. I refers to chylomicra labeled in vivo with [I-W]oleic acid and [3H]glycerol. refers to chylomicra labeled in vitro with percent of recovered radioactivity.
[3H]cholesteryl
Expt.
Cholesteryl ester Triglyceride Free fatty acid Cholesterol + diglyceride Monoglyceride + phospholipid
Expt.
I aHI’%
1%
0.9 90.7 3.6 3.8 I.0
palmitate.
I .oo I.12
3H
97.7
2.3
I.20
TABLE II FATTYACIDCOMPOSITION OF LABELEDCHYLDMICRON LIPIDS See Table I for identification of experiments. Percentages by weight. Fatty acid carbon numbw
Exfit.
1‘+:0
2.3 13.5
16:o 16:1
Expt.
12.9
4.9
18:o
3.0 26.2 56.0 0.7 0.7
4.1
56.0 ‘4.4 2.5 2.5
18:1 18:2 18:3 20:4
Biochim.
I
Biophys.
Acta,
202
(1970)
259-258
II
Expt. Values are expressed
II
IJ as
PLASMALIPIDMETABOLISM
261
buffer (pH 7.4) were added and the mixture shaken. Dispersion was completed by repeated aspirations through a fine needle. No visible fat droplets were present in the final dispersion. The mixture was administered through a gastric fistula to a thoracic duct cannulated rat. Chylomicra were collected overnight according to ONTKOAND ZILVERSMIT~~. The lymph was filtered to remove fibrin clots and the chylomicra were isolated by centrifugation through o.go/o NaCl. The chylomicra were used within 24 h of their collection. The final chylomicron suspension contained 40-80 mg lipid per ml. 2-4 mg lipid were administered to each rat. Another batch of chylomicra isolated in a similar way, was labeled ilz vitro according to QCARFORDT AND GOODMAN'~ Typical analyses of these preparations are shown in Tables I and II. Animal procedures
Male Sprague-Dawley rats (AB Anticimex, Stockholm, Sweden) weighing about 200 g were used in all experiments. Partial hepatectomy was performed 13. Control rats were subjected to laparotomy according to HIGGINSAND ANDERSON and handling of the liver. Experiments were performed at a number of times 5 min51 h after operation. In the experiments which were done within 24 h after the respective operations, the preoperative fasting period was adjusted so that total fasting periods of about 24 h were obtained in all these groups. The 51-h postoperative groups were fasted from the operation to the time of killing. design The disappearance from the circulating blood of intravenously injected labeled chylomicra was studied in separate experiments. In other experiments the tissue distribution of the various isotopically labeled substances was determined. Tissue distribution of labeled oleic acid was studied 2 min after intravenous injection, distribution of [3H]oleic acid labeled or [W]oleic acid [3H]glycerol labeled chylomicron lipids 20 min after intravenous injection, distribution of chylomicra labeled with [3H]cholesteryl palmitate 60 min after intravenous injection, and distribution of [3H]glycerol 30 min after intravenous injection.
Experimental
Sampling of tissues and extraction of lipids
At appropriate times the animals ‘were anaesthetized with ether and the labeled material injected into the femoral vein. The disappearance of labeled chylomicra from the circulating blood was studied by withdrawing several small blood samples from an exposed neck vein at various intervals up to 20 min after injection. The blood samples were immediately transferred to preweighed tubes containing 0.5 ml of 0.1 M sodium oxalate and the tubes weighed again. Usually about 5 samples were taken from each rat. The total amount of blood withdrawn was less than I ml. The blood lipids were extracted with IO ml chloroform-methanol, 2:1 (v/v). 0.4 vol. of 2% KH,PO, was added and the tubes were shaken. After separation of the phases overnight, the lower, chloroform phase was collected. In the study of the disappearance of [3H]cholesteryl palmitate labeled chylomicra, these chloroform extracts were filtered directly into counting vials. In the experiment with fatty acid labeled chylomicra, the blood lipids were separated by thin-layer chromatography. Tissue distribution of various isotopically labeled lipids was studied in the following way: At appropriate times after injection the rats were killed by exsanguinaBiochim.
Biophys.
Acta,
202 (1970) 259-268
T. OLIVECRONA,
262
tion through
the abdominal
aorta under ether anaesthesia.
