The role of the liver in the uptake of plasma and chyle triglycerides in the rat

BIOCHIMICA ET BIOPBYSICA ACTA BBA

55271

THE

ROLE

OF THE

TRIGLYCERIDES

MICHAEL

LIVER

IN THE

C. SCHOTZ*,

4%

IN THE

UPTAKE

OF PLASMA

AND CHYLE

RAT

I30 ARNESJd

AND

Radioisotope Research, Veterans Administration Center. LLS Angeles, Calif. (U.S.A.) me&s of Physiological Chemistry and Surgery, University of Lund, Lund (Sweden)

and Depaut-

(Received March jr-d, 1966) (Revised manuscript received June zznd, 1966)

SUMMARY

I. Recently it has been proposed that chylomicron triglyceride cannot be metabolized directly by the liver. In order to test this proposal, two experiments were performed. Chyle and serum lipoproteins, respectively, labeled with [3H]palmitic acid in the glyceride moieties, were injected into unanesthetized fasted and carbohydraterefed rats. It was assumed that liver phospholipids would be mainly labeled only through the incorporation of fatty acids liberated upon hydrolysis of the labeled chyle or serum lipoprotein glyceride. The liver phospholipid radioactivity thus gave a measure of the incorporation of liberated fatty acids into the liver lipid esters. This was compared with the calculated flux of label through the plasma free fatty acid pool into the liver. It was found that this flux was insufficient to explain the entire incorporation of label into the liver lipid esters except perhaps in the fasted group of rats injected with chyle. This indicates that some of the chyle or serum lipo-protein triglyceride was hydrolyzed in the liver. 2. Labeled serum lipoprotein triglyceride initially disappeared from the circulation faster than labeled chyle triglyceride. With the serum lipoprotein triglyceride, but not with the chyle triglyceride the initial rate of disappearance was more rapid in fasted than in carbohydrate-refed rats. 3, No major differences were found between anesthetized and unanesthetized rats with regard to rate of removal of chyle label or uptake of this labeled material by the liver.

INTRODUCTION

The disappearance of chyle triglyceride from the circulating blood has been extensively studied in recent years. The experimental evidence supports the view I_____ * Permanent address: Calif. (U.S.A.)

Radioisotope

Research,

Veterans

Administration

Riochim. Biophys.

Center, Los Angeles,

Acta, 125 (1966) 485-495

M. C. SCHOTZ,B. ARNESJB, T. OLIVECRONA

486

that in adipose tissue hydrolysis precedes the uptake of the triglyceride fatty acids into this tissue’. It has been suggested that lipoprotein lipase is involved in this hydrolysis213. In the liver, however, it seems that the triglycerides can be taken up without previous hydrolysis *. Although anatomical details of this process in the liver are not clear, some investigators favor the suggestion that these triglycerides do not penetrate into the liver cells, but are “trapped” in extracellular spaces within the livers. FELTS AND MAYES~ recently proposed that although the liver can “trap”, it cannot directly utilize chylomicron triglyceride. This proposal was based upon experiments in vitro on chylomicron uptake and metabolism in perfused livers obtained from fasted rats. According to this view chylomicron triglyceride utilization would depend on transfer to extrahepatic tissues where hydrolysis occurs. BELFRAGE* has tested this proposal in vivo in carbohydrate-fed rats and concluded that transfer via the plasma free fatty acid compartment alone could not account for the incorporation of injected chyle triglyceride label into the hepatic lipid esters but that hydrolysis of the “trapped” triglyceride must have occurred in the liver. Comparison of the results of FELTS AND MAYES~ and of BELFRAGE* is difficult since the experiments in vitro and in vivo were performed on animals in different nutritional states. In the present study we have extended BELFRAGE’S experiments to unanesthetized fasted and carbohydrate-refed rats. In addition, we have investigated the role of the liver in the uptake of endogenously labeled serum glycerides. MATERIALAND METHODS Animals

Male Sprague-Dawley rats (AB Anticimex, Stockholm) were allowed free access to tap water but no food for 24 h. The animals designated fasted were continued on this regimen overnight, whereas those designated carbohydrate-refed were given free access to ZO~/~ glucose (w/v) dissolved in 0.45 o/oNaCl (w/v) overnight. Isotopes

