Metabolism of chyle triglycerides in the liver

474 BBA

BIOCHIMICA

ET BIOPHI’SICA

ACTA

55263

METABOLISM I. STUDIES NOUSLY IN THE

OF CHYLE ON THE

INJECTED,

TRIGLYCERIDES

MEC~ANIS~~S GLYCEROL-

CARBOHYDRATE-FED

FOR AND

IN THE LIVER FATTY

LIVER

UPTAKE

OF INTRAVE-

ACID-LABELED

CHYLE

RAT

SUMMARY

I. Blood and liver radioactivity was studied after the intravenous injection of chyle, containing triglycerides labeled with [r-l*C]glycerol and ~q,~o-3H~]palmiti~ acid or [9,ro-3H,joleic acid into carbohydrate-fed male rats. 2. More than 25% of the fatty acid radioactivity in the livers IO min after the injection of chyle could be washed out by an immediate perfusion in situ with saline, A major part of this radioactive material consisted of chyle triglycerides. 3. Labeled palmitic acid liberated by the hydrolysis of chyle triglycerides was distributed in approximately equal amounts in liver endogenous non-phospholipids and phospholipids. Labeled oleic acid was found to a higher extent in the nonphospholipids. 4. It is concluded that, after the intravenous injection of labeled chyle into carbohydrate-fed rats, chyle particles are removed by the liver intact and deposited mainly in liver extracellular spaces. The chyle triglycerides are then hydrolysed and the fatty acids and, to a smaller extent, the glycerol are used for the synthesis of liver lipids. Labeled fatty acids recirculated via the blood after hydrolysis of chyle triglycerides at peripheral capillary beds contributed less than 20% to the labeled endogenous liver lipids.

INTRODUCTION

The mechanisms by which chyle lipids are removed from the circulating blood and incorporated into tissue lipids have been extensively studied in the rat during recent years in this laboratoryl-p and elsewhere 7~~.Since the liver plays a quantion this tatively important role, most of our recent work 5y6has been concentrated organ. It is generally believed that the rat liver, in contrast to adipose tissue, takes up intact chyle triglycerides 1- B, but the mechanism for this uptake is by no means completely known. B&him.

Biophys. Ada,

12s (1966) 474-484

475

METABOLISMOF CHYLE TRIGLYCERIDES IN THE LIVER

In a previous papers it was suggested that much of the intact chyle triglycerides generally found in the livers of rats at short times after the injection of chyle might not be located inside the liver cells but may be trapped extracellularly, e.g. in the vascular spaces and the spaces of Disse in the liver. It became clear to us that the data on liver radioactivity in experiments with injected labeled chyle showed the existence of two radioactive pools: (I) a pool of intact chyle lipids mainly located extracellularly, and (z) a pool of endogenous liver lipids containing labeled fatty acids derived from the hydrolysis of chyle triglycerides. The data of GREEN AND WEBB~ which showed that chyle triglycerides could be hydrolysed in vitro by liver parenchymal cells and that the hydrolysis occurred at the cell plasma membranes were in agreement with the proposed mechanism for the hepatic uptake of chyle particles stated above. This proposed mechanism was used in the calculations of the data from another and the results of that experiment seemed to experiment from this laboratory6 substantiate this hypothesis further. The present paper presents the results of a series of experiments which were undertaken further to elucidate the mechanisms for the liver uptake of injected chyle. In one experiment the chyle contained triglycerides labeled with [Klglycerol and [3H]palmitic acid (hereafter called “palmitic acid” experiment). In the other experiments in this study the chyle used for injections contained triglycerides labeled with [%]glycerol and [3H]oleic acid (hereafter called “oleic acid” experiments). MATERIALSAND METHODS Prefiaration

of’ labeled chyle

All labeled compounds were obtained from the Radiochemical Centre, Amersham, England. The labeled fatty acids were purified by preparative thin-layer chromatography before use. The [3H]oleic acid was tested by fractionation of its methyl esters on an AgNO,-50, column as described by ELOVSON~O.More than 99% of the radioactivity was found in the monoene fraction. The [14C]glycerol was used without further purification. The doubly labeled triglycerides were synthesized from [Xlglycerol and unlabeled oleyl chloride with traces of [3H]palmityl or [3H]oleyl chloride, essentially according to BORGSTR~~MAND JORDAN’. The labeled fatty acid chloride was synthesized as reported previously from this department”. The [3H]palmitic acid-labeled chyle was obtained as previously reported5. The i3H]oleic acid-labeled chyle was obtained in a similar manner but from a donor rat in which a thoracic duct cannulation via the jugular approachI had been performed. The chyle was collected during the night at room temperature and all experiments were performed during the following day. The chyle contained 18.0 mg of total lipid (1.0 mg of phospholipids) and 1.8 ,uC 14C and 26.2 ,uC 3H per ml. In all experiments undiluted chyle in doses of 0.5 or 1.0 ml was administered to the experimental animals. Treatment of animals,