G. FEX
The livers were quickly
excised, rinsed in water, blotted dry, weighed, and the lipids extracted by homogenization in 20 vol. of chloroform-methanol, 2 : I (v/v). In some experiments 2 ml of whole blood were also taken
and extracted
in the same manner
as described
for the liver.
The extracts were filtered into separatory funnels. 0.4 vol. of 2% KH,PO, and the funnels were shaken. After separation of the phases, the chloroform
was added phase was
collected. The remains of the rats were transferred to 1-1 erlenmeyer flasks containing 30 ml 30% ethanolic KOH. The flasks were warmed at 65” overnight, the volume made up to IOOO ml with 95% ethanol, and the fatty were extracted1 and assayed for radioactivity.
acids in aliquots
of the digest
Liver lipids were separated into two fractions on silicic acid columns14. Chloroform was used as eluent for the neutral lipids and methanol for the phospholipids. Blood
and chylomicron
lipids were separated
the spots were visualized
by spraying
z,5-dichlorofluorescein and eluted with ether.
in ethanol
0.2%
by thin-layer
chromatography’s,
with 1% iodine in methanol (w/v). Appropriate
spots
and
(w/v) or with
were scraped
off
To prepare methyl esters of chylomicron lipids, aliquots of the lipid extracts were evaporated to dryness under a gentle stream of nitrogen. 2 ml of 2% H,SO, in dry methanol were added, the tubes were flushed with nitrogen, stoppered, and warmed at 65” overnight. The methyl esters were extracted with light petroleum and purified on silicic acid columns as previously described’. The fatty acid methyl esters were separated by gas-liquid chromatography on a Perkin-Elmer model 81 gas chromatograph, as described in an accompanying paper’. The radioactivity of the various lipid extracts was measured model
2002
liquid scintillation
for by the channels
spectrometer.
Quenching,
in a Packard
when present, was corrected
ratio method.
RESULTS
Fig. I shows the disappearance acid labeled chylomicron triglyceride
3
6
9
3 12 Time after
6 9 12 iqectlon (mlnl
from blood of intravenously injected [3H]oleic at 4, 20 and 49 h after partial hepatectomy
3
6
9
12
Fig. I. Disappearance from blood of intravenously injected [3H]oleic acid labeled chylomicra at 4, 20 and 4g h after laparotomy or partial hepatectomy. Each curve represents the mean of values from 5 rats. The injected dose contained 3.5.106 counts/min. The half-lives & S.D. for the injected label were 4.75 + 1.06 and 2.79 & 0.79 min (I’ < 0.01); 6.64 + 1.45 and 4.37 + 0.96 min (P < 0.01) ; and 6.28 + 1.34 and 3.00 + 0.49 min (P < 0.01) for partially hepatectomized and laparotomized rats at 4, 20 and 49 h after operation. O-O, laparotomized rats; O---O, partially hepatectomized rats. Riochim.
Biophys.
Ada,
202 (1970) 259-268
PLASMA LIPID METABOLISM
01)
0
20
Time
after
40
operation
263
60
(h)
Fig. 2. Radioactivity in total liver lipids zo min after intravenous injection of [sH]- or [r4C]oleic acid labeled chylomicra and z min after intravenous injection of [sH]oleic acid complexed to albumin. Each point is the mean of values from 3 rats. The labeled chylomicra contained 3.7.106 complex contained counts/min sH or 0.6.106 counts/min 1% per dose; the fatty acid-albumin (% of injected [SH]oleic acid radioactivity in liver 29.4’ 106 counts/min per dose. O-O, lipids of partially hepatectomized rats/% of injected [3H]oleic acid radioactivity in liver lipids of laparotomized rats) x roe; /---I, (% of injected chylomicron radioactivity in liver lipids of partially hepatectomized rats/% of injected [sH]oleic acid radioactivity in liver lipids of laparotomized rats) x 100; O---O, liver wet weight after partial hepatectomy as 74 of preoperative liver weight.
or laparotomy. At each of the times studied the disappearance rate was slower in the partially hepatectomized rats. In the laparotomized control rats the half-lives for the injected intact
animal+.