[9,ro-3H,]Palmitic acid (51 mC/mmole, Batch 3) and [I-%]palmitic acid (31.2 mC/mmole, Batch 28) obtained from the Radiochemical Centre, Amersham, England were purified by reverse-phase chromatography 8 and alkaline extraction 7. Preparation

of the labeled

injection

material

Approx. I mC of [9,10 3Hz]palmitic acid was mixed with 2 ml of unlabeled fat emulsion** and administeredvia a stomach fistula to a rat cannulated in the cisterna chyli. The labeled chyle was collected within the next IO h, then filtered and used within 6 h. The chyle was collected and stored at 37’. The labeled endogenous serum triglycerides were prepared by injecting into the tail vein of 6 ad lib. fed rats 3 mC of [9,10 3Hz]palmitic acid complexed to rat serum*. The rats were ether anesthetized 25 min later and blood was collected by severing the aorta. The blood was allowed to coagulate and serum was obtained by centrifuga* P. BELFRAGE,

unplublished observations. ** IntralipidR (AB Vitrum, Stockholm, Sweden) containing zo O,Osoya-beanoil(w/v), (w/v) and 2.5Ojo glycerol (w/v). Biochim.

Biophys.

Acta,

125 (1966) 485-495

1.2 o/o lecithin

LIVER UPTAKE

487

OF CHYLE AND BLOOD TRIGLYCERIDES

tion. 0.2 ml of [r-14C]palmitic acid complexed to rat serum containing 0.05 ,uC was added to 13 ml of *H-labeled serum and the doubly labeled mixture was then stored at 37O. This material was used within 5 h after its preparation. Injections and sanzpling Both the labeled chyle and the endogenously labeled serum were injected into the tail vein of unanesthetized, fasted and carbohydrate-refed rats. Two groups, each containing 3 fasted rats were anesthetized before injection of labeled chyle. One of these groups was m~ntained under ether anesthesia and the other under intraperitoneal pentobarbital anesthesia (about 60 mg/kg body weight) for j and 20 min, respectively, before injection of labeled chyle, and continued to the end of the experimental time., At varying intervals (see tables) after administration of the labeled materials the animals were decapitated and blood was collected into weighed flasks containing chloroform-methanol (z : I, v/v). After severing the portal vein the livers were perfused free of blood i?z situ by infusing 20 ml of warm 0.9% NaCl (w/v) into the inferior vesa cava, cranial to the entrance of the hepatic vein. The livers were then excised, rinsed, blotted, homogenized, and extracted with c~orofo~-methanol (2: I, v/v). The extracts were washed and the phospholipids separated from the nonphospholipids on silicic acid columns $. Aliquots for radioactivity assay were dried in vials. The whole blood lipid extracts were filtered, washed, dried and quantitatively streaked on a thin-layer plate of silicic acid 9. The plates were developed and then exposed to iodine to visualize the lipid fractions. The free fatty acid and the triglyceride fractions were then quantitatively scraped into vials. The radioactivity was determined in a Packard liquid scintilfation spectrometer. Internal standards were added to all samples to correct for quenching. RESULTS

Table I shows the distribution of radioactivity in the injected chyle and the endogenously labeled serum of the two experiments. The data show that in the case of the endogenously labeled serum about 70% of the %I radioactivity was in the triglyceride fraction and 2oOj in the free fatty acid fraction. In the case of chyle 90 y0 of the radioactivity was in the triglyceride fraction. The added 1% radioactivity in the endogenously labeled serum was exclusively in the free fatty acid fraction as determined by thin-layer chromatography. The injected dose of chyle and endogenously labeled serum contained 6 mg and I mg triglyceride, respectively. Since the endogenously labeled serum contained appreciable amounts of aHlabeled free fatty acid which would contribute to the tissue radioactivity measured in subsequent analysis, it was necessary to “correct” the distribution of radioactivity in the serum injected and the amount found in the tissue lipid fractions. The latter correction was needed in order to obtain the uptake of radioactivity from the injected triglyceride fraction. The correction of the injected dose consisted of subtraction of the contribution of the free fatty acid label and recalculation of the percent distribution of the label in the other fractions (Table I). The correction for the tissue uptake was based on the amount of i14C]palmitic acid label found in each individual tissue fraction after injection of the doubly labeled serum (Table II). We calculated the SH content in that fraction that could have originated from the SHBiOChi?%

BdOphyS.