injection and sampling

Male Sprague-Dawley rats (AB Anticimex, Sweden), weighing approx. 220 g and reared on a standard pellet diet, were used in all experiments. At about 5 p.m. Biochim. Biophys.

Acta,

125 (1966) 474-484

476

P.%ELFRAGE

the day before the experiment they were given ~0% glucose (w/v) dissolved in 0.45% (w/v) NaCl ad Z’b z i twn for the night. Approx. 2 h before the injection of the chyle the rats were given, by intubation, 5 ml of this glucose solution and then allowed free access to the glucose solution during the entire experimental period. Each animal drank approx. 4o ml of the glucose solution during the night. Under light ether anesthesia an appropriate volume of the fabeled chyle was injected into the exposed right jugular vein. The injection time was 15 sec. The sampling procedures in the “palmitic acid’ experiment have been reported previously s.The sampling procedures in the “oleic acid” experiments varied according ta the specific purpose of the different experiments and therefore the details for each of these have been presented in their respective table headings under RESULTS.

The perfusion of the liver was shown to be an important step and therefore deserves some further description. The rats were always partially exsanguinated from the abdominal aorta for a period of about I: min. If the livers were to be perfused, a camlula was then inserted into the portal vein immediately prior to where the vein entered the liver. The inferior vena cava was then cut between the liver and the diaphragm and the portal vein was clamped wit,h a hemostat immediately distal to the insertion point of the needle. Usually IO ml of 0.9% NaCl (w/v) at 37” was then injected over a period of 1-1.5 min. The erythrocytes in the liver seemed to be flushed out with only 3 or 4 ml of the perfusion fluid. It appeared important to start the perfusion procedure as fast as possible after the exsanguination as judged by the disappearance of the erythrocytes from the liver. In a few cases the liver was rapidly cut out before the start of the perfusion and transferred into a filter funnel plugged with a small piece of gauze. The perfusate was then collected fractionally in graduated test tubes. In these cases larger volumes of perfusion fluid were injected. The whole procedure from the insertion of the cannula to the end of the perfusion generally lasted 4-5 min. Rowever, considerable difhculties were encountered in performing this procedure rapidly enough to obtain an efficient wash-out, as judged by the rate of disappearance of the erythrocytes from the liver. Therefore it seems reasonable to assume that the direct perfusion of the livers ilz situ removed more radioactive material than was the case with the second procedure.

ofZifd samples E~~~~~~~~~. The excised livers or liversamples in ah experiments were rinsed in water, blotted dry, weighed and homogenized in 150 ml of chloroform-methanol (z : z, v/v). The tissue extracts were left for at least 6 h at room temperature before being filtered. Blood samples were weighed and extracted with 20 volumes of the same solvent. Samples of perfusate from the perfusions of livers were extracted in the same manner as the blood samples. The chloroform-methanol extracts were equilibrated against a salt solution and the chloroform phase was dried with anhydrous sodium sulphate to obtain a primary chloroform extract as previously described*. ~~~~~~~~~~~s 0% siticic a& ~~~~~~~~s. The separation procedure in the “palmitic acid” experiment has been reported previously 6* In the “oleic acid” experiments the lipids were generally separated into non-phospholipids and phospholipids with 1.5% (v/v) methanol in chloroform and methanol, respectively, as previously described13. Adysis

B&whim.

Biophys.