label were 3-5 min, which is about the same as is usually This means that the disappearance
of chylomicron
found in
triglyceride
was
retarded by partial hepatectomy. The uptake of the labeled lipids by the liver is shown in Fig. 2. Liver radioactivity was measured 2 min after the injection in the experiments with [3H]oleic acid and 20 min after the injection in the experiments with [3H]oleic acid labeled chyle. These times were chosen so that most of the radioactivity had disappeared from the blood and were kept as short as possible to minimize redistribution. 2 min after injection of labeled fatty acid less than 4% of the radioactivity remained in the blood of laparotomized rats and less than 10% tectomized rats. 20 min after injection of labeled
in the blood of partially hepachylomicra, the corresponding
figures were less than 4% in all laparotomized groups and less than 6, 16 and 13% in the partially hepatectomized groups at 4,20 and 49 h after operation, respectively. In Fig. 2 the radioactivity in the liver of the partially hepatectomized rats is expressed as a percent of that in the liver of control rats. Thus, if the uptake of radioactivity by the liver is strictly proportional to the liver weight, this curve should coincide with that for liver weight as a percent of its preoperative value. The uptake of radioactivity by the residual liver was less in the partially hepatectomized rats than in the control rats, i.e. the values in Fig. 2 are less than IOO, but the radioactivity was not decreased in proportion to the liver weight during the first 18 h. Actually, at 4 h after the operation more than twice as much label was present per g liver in the partially hepatectomized rats than in the corresponding laparotomized group, regardless of which labeled precursor had been injected. During the second postoperative day, labeling of the liver lipids in the partially hepatectomized rats became proportional to the liver weight. Table III shows the distribution of label between liver lipid fractions 20 min after the intravenous injection of doubly labeled chylomicra at three separate times Biochim.
Biophys.
Acta,
202 (1970)
259-268
T. OLIVECRONA, G. FEX
264 TABLE
III
R*~~ci*cr~vrTv IN r_rvRR mmn cL*ssRs 20 min AFTER INTRAVRNOU~rNJRcT*ON OF cnvLOivircR* LABELED WITH [~K]OLEIC ACID AND [3H]~~~~~~~~ INTO PARTIALLY HEPATECTOMIZEDOR LAPAROTOMIZEDRATS 4, 20 AND 4g h AFTER OPERATION Values are mean f S.D. of 5 rats in each group. The injected dose contained 1% and 3.4.106 counts/min 3H. Treatment
Time after operation
Radioactivity Neutral
( yO of injected
dose pev g liver)
lipids
Phospholipids
1%
(hJ -
0.6.106 counts/min
3H
w
3H
4
Laparotomy Partial hepatectomy
0.1
I.2
5.9 f
0.6
3.9 + 0.3
0.40 0.39
& 0.06 + 0.13
0.39 & 0.12
20
Laparotomy Partial hepatectomy
I.5 + 0.2 1.8 i 0.3
0.9 4 0.2 I.1 + 0.2
0.35 _c 0.11 0.18 * 0.05
0.24 f 0.04 0.20 & 0.07
49
Laparotomy Partial hepatectomy
I.4 5 0.2 I.4 + 0.3
0.8 & 0.3 1.0 f 0.3
0.29 h 0.06 0.23 + 0.07
0.26 & 0.03 0.29 * 0.11
after operation. activity
2.0
*
At 4 h after the operation
of the liver neutral
*
0.1
0.34
both fatty acid 14C and glycerol
lipids were significantly
higher
*
0.04
3H radio-
in the partially
hepa-
tectomized rats than in the controls. At 20 and 4g h after the operation, there was no significant difference in the labeling of the neutral lipids. Radioactivity in the liver phospholipids
was similar in partially
hepatectomized
and laparotomized
rats
at all three times studied, with regard to both glycerol and fatty acid label. Table IV shows the incorporation of [3H]glycerol into liver lipids 30 min after intravenous injection at two times after the operation. The radioactivity in liver neutral lipids, expressed per g tissue, was much higher in the partially hepatectomized rats than in the corresponding controls at 4 h after the operation. At zg h the values were less than at 4 h after the operation, but there was still a significant difference between the two groups. Labeling of liver phospholipids was also higher in the partially
hepatectomized
than in the laparotomized
rats at 4 h but not at 29 h after
the operation. TABLE
1V
RADIOACTIVITY
Is
GLYCEROL INTO
LIVER LIPID FRACTIONS 30 min AFTER INTRAVENOUS INJECTION OF [3H]HEPATECTOMIZED OR LAPAROTOMIZED RATS AT 4 AND 29 h AFTER
PARTIALLY
OPERATIOX
Data from individual Time
aftev
operation
rats. The injected dose contained Treatment
(h)
8.7’106
counts/min.