A&&,

I%?5

(1966) 485-495

M. C. SCBOTZ, W. AKNESJii, T. OLlVECKONA

488 TABLE

I

DISTRIBUTION OF CNROMATOGRAPHY

RADIOACTIVITY

IN TNE

INJECTED

MATERIAL

DETERMINED

BY

THIN-LAYER

The values shown in each fraction are expressed as percent of the total radioactivity. represents the mean of three determinations.

.~~_-.-.-

.-..

._

FY&iUZt

cf@e

serwm

fn?e fiiqt ~-x&L acid subtraction I-~-

fvee j&y mid subhactima _...__~~ _._..__--.

Befbre

Cholesterol esters Triglycerides Free fatty acids Uiglycerides Phospholipids -i_ Monoglycerides

TABLE

Each value

.. ._.-1.1

2.2

9r.o 2.6 I.0

66.6

4-3

4.8

_

2.8

84.9

21.5

--

4.8

6.1 6.1 . ___ _..--

--._-__-

.__-

II

INCORPORATION INJECTED WITH

OFRADIoACTIVITY '%LABELED FREE

INTO LIVER AND BLOOD COMPLEXED

PATTYACID

LIPIDS OF FASTED TO RAT SERUM

AND

CARBOHYDRATE-REFED

RATS

Donor rats were injected with [W]palmitic acid and 25 min later the blood was collected. Strum from this blood containing SH-labeled triglycerides and free fatty acids was mixed with W!palmitic acid complexed to rat serum. The mixture was then injected intravenously into unanesthetized rats. The blood weight was assumed to be 7.5 y0 of the mean body weight of the carbohydrate-refed and the fasted rats, 229 g and 208 g, respec.tiveIy. _..-_ -__._ ---._ _..yz qf i%j&ed ~~~-~~~~~~dfmz fat& mid ~~d~~~~~~v~t~i fiat No. Min after T~~~t~~~t -injectiatl Liiler 3Eood ---

--_I Carbohydrate-refed

Fasted.

Non-phospholipids __-... .._-_ ___12.4

Phosph;: lipids

_--_

Tn:&cerides

I”vcc:futty acids -- . ..--- -

T

2

2 3

2 2

12.8 12.1

10.2 IO.2 II.3

4 c

6 6

12.9 15~0

10.7 ro.3

0.04 Cl.04

0.32

;

6

12.0

IO.7

0.05

o.i9

;

2 2

6.2 8.1

8.8 7.4

0.01 -.

3.10 --

0.07 0.03 o.oh

I.10 0.78

I.44 or4

9

2

4.5

8.9

0.02

2.06

IO

6 6 6

6.8 6.0 8.0

8.6 7.2 7.5

0.02 0.01

0.44 0.37

0.02

0.56

IX 12

.._

~-

labeled free fatty acid fraction if this behaved identically to the 1*4G]palmitic acid. This amount was then subtracted from the total 3H radioactivity measured in that fraction. Finally, all data were expressed as percent of the injected 3H radioactivity in all fractions except the free fatty acids. In the chyle experiment approx. ~5% of the injected radioactivity was found in the liver lipids after 2 min and this increased to about 30% within 6 min in the carbohydrate-refed rats. A similar amount of radioactivity was also found after 12 and 20 min (Tabte III), chromatography of the total liver lipid extracts of these animals indicated that the radioactivity was mainly in the non-phospholipid fraction. However, small amounts were found in the phospholipid fraction z min after injection of the Biochim.

Biophys.