Acta,

125 (1~66) 474-484

IMETABOLISM OF CHYLE TRIGLYCERIDES

IN THE LIVER

477

The separations on the silicic acid columns were always checked by thin-layer chromatography of the eluted fractions. Silicic acid thin-layer chromatography. Methods described previously5y6 were used. All l*C radioactivity values in the experiments have been corrected for l*Cdata thus represents labeled fatty acids as previously described 4. l*C radioactivity only [14C]glycerol. Assay of radioactivity. Radioactivity was measured in a Packard liquid scintillation spectrometer Model 3003 in a dioxane-scintillation solution. Quenching was corrected with internal standards and 14C and 3H radioactivity was calculated as disintegrations per min. RESULTS

Radioactivity values are presented as percentages of injected l*C and 3H radioactivity unless otherwise stated. l*C values represent [14C]glycerol, i.e. they have been corrected for l*C radioactivity incorporated into fatty acids. Blood data have been calculated as previously described *. Composition of the labeled chyle. Distribution of label and ratios 14C/3H in different lipid fractions are presented in Table I. More extensive data on the chyle used in the “palmitic acid” experiment have been reported previously (ref. 3; Table I). TABLE

I

DISTRIBUTION ACID

COLUMN

OF RADIOACTIVITY AND

THIN-LAYER

AND

RATIOS

IN

CHYLE

LIPID

FRACTIONS

SEPARATED

BY

SILICIC

CHROMATOGRAPHY

The ratio 1aC/3H in the total chyle lipids was taken as 1.00.

“Palmitic acid” experiment Non-phospholipids Free fatty acids Phospholipids “Oleic acid” experiment Triglycerides* Free fatty acids Diglycerides Phospholipids * Including

a small amount

Percent of t&l 3H radioactivity

14C13H

97.8 1.0

I.04 -

2.2

0.78

90.8 0.3 3.2 5.9

0.99 -

of cholesterol

I.73 I.00 ester radioactivity.

Less than 4% of the 3H radioactivity was found in partial glycerides and free fatty acids in any of the chyle batches. Blood. As the main interest of this paper was the mechanisms for the uptake of chyle lipids into the liver, no data on blood values are presented. However, the data obtained indicated that intact chyle triglycerides were rapidly removed from the circulating blood as is usually found after the intravenous injection of labeled chyle into carbohydrate-fed rats. For the “palmitic acid” experiment, data on radioactivity in blood partial glycerides and free fatty acids have been published elsewhereb. Liver. In the “palmitic acid” experiment the amount of 3H radioactivity inBiochim. Biophys. Acta, 125 (1966) 474-484

478

P. BELFRAGE

creased approximately linearly with time (see Fig. I, “total lipids’“). Table II shows that the main part of the 3H label was found in the non-phospholipid fraction but increasing amounts were found in the phospholipids at later times. In the nonphospholipid fraction the l*C radioactivity and the 3H radioactivity were similar at the early times but considerably more 3EIlabel was found after IO min. In the phospholipids there was always much more VI than 1% radioactivity.

Time fminl Fig. I. Uptake and metabolism in the liver ot chyle containing triglycerides labeled with [‘*C]glycerol and [aH]palmitic acid, Livers have been perfused with NaCl solution before excision” The data are calculated as described in the text. TABLE

II

DlSTRXBUTION CONTAINING

0 OF LABEL

IN LIVER

TRIGLPCERIDES

XYDRATE-FED

MALE

TOTAL

LABELED

LIPID

WITS

EXTRhCT [‘4cj-GLYCER5L

AFTER

INTRAVENOUS

&ND

[3H]PAL3%iTIC

INJECTION ACID

OF CHYLE

INTO

CARRO-

RhTS

The livers were perfused via the portal vein with ro ml of 0.9% NaCl (w/v) at 37’ immediately before excision. 1% values are corrected for l&C incorporation into fatty acids and represent [Wlglycerol. Each value represents a single rat. Time (miw)

Percent oj injscted radioactivity ~~-. Non-phospholifiids SH ‘PC

Pxospholipids ‘4C SH

50 2-4

5.5 2.8

0.1

2.2

223

0.X

3.5 6~5 6,7 8.8 7.0 13.8 X4.4

0.X

2.8 5.7

4.8 7.7 4.3 6.5 7.2 _I__-~

0.2 O”4

0.4 0.3 o-4 o-4 0.7 I.9

0.2

0.8

0.4 0.7 0.8

4-7 4.3

0.1

-

I.8

When compared with the results of previous experiments4~@it was obvious that the total amount of %I radioactivity in the livers in the “palmitie acid” experiment was much smaller, However, in this experiment, the livers were perfused with saline Biochim.