Radioactivity liver) Neutral
lipids
( yO of injected
Phospholipids
4
Partial hepatectomy
I .8 2.4 2.3
0.42 0.35 0.44
4
Laparotomy
0.15 0.13
0.21 0.25
29
Partial hepatectomy
0.22 0.26 0.36
0.13 0.09 0.14
29
Laparotomy
0.09 0.07 0.06
0.17 0.16 0.17
Biochim.
Biophys.
Acta,
202
(1970)
259-268
dose per g
PLASMA
LIPID
TABLE
V
METABOLISM
IN
RADIOACTIVITY CHOLESTERYL ROTOMIZED
LIVER
PALMITATE
BLOOD
LABELED
LIPIDS
60 min
CHYLOMICRA
after opevation
rats. The injected dose contained (h)
Treatme?zt
INJECTION
HEPATECTOMIZED
OF
i3H]-
OR
LAPA-
z.6.10@
counts/min.
Radioactivity
(“/b
ofinjected dose) Ester Free
(per whole livev) Partial hepatectomy Laparotomy
4 Blood
INTRAVENOUS
PARTIALLY
Total cholesterol
-_Liver 4
AFTER
INTO
RATS
Data from individual Time
AND
2%
80 74 85 80
0.56 0.79 0.30 0.30
(per ml blood)
4
Partial hepatectomy
4
Laparotomy
0.39 0.3r
5.2 I.8
0.6 0.7
0.22
0.74
Timeafter injectionimin)
Fig. 3. Disappearance from blood of intravenously injected [3H]cholesteryl pahnitate labeled chylomicra 4 h after partial hepatectomy or laparotomy. Curves from individual rats are plotted. The injected dose contained 2.4.106 counts/mm O-O, laparotomized rats; a------0, partially hepatectomized rats.
The disappearance from the circulating blood of intravenously injected chylomicra labeled with [3HJcl~olesteryl palmitate 4 h after the operation is shown in Fig. 3. The disappearance curve consists of at least two exponentials, the first one much more rapid than the second. The slope of the second part of the curve is less for the partially hepatectomized rats. The uptake of labeled cholesteryl palmitate by the liver is shown in Table V. Total uptake of radioactivity was similar in the two groups and represented about 8074 of the injected radioactivity. However, in the partially hepatectomized rats a lower fraction of label was present as free cholesterol, indicating that the cholesteryl esters were hydrolyzed more slowly in these rats. Very little radioactivity was present in the blood 60 min after injection in either group (Table V). In the partially hepatectomized rats this radioactivity was mainly in the cholesteryl esters, whereas in the laparotomized rats most of it was in the free cholesterol fraction. DISCUSSION
A current view of the metabolism of chylomicra and other very low density lipoprotein triglycerides by the liver is that they are first “trapped” in the spaces of Disse311’. Here they are hydrolyzed by a lipase, and the liberated fatty acids Biochim. Biophys.
Acta,
202
(1970)
259-268
T. OLIVECRONA, G. FEX
266
are taken up into the liver cells where they are further metabolized”,l*,l9. At this stage their metabolism is probably indistinguishable from that of plasma free fatty acidsSO. However, FELTS AND MAYES~~ have suggested that chylomicron triglyceride fatty acids enter the liver only after hydrolysis of chylomicron triglycerides in extrahepatic tissues. During at least the first day after partial hepatectomy, the portal pressure is increased and blood flow per g liver is higher than in the normal liverapg. This means that the force that filters chylomicra
out into the spaces of Disse
increases,
in these
and an increased
“trapping”
of chylomicra
spaces
appears
a
logical consequence. The present data show that the radioactivity in the liver after injection of fatty acid labeled chylomicra is considerably higher than corresponds to the liver weight during the first 20 h after partial hepatectomy. becomes approximately proportional the above hypothesis on chylomicron
Later the uptake
to the liver weight. These results agree with metabolism. They also concur with the data
of MoRRW~, which show that the utilization of chylomicron lipids by the perfused rat liver increases with the perfusion rate. The liver is not essential for the clearing of chylomicra from the circulation, and probably a large proportion of chylomicron triglyceride is normally taken up that by extrahepatic tissues. NESTEL, HAVEL AND BEZMAN~~ have demonstrated chylomicron lipids are removed from the circulation in dogs with the liver circulation excluded and BEZMAN-TARCHER, OTWAY AND ROBINSON~~ have shown that injected labeled chylomicra disappear from the blood at similar rates in the isolated supradiaphragmatic portion of the rat and in the intact rat. Our data show that the disappearance was retarded
from the circulating in the partially
blood of the injected
hepatectomized
fatty acid labeled chylomicra
rats. This slower removal
rate can be
partially explained by the decreased total capacity of the liver to take up chylomicra. However, this may not be a sufficient explanation, and the extrahepatic uptake of chylomicron triglyceride from the circulation may also be retarded in the partially hepatectomized rats. Our data are in good agreement with those of NESTEL et aLz3 who showed that partial exclusion of the liver from the circulation leads to a retardation in theclearing
of labeled chylomicron lipids from the blood. In previous studies 3,17920various techniques have been used to evaluate the chylomicron triglyceride as presence in the liver of “unhydrolyzed” or “intact” distinguished from “hydrolyzed” chylomicron triglyceride, also called “newly synthesized”
triglyceride.