Acta,

~25 (1966) &3y-495

LIVER UPTAKE OF CHYLE AND BLOOD TRIGLYCERIDES TABLE

489

III

~~~~~~~~~=~~~

OF RADIOACTlVlTY

INTO

LIVER

AND

BLOOD LIPIDS OF FASTED AND CARBOHYDRATE-REFEDRATS

INJECTEDWITH *H-LABELED CHYLE LIPIDS Chyle was collected overnight from a donor rat fed [SH]pahnitic acid. Unanesthetized and anesthetized rats were injected with the labeled chyle and decapitated after various times. All livers were perfused with a saline solution immediately after decapitation. Carcass designates the radioactivity remaining in the rat after draining the blood and excising the liver. Recovery represents the sum of radioactivity in the blood, liver and carcass. and the fasted The blood weight was assumed to be 7.5 76 of the mean bodv weight of the carbohydrate-refed rats, 229 and 208 g, respectively. -~ ~______.. -carcass fZecovery o/O of imjected radioactivity Rat No. Miz after Livev Blood -. injectioE

Unanesthetized

carbohydrate I

2

3

2 2

4

G

2

Free fatty

Eipids

cerides

acids

I.0

1.24 1.58 1.7’

refed 17.6 15.7 IO.7

1.0

45.9 45.2 39-8

2.7 2.5 4,6

12.8

0.95

34-7 ro.4

1.27

7,3 $4.9 3.4

0.46

4.5 3.6

0.9 0.9 1.4

0.14 0.44 0.37

2

6

; 9

12 I2

18.3 15.7

IO II I2

20 10 20

1.1

I.31 1.00

1.15

19.7 15.6

6.4 5.8 6.5

8.4 II.8

0.5

‘4.7

1.6

32.3 45.9 29.2

3.5’2 3.75 3.60

16.2

2.7

18.8 23.2 27.0

I.95 3.05 2.44

3.6 5.3 4.8

7.9 2.2 2.9

I.37 0.76 I.Oj

4.9

5.2 5.1

0.7 I.2 2.3

0.21 0.42 0.38

4.7 3.5 5.0

6.6 4.5 3.8

1.51 I.67 1.04

7-3

1.50 I.69 1.05

22.3

48.5 56.2

77.9

30. I 29.3 38.7

49.2 55.4 40.9

80.5

fasted

‘3 14 =s

2 2 2

16 ‘7 I8

6 6 6

I9 20 21

12 I2 I2

22 23 24

20 20 2.0

16.4 13.6 27.3 16.4 14.2 16.6 74.7

1.1

2.1

fasted, ether 2.5 26 27

Anesthetized

Trig&-

lipids

24.5 19.4 29.3

Unanesthetized

Anesthetized

L~~o?a_phospko~-‘- PhOSphO-

I2 12 I2

18.5 12.9 16.1

fasted, pentobarbital 28

I2

29 30

I2 12

21.3

24.1 24.5

6.2 5.0 5.4

IO.1

3.5

-_

labeled chyle and this progressively increased with time to tiq/, of the injected dose at 20 min. Similar values were also found in the fasted animals. No direct measurement was made of the radioactivity washed out by the perfusion of the livers. In the endogenously labeled serum experiment 30-50 “/bof the injected dose was ~~~c~~~.Biophys.

Acta,

125 (1966) 485-495

M. C. SCHOTZ, B. ARNESJ6,

490

T. OLIVECRONA

found in the liver non-phospholipids at 2 and 6 min (Table IV). Lesser amounts of radioactivity were found in the liver phospholipids. These results are difficult to interpret because

of the scatter

of the data;

however,

there was definitely

more radio-

activity in the phospholipids in this experiment than in the chyle experiment. The individual data for the amount of radioactivity in the blood triglyceride and free fatty acid fraction are shown in Table IV. Mean values for these data are plotted in Fig. I. TABLE

IV

INCORPORATION OF RADIOACTIVITY

INTO

INJECTED

SERUM

WITH

$H-LIPID-LABELED

LIVER

AND

BLOOD

LIPIDS

OFFASTED

AND

CARBOHYDRATE-REFED

RATS

LIPOPROTEINS

Donor rats were injected with [SH]palmitic acid and 25 min later the blood was collected. Serum from this blood containing 3H-labeled triglycerides and free fatty acids was mixed with [i%]palmitic acid complexed to rat serum. The mixture was then injected intravenously into unanesthetized rats. The results are expressed as o/0 of the $H-labeled serum radioactivity after subtraction of the 3H-labeled free fatty acid contribution. Also the contribution of the $H-labeled free fatty acid to the tissue and blood lipids was corrected based on the i4C data shown in Table II. The blood weight was assumed to be 7.5 ;/, of the mean body weight of the carbohydrate-refed and the fasted rats, 229 g and 208 g, respectively. Treatment

Rat No.

Min after injection

o/0 of the injected radioactivity Blood

Liver Non-phospholipids

Carbohydrawrefed

I L 3

2 2 2

34.8 38.1 40.1

0.6 2.9 I.0

4

6 6 6

31.5

16.6

46.1

6.0

52.3

9.7

; Fasted

PhosphoLiPids

s7

2

9

2

42.7 44.7 50.5

‘3-3 6.7 8.9

6 6 6

49. r 49.4 46.5

8.6 8.3 6.1

IO

II I2

Trigl~lcerides

Free fatty acids

39.9 30.0 43.8

0.67 0.84 1.7I

I5.2 16.5 12.6

0.64 1.23 I .08

21.4