Biophys.,Acta.

x25 (1966)

474-484

METABOLISM

OF CHYLE TRIGLYCERIDES

IN THE LIVER

479

and, as suggested previously5, this has supposedly removed much labeled material contained in the sinusoids and spaces of Disse. The “oleic acid” experiments were undertaken to study the amount and nature of the radioactive material washed out from the livers by the perfusions. In Table III TABLE

III

RADIOACTIVITY IN PERFUSED AND NON-PERFUSED LIVERS JECTION OF 0.5 ml OF CHYLE CONTAINING TRIGLYCERIDES [*H]OLEIC ACID INTO CARBOHYDRATE-FED RATS

IO min AFTER THE INTRAVENOUS LABELED WITH ['*C]CLYCEROL

INAND

The livers were perfused with IO ml of saline via the portal vein as described in METHODS. Nonperfused liverswere sampled exactly IO min after the end of the injection.For Rats 3 and 4 the livers were perfused between 9.5 and 10.5 min after the end of injection. In all cases, the rats were partially procedures. Rat

exsanguinated

from the abdominal

before

any perfusion

and sampling

_________Percent of injected vadioactivitr’

Treatment of livers

No. I 2 3 4

aorta

Not perfused Not perfused Perfused Perfused

in total lipids 1%

3H

9.3 IO.4 7.3 4.8

14.8 14.7 12.3 8.8

-_____.

data are presented showing the differences between the radioactivity in livers that have been perfused and that in livers that have not been perfused, IO min after the injection of labeled chyle. The perfused livers contained less 14C and 3H radioactivity than the unperfused ones and these results will be discussed later. Table IV shows that the radioactive material washed out from the livers of TABLE

IV

sH RADIOACTIVITY

AND RATIOS laC/SH IN PERFUSATR OBTAINED By PERFUSION 0F LIvFRs via PORTAL VEIN APPROX. IO min AFTER INTRAVENOUS INJECTION OF IO ml OF CHYLE CONTAINING TRIGLYCERIDES LABELED WITH [%]GLYCEROL AND[~H!OLEIC ACID

The ratios 14C/SH were expressed relative to the ratio 14C/3H in the injected chvle. The rats were partially exsanguinated from the abdominal aorta starting 8 min after the end of the injection and lasting approx. I min. The perfusion of the livers and collection of fractions generally lasted until approx. I2 min after the end of the injection. Volumes of the perfusate are given in the table.

Rat

Perfusate

Percent of injected radioactivity in

No.

!mU

3H

I

3.0 3.2 3.9 3.2 8.8

0.88 0.15 O.I5 0.38 0.18 0.11

Sum 26.2

1.85

4.'

3.8

O.I2

2.7 3.0 3.’ 3.3

0.02 0.40 0.67 0.25

Sum 15.9

1.46

total lipids

%pH

0.83

0.82 Biochim. Biophys. Acta, 125 (1966) 474-484

480

P.BELFRACE

rats that have been injected with labeled chyle did not appear in any regular manner in fractions of the perfusate (probably due to technical difficulties), even if the amounts of radioactivity in these fractions tended to decrease at the end of the perfusions. Unpublished experiments indicated that label could be washed out even after perfusion with 40 ml of saline. The ratio 1*C/3Hfor each individual fraction could not be measured due to the statistical inaccuracy in the counting of the ‘K values. The ratios l*CJ3H given in the table represent the pooled values for all the fractions. Dejinitions and calculations made from the data. The results of previous experiments51B had indicated that the radioactivity found in the liver lipid extracts (called “total lipids”) after the intravenous injection of labeled chyle did not represent a single radioactive pool but was composed of two components: (I) radioactivity in intact chyle esters (= entrapped chyle lipids) and (2) radioactivity in liver esters (= glycerides, phospholipids and cholesterol esters synthesized from labeled fatty acids, and to some extent glycerol, which had been derived from hydrolysis of chyle lipids). It should be noted that these definitions do not have any morphological implications. For example, intact chyle esters can be found inside liver cells, being brought there by pinocytosis and liver esters can be found extracellularly, e.g. as very low density plasma lipoproteins. In order to calculate the relative amounts of radioactivity in intact chyle esters and liver esters the following assumptions have been made: Labeled liver esters are formed from labeled fatty acids and glycerol derived from the complete hydrolysis of chyle lipids. If the total liver lipid extract has not been separated into non-phospholipids and phospholipids the total l*C values are assumed to represent intact chyle esters. As the ratio 14C/3Hin the intact chyle esters is approximately unity the 3H values in this fraction will have the same values as the 1% values. Any excess 3H radioactivity (total 3H-intact chyle ester 3H) is thought to represent liver esters. This definition does not take into consideration any [Xlglycerol that is incorporated into liver esters and thus gives slightly low values for the 3H radioactivity of this fraction. If the total liver lipid extract is separated into non-phospholipids and phospholipids, the non-phospholipid l*C values are assumed to represent intact chyle esters. The excess non-phospholipid 3H label+the phospholipid 3H label represent the liver esters. This definition takes into consideration the [14C]glycerol incorporated into the phospholipid fraction of the liver esters and thus gives truer liver ester values. However, as the [14C]glycerol incorporated into the non-phospholipid fraction of the liver esters is still not considered, this calculation also gives slightly low liver ester values and slightly high intact chyle ester values. In Fig. I the 3H radioactivity in liver total lipids (the original liver extract) has been separated into the calculated intact chyle ester and liver ester fractions and these three fractions have been plotted against time. The total lipid 3H radioactivity curve that increases approximately linearly with time can be divided into an intact chyle ester fraction that increases rapidly during the first 6 min after injection but then shows only a slight additional increase during the rest of the experimental period, and a liver ester fraction that initially rises at a low rate but after IO min is responsible for practically all the increase in the total lipid 3H radioactivity. The total lipid 3H radioactivity in Table III can be separated into an intact chyle ester fraction and a liver ester fraction, as described above. It then appears as Riochim.