Provided
some assumptions
are made,
these
techniques
allow an approximate calculation of the amount of trapped chylomicron triglyceride present in the liver. It would be logical to apply some of these techniques to the present experimental situation. Our data from the experiment with chylomicra labeled in both the glycerol and fatty acid moiety suggest the presence of “trapped” chylomicra in the livers of the partially hepatectomized rats 4 h after operation. However, no rigorous interpretation of these data is possible, since other changes. in lipid metabolism occur after partial hepatectomy, which make such calculations. difficult. Thus, we find that the incorporation of free glycerol into liver triglyceride is increased several fold during the early stage of liver regeneration. AS previously demonstrated6,’ and extended in the present publication, the uptake of plasma free fatty acids per g liver is also increased, and the distribution of both labeled percursors. between liver lipid classes is different from that in the controls. Biochzm.
Biophys.
Acta,
202 (1970)
2.59-268
PLASMA
LIPID
METABOLISM
267
The uptake of plasma free fatty acids by the liver is considered to be governed mainly by the plasma concentration of free fatty acids and the rate of blood flow through the liverz4. Hormonal or nutritional manipulations do not influence the uptake of plasma free fatty acids by the liver a5. Blood flow per g liver tissue is increased during the early stages of liver regeneration*yg. Conditions for an increased uptake of plasma free fatty acids would thus be expected to prevail. The data suggest that this was the case during the first 18 h after operation. Actually, the relative uptakes of labeled plasma free fatty acids and of labeled chylomicron triglyceride were increased to a similar degree during the first 18 h after the partial liver resection (Fig. z), suggesting that the two changes may be caused by the same factors. Chylomicron cholesteryl ester is removed from the circulation almost exclusively by the liver4y26.The present data show that this is also true after partial hepatectomy. Thus, even when the liver parenchyma is reduced to about r/3 of its original weight, no other tissue can substitute to any great extent for the liver in the removal of chylomicron cholesteryl ester from the circulation. The exact mechanism by which the chylomicron cholesteryl esters are metabolized is not well-defined. We favor the following hypothesis which is consistent with the present data. Most of the chylomicron triglyceride is removed from the circulation by extrahepatic tissues, probably by the action of lipoprotein lipase. The residue of the lipoprotein then appears in the plasma as a higher density lipoprotein which is subsequently metabolized mainly by the liver. The first steep part of the disappearance curve would thus represent the removal of chylomicron triglyceride and the subsequent rapid removal of the lipoprotein residues by the liver, while the second slower part of the disappearance curve represents the metabolism of cholesteryl esters mixed into a liver plasma pool of cholesteryl esters. The present data show the main difference between laparotomized and partially hepatectomized rats to be a slower second part of the disappearance curve in the partially hepatectomized rats. No detailed determination was made of the first part of the slope. It is possible that the initial disappearance of radioactivity was retarded in the partially hepatectomized rats to the same degree as the disappearance of fatty acid labeled chylomicra, although this cannot be definitely stated from our data. ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Medical Research Council (B68-13x-7z7-o3A) and the Swedish Cancer Society (245-K69-01X). The skilful technical assistance of Miss K. Lindberg is gratefully acknowledged. REFERENCES
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Biophys. Acta, 202 (1970) 259-268