1.36

21.7

I .04

6.1

5.7 6.2

1.50 0.97 1.18

It appears that the endogenously labeled serum triglyceride radioactivity initially disappeared faster from the circulating blood than the radioactivity of injected chyle triglycerides. Since data were obtained at only two times the shape of the curve cannot be decided. We have arbitrarily connected the points with a straight line. At both times studied there was less radioactivity in the blood of the fasted rats compared with the glucose-refed rats. Recently we reported that the tissue distribution of injected labeled free fatty acid is quite different in unanesthetized compared with anesthetized ratslo. Since most of the previous experiments concerned with chylomicron metabolism were performed on anesthetized rats, we included in this investigation ether- and pentobarbital-anesthetized rats at one time interval. No major differences were observed either in the incorporation of label into the liver lipids or in the amount present in the blood lipids (Table III). Calculations The total

liver lipid

B&him.

Acta, 125 (1966) 485-495

Biophys.

radioactivity

can be due to at least three

constituents:

LIVER UPTAKE

OF CHYLE AND BLOOD TRIGLYCERIDES

491

Time (min) Fig. I. Radioactivity in the blood triglyceride fraction in fasted (- - - -) and carbohydrate-refed ) rats after intravenous injection of *H-labeled chyle (circles) or 8H-labeled serum (squares) (-triglyceride. o-0, carbohydrate-refed animals injected with chyle: O-C, carbohydraterefed animals which received endogenously labeled serum; l - - -0, fasted animals injected with chyle; n - --¤, fasted animals injected with endogenously labeled serum. See text for further details. Each value plotted is the mean of three values.

(I) residual blood (2) “trapped” and unmetabolized chyle lipids and (3) “newly synthesized” lipids formed from injected ester fatty acids. The major aim of the present investigation was to compare the incorporation of injected triglyceride radioactivity into the “newly synthesized” liver lipids with the incorporation of radioactive free fatty acids into this fraction. The following assumptions and calculations were made to obtain measures of these parameters. First, it was assumed that incorporation of radioactivity into liver phospholipids can only occur when fatty acids have been liberated from chyle esters by hydrolysis. Secondly, it was assumed that these liberated fatty acids would be distributed between the liver non-phospholipid and phospholipid fractions in the same manner as injected free fatty acid and that it would not change between 2 and 20 min. Data for this distribution were obtained from the incorporation of [Xlpalmitic acid into the liver in the endogenously labeled serum experiment (Table II). The mean values for the fasted and carbohydrate-refed rats were used. Thus, the radioactivity in newly synthesized liver lipids equals :

o/O3H in liver phospholipids

plasma

x

o/o 14C in liver total lipids o/o l*C in liver phospholipids

The influx of radioactivity into the “newly synthesized” free fatty acid compartment was calculated as follows: Biochim.

Biophys.

liver esters from the

Acta,

125 (1966) 485-495

o/o3H in plasma free fatty acids x fractional turnover rate of plasma free fatty acids x o/o *K in liver total lipids. Such calculations were made for each minute from o to 20, and the products summed. The 3H-labekd plasma free fatty acid values at each minute were obtained from a curve of the mean experimental values plotted in a coordinate system in which the data points were connected by straight Iines. The fractional turnover rate of plasma free fatty acids in unanesthetised rats was assumed to be x.5 min+z*. Since the data on liver total AK radioactivity were similar at z and 6 min after injection in the fasted and the carbohydrate-refed rats, respectively, the mean values were used, and in addition it was assumed that these values remained constant until 20 min after injection These calculations are based on the assumption that incorporation of radioactivity in the liver p~os~holj~i~ can onfy occur when the fatty acids have bee23 liberated from cbyie triglycerides by