Biophys.

Acta,

125 (1966)

474-484

METABOLISM

OF CHYLE TRIGLYCERIDES

IN THE LIVER

481

if the larger amount of total lipid 3H radioactivity found in the non-perfused livers could be explained by a larger amount of intact chyle ester 3H radioactivity, while the liver ester 3H radioactivity would be similar in both non-perfused and perfused livers. The mean intact chyle ester 3H radioactivity is 9.9% for Rats I and z and 6.1% for Rats 3 and 4 which would mean that more than one-third of the intact chyle esters had been washed out by the perfusion procedure. In Table IV the total lipid 3H radioactivity in the sum of the fractions of perfusate can be separated into intact chyle esters and liver esters as described above. Rat I contained 4.6% 14C and 7.6% 3H radioactivity and Rat z had 4.1($/o 1% and 6.9% 3H radioactivity in the livers after the perfusion. It can be calculated that approx. 1/k of the intact chyle esters had been washed out of the livers by perfusion. This figure is less than the figure obtained in the experiment presented in Table III even if the volume of perfusate was larger. It appears as if the perfusion of the livers in situ, immediately after the partial exsanguination, washes out the livers more efficiently, perhaps due to the rapid clotting of the erythrocytes in the liver sinusoids. The ratios %/3H for the pooled perfusate fractions were not unity as would have been expected if only intact chyle esters had been washed out. This finding is interpreted as an indication of the presence of some liver ester 3H radioactivity in the perfusate e.g. in very low density plasma lipoproteins secreted by the liver parenchymal cells into extracellular spaces. As chyle, containing triglycerides labeled with [3H]palmitic acid, and chyle, containing triglycerides labeled with [3H]oleic acid were used in comparable experiments during this investigation it was possible to study the distribution of these respective labeled fatty acids between the non-phospholipid and phospholipid fraction of the liver esters in the livers after the injection of chyle. Table V shows that [3H]oleic acid was distributed to the non-phospholipid fraction to a much higher degree than the [3H]palmitic acid. TABLE

V

DISTRIBUTION OF FATTY ACID RADIOACTIVITY BETWEEN DIFFERENT FRACTIONS OF LIVER ESTERS IO min AFTER THE INTRAVENOUS INJECTION OF[~%]GLYCEROL AND [3H]~~~~~~~~ ACID (A AND B) AND[~%]GLYCEROLAND[~H]OLEICACID (CAND D)LABELED CHYLE Values are expressed as fractions of total liver ester SH radioactivity foundinthephospholipids.

Rd A B C D

$H in phospholipidsj3H (16:o) (16:o) (18:1) (18:1)

in liver esters

0.4 0.4 0.2 0.2

DISCUSSION

Numerous studies have reported the rapid accumulation of chyle lipids in the liver after the intravenous injection of labeled chyle into experimental animals. Experiments using chyle containing glycerol- and fatty acid-labeled chyle have indicated that the liver initially contains mainly intact chyle triglycerides while at later times their constituent fatty acid moieties are found in endogenous liver lipids. There is not much information on how this transformation is achieved. Based on electron-microscopical studies it has been suggested that chylomicrons are taken up Biochim. Biophys.