hydrolysis. However, radioactive r,z-diglycerides in the chyle could possibly be incorporated directly into the liver phospholipids. Judging from previous experiments it is probable that only half of the chyle diglycerides were of the I,Z varietvr’. Furthermore, only part of these would be expected to be taken up by the liver and of these only one-third would form phaspholipids; the rest would be converted to triglycerides ** . We therefore feel that the possible contribution to the liver Fhosp~~~i~ids from chyle diglycerides is quite Iow and therefore we have not in&ded this in our ~~~u~ation~. It is also possible that part of the labeled chyk ~hos~ho~~ids could be incerporated into the liver phospholipids without prior hydrolysis. Little data is available on the metabolism of chyle phospholipids. STEXNAND STEIN 12have recently reported a maximal uptake of x4 “/o of labeled lipoprotein phospholipids into the liver phospholipids. This factor could have been taken into account in the present calculations but would have made only slight changes and would not alter the conclusions. Since the validity of the figure of 14% chyfe ~ho~~holi~id uptake into the liver is unknown, we have decided not to use it in our cakulations, Another possibility is that chyle triglycerides, in or outside the liver cell can be partially hydrolyzed to diglycerides. The latter could then be utilized without further hydrolysis in the formation of liver phospholipids. Using doubly labeled chyle triglycerides it has been shown that only small amounts of the glycerol moiety is found in the liver phospholipid fraction suggesting that this pathway is of minor in~portance4*13.

It is well established that triglyceride removal is not accomplished by hydrolysis in the bloodi4. Also, as a working hypothesis, it is generally assumed that hydrolysis of the triglyceride moiety precedes the entry of its constituent fatty acids into adipose tissuetF3. Most investigators envisage this hydrolysis to occur at or in the ca$Iary endothelium, catalyzed by the enzyme lipoprotein lipase1+ IS. The liver, however, has been shown by several techniques to remove ~h~dro~~~ed triglyceride * M. C. SCFIQTZAND N, RAKER, unpublished okmrva2ions. ** J. ELOVSON, unpubliabed observations. Mwhim.

Riophys. Act& rzj (x966) 485-495

LIVER UPTAKE OF CWYLE AND BLOOD TRIGLYCERIDES

493

from the bloodWft+~-18. Recent evidence suggests that a large part of this triglyceride is “trapped” in the extravascular spaces in the Ever, hydrolyzed there and that the fatty acids are incorporated into liver lipid esters or oxidized to CO, (ref. 6). However, direct evidence for this hydrolysis has not been presented. On the contrary, the data of FELTS AND MAVES~,~@ have indicated that in the isolated perfused liver from rats such hydrolysis does not occur. One possible suggestion consistent with both studies is that the “trapped” triglyceride can be transported to extrahepatic tissues where hydrolysis occurs. During the clearing from blood of injected labeled triglyceride, part of the label is retransported in the plasma as free fatty acids 211.According to FELTS AxD MAYEs~~@ these would be the only fatty acids available to the liver for the incorporation into liver lipid esters or for oxidation. In the present study, we have obtained a measure of the flux of label derived from the injected triglycerides through the plasma free fatty acid compartment and of the incorporation of this label into “newly synthesized” liver lipid esters (see RESULTS). In all cases except, perhaps, the fasted rats injected with chylomicrons, the free fatty acid Aux was too small to explain the amount of label found in the “newly synthesized” liver lipid esters (Figs. z and 3). These results