Ada,

125 (1966) 474-484

BELFRAGE into liver cells by the process of pinocytosis 14-18.Thus, the intact chyle lipids found in the liver at short times after the injection would be present intracellularly, in pinocytotic vesicles. On the other hand, ROBINSONI7 has suggested that the intact chyle lipids found in the liver might not have accumulated intracellularly but may have been trapped in extracellular spaces, such as the spaces of Disse and the sinusoids. Accumulation.of chyle particles at these sites had actually been observed by several authors using histological techniques 16p1*,but no quantitative information can be obtained from these experiments. The results of the “palmitic acid” experiment reported in this paper showed considerably less liver uptake of labeled chyle than had been found in a previous experiment under similar conditionsa. The difference was tentatively attributed to the removal of intact chyle esters from extracellular spaces of the liver by the perfusion procedure used in the present experiment (cf. Fig. I this paper and Fig. I of ref. 6). The “oleic acid” experiments in this paper were performed to study further the changes introduced by the perfusion of the livers. The results confirm the conclusions from the “palmitic acid” experiment and the experiment published previously6 and indicate that a large fraction of the labeled chyle lipids in the liver after an intravenous injection of chyle are really located extracellularly and can be washed out by perfusion. It is also shown that the main part of the labeled material has a ratio 14C/3H that is similar to the ratio for the chyle lipids and higher than the ratio for the lipids remaining in the liver. During the preparation of this manuscript the experiments of FELTS~~ became known to us. His data indicate that injected chyle lipids that have been taken up by liver to a large extent can be perfused out of this organ again, showing their extracellular deposition. Thus, his data seem to be in agreement with ours on this point. However, from these findings and from perfusion experiments in vitro it is suggested by FELTS’s that the liver does not take any part in the direct metabolism of chyle lipids. He suggests that the presence of labeled lipids in the liver at short times after the injection of labeled chyle can be explained by (I) the deposition of chyle lipids in extracellular spaces in the liver, which would not represent any true uptake by the liver and which might possibly be an artifact caused by the “single-shot” injection of labeled chyle and (2) the transport of labeled fatty acids to the liver from peripheral capillary beds. This latter contribution to the labeled lipids of the liver would be derived from the lipoprotein lipase-catalyzed hydrolysis of chyle triglycerides in the capillary beds of adipose tissue with a subsequent “leak” back into the bloodstream of part of the liberated labeled fatty acids. Thus, essentially no hydrolysis of chyle triglycerides would occur in the liver. According to this hypothesis by FELTS, the labeled albumin-bound free fatty acids in the blood would be the precursors for the labeled liver esters found in the “palmitic acid” experiment of the present communication. In the latter study the free fatty acid fraction of the extracted blood lipids was isolated by thin-layer chromatography on silicic acid and further purified from triglycerides by a liquid partition procedure 5. The data obtained for this fraction have been published previously (ref. 5, Table II). A recovery of 800/Oor more of added [3H]oleic acid was obtained in test experiments. Knowing the amount of 3H in the blood free fatty acids at each time interval, the fractional turnover rate for albumin-bound palmitic acid in carbohydrate-fed Biochim.

Biophys.

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125 (1966) 474-484

METABOLISMOF CHYLE TRIGLYCERIDES IN THE LIVER

483

rats20 and the fraction of albumin-bound blood palmitic acid that is maximally taken ZO, it is possible. to calculate the maximal accum~llation of labeled liver up by the liver esters synthesized from these free fatty acids. As an example the interval 3 to 5 min is chosen. During this time the free fatty acid label of the blood has decreased from 0.16 to o.II~/~ of injected 3H. The fractional turnover rate for palmitic acid under similar conditions as in the “palmitic acid” experiment is 1.5 min-l (ref. 20). The liver during each time interval also takes up 33% of the free fatty acid that is removed from the circulation2*. The amount of free fatty acid label taken up by the liver during this time interval can then be calculated as: 0.14 - z - 1.5 * 0.33 = 0.11 where 0.14 - z represent the integrated values for the free fatty acid label, x.5 is the turnover rate and 0.33 the fraction taken up by the liver. Thus the maximum liver uptake of free fatty acid label can be calculated for each time interval and these values summed to give the cumulative curve in Fig. 2. The values for the liver ester radioactivity are the same as in Fig. I. The true values for the first time interval cannot be calculated