Fig. 2. Per cent of the injected W-labeled chvle radioactivity in “newly synthesized” liver esters calculated (.&--A) fromliverphospholipids-is plotted ZW.time. This curve is compared with the curve of possible contribution f +- - -+) of the plasma free fatty acids to the “newly synthesized’* liver esters. “ Refed” and “fasted” represents carbohydrate-refed and fasted rats, respectively, For further details and calculations see text. Each value is based on a mean of three values. Fig. 3. Per cent of the injected endogenously *W-labeled serum radioactivity in “newly synthosized” liver esters calculated (n-a) from liver phospholipids is platted vs. time. This curve is compared with the curve of possible contribution (+- - -+) of the plasma free fatty acids to the “newly synthesized” liver esters. “Refed”’ and “fasted” represents carbohydrate-refed and fasted rats, respectively. For further details and calculations see text. Each valne is based on a mean of three values.

agree with similar data obtained by BELFRAGE* in this laboratory using doubly labeled chylomicrons in carbohydrate-fed rats. Both the latter and our results are in agreement with the hypothesis that the liver can hydrolyze the “trapped” triglyceride. * P.

BELFRAGE,

unpublished observations.

M. C. SCHOTZ,B. ARNESJij, T. OLIVECRONA

494

Our data in vivo does not correlate with the data of FELTS AND MAYES~~ obtained from perfused liver experiments. This discrepancy may be due to technical problems associated with perfusion in vitro or perhaps to the absence of extrahepatic tissues in the perfusion experiments. In the latter case passage of the chylomicron through extrahepatic tissue may be necessary to induce some change in its structure so that it can subsequently be hydrolyzed by liver enzymes. One possible change could be the attachment of lipoprotein lipase to the chylomicron surface. Thus, the chylomicron particles would carry their own enzymes with them to the liver. Another possibility is that hydrolysis of the triglyceride proceeds very actively in the splanchnit vascular bed, so that the blood entering the liver in the portal vein could have a much higher content of labeled free fatty acid than the blood of the systemic circulation, which was sampled. These possibilities will have to be investigated before any definite conclusion with regard to the direct hydrolysis of the triglycerides by the liver can be reached. Previous investigations using endogenously labeled plasma triglycerides have usually been interpreted to show that these are metabolized similarly to chylomicronszr. In the present investigation several distinct differences were noted. The endogenously labeled triglycerides were initially removed from blood faster than the labeled chylomicron triglycerides. This may have been due to a smaller amount of injected lipid. However, the endogenously labeled triglycerides showed a faster initial disappearence from the blood in the fasted than in the carbohydrate-refed rats, whereas in the chyle experiment the same disappearance rate was found in both nutritional states. Also, a considerably higher fraction of the endogenously labeled triglycerides was removed by the liver than the chylomicrons. In addition, it appears that radioactivity from the endogenously labeled triglyceride was more rapidly incorporated into the “newly synthesized” liver esters. This may also be due to the small amount of lipid in this case. The present results thus reveal distinct metabolic differences between chylomicron triglyceride and plasma lipoprotein triglyceride in the rat. ACKNOWLEDGEMENT We wish to thank Dr. P. BELFRAGE for his cooperation during the planning and execution of this investigation. We are also indebted to BARBRE GOETHE,INGRID ANDERSSON, and INGA JOHNSSON for their skillful technical assistance. This investigation was supported in part by the Swedish Medical Council and U.S. Public Health Service Research Grant 4706 from the National Institutes of Arthritis and Metabolic Diseases.

REFERENCES I R. J.

HAVEL, in

Washington

A. E.

RENOLD AND G. F. CXHILL, Handbock

D.C., 1965, p. 499.

of Physiology,

2 E. D. KORN, Colloq. Intern. Centre Nat. Rech. Sci. Paris, gg (1961) ‘39. 3 D. S. ROBINSON, Am. J. Clin. Nutr., 8 (1960) 7. 4 B. BORGSTRGM AND P. JORDSN, Acta Sot. Med. Upsalien., 64 (1959) 185. 5 J. M. FELTS AND P. A. MAYES, Nature, 206 (1965) 195. 6 J. ELOVSON, Biochim. Biophys. Acta, 84 (1964) 275. 7 R. BORGSTR~M, Acta Pkysiol. Stand., 25 (1952) III. Biochim.

Biophys.

Acta,

125 (1966)

485-495

Am. Physiol. Sot.

LIVER UPTAKE 8 G. g N.

OF CHYLE AND BLOOD TRIGLYCERIDES

495

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