IO-

Time (mid

Fig. 2. Radioactivity in liver esters calculated from the liver data and calculated from the influx of labeled blood free fatty acids after the intravenous injection of chyle containing triglycerides labeled with [Wlglycerol and [3H]palmitic acid. Details of the calculations are given in the DISCUSSION.

due to the fact that the changes in the amount of free fatty acid label in the blood are not known. As shown by Fig. z the accumulated label in the liver esters derived from the free fatty acids of the blood is not sufficient to account for the actual minimum amounts of liver ester label except for the very first time intervals. Thus the results of this experiment are not compatible with the view that the labeled liver esters are mainly synthesized from albumin-bound labeled free acids fatty derived from the hydrolysis of chyle triglycerides in peripheral vascular beds as’suggested by FELTSIS. However, it should be observed that FELTS’ experiments were performed with fasting rats while in the present experiment carbohydrate-fed rats were used. The concentration of free fatty acids in the circulating blood is known to be higher in the fasting than in the carbohydrate-fed state PI, and experiments in our laboratory xausing fasting rats seem to show that in this nutritional state a large fraction of the Iabebd liver esters are formed from labeled free fatty acids in the blood after the injection of labeled chyle, as suggested by FELTS lg. Also the finding by both groups of an extracellular pool of intact chyle esters in the liver seems to show that the role of the liver in the direct metabolism of chyle triglycerides may have been overestimated in previous reports from our laboratory l-4. Biochim. Bio$kys.

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125 (x966)

474-484

484

P. BELFRAGE

ACKNOWLEDGEMENTS

Miss K. HEDMYR has given excellent technical assistance. This work was supported by grants from the Medical Faculty, University of Lund, and by United States Public Health Service, Grant No. HE-05302-04 (Metabolism). REFERENCES I B. BORGSTR~M AND P. JORDAN, Acta Sot. Med. Ups&en., 64 (1959) 185. 2 T. OLIVECRONA,J. LipidRes., 3 (1~62) 439. 3 P. BELFRAGE, B. BORGSTROM AND T. OLIVECRONA,Acta Physiol. Stand., 58 (1963)

III. 4 T.~LIVECRONA AND P. BELFRAGE,Biochim.Biophys.Acta.98 (1965) 81. 5 P. BELFRAGE. J. ELOVSON AND T. OLIVECRONA,Biochim.Biophys. Acta, 106 (1965) 45. 6 1. ELOVSON.T. OLIVECRONA AND P. BELFRAGE,~~och~rn.B~~~ys. Acta, 106(1965)34. j.H. BRAG~ONAND R. S. GORDON, J, C&z. Invest., 37 (rg58)*574. ;: B. MORRIS AND J. E. FRENCH, @art. J. ExptE. Physiol., 43 (1958) 180. 9 C. GREEN AND J. A. WEBB, Biachim. Biophys. ;4cta, 84 (1964) 404. IO J, ELOVSON,Biochim. Biophys. Acta, 106 (1~65) 291. II B. BORGSTRGW AND L. KRABISCH,J. LipidRes., 4 (1963) 357. 12 T. SALDEEN AND E. LINDER.Acta Pathol., 49 (1961) 471. .II rj T. OLIVECRONA,Acta Physik. &and., 54 (I&) ;g5. 14 R. G. MURRAY ANU S. FREEMAN, J. Lab. Cliw Med., 38 (1951) 56. 15 C. T. ASHWORTH,~. A. STEMBRIDCE AND E. SANDERS,Am.J.Physiol., rg8(1960) 1326. 16 H. F. PARKS,Anat. Record, 142 (1962) 320. I7 D. S. ROBINSON ,inH. C. MENG, Lipid Transport, Thomas. Springfield, Ill., 1964, p. 195. I8 J. E. FRENCH, in A. C. FRAZER, ~~oc~ern~cal Problems

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p. 296. xg J. M. FELTS,Ann. N. Y. Acad. Ski., 131 (1965) 24. 20 G. GBRANSSON AND T. OLIVECRONA,Acta Physiol. Stand., 62 (1964) 224. 21 V. P. DOLE, J. CEin. Invest., 35 (1956) 150. _ 22 M. SCHOTZ,B.ARNESJBAND T.~LIVBCRONA,Biochem.Biophys. Acta, 125(1966)485. Biochim. Biophys. Acta, 12.5 (x966)474-484

1963,