The metabolism of [3H]oleic acid in the partially hepatectomized rat

237

BIOCHIMICA ET BIOPHYSICA ACTA

nnA 55432

THE

METABOLISM

HEPATECTOMIZED

GORAN

FIX*

AND

OF pH]OLEIC

ACID

IN THE

THOB~AS OLIVECRONA*

De~ay~~e~t of Phys~oZogic~ Chemistry, ~~~~ers~~y of Lud, (Received

PARTIALLY

RAT

October njrd,

Luad ~S~eden~

1967)

SUMMARY

[3H]Oleic acid complexed to rat serum or 5% bovine serum albumin was injected intravenously into partially hepatectomized or laparotomized rats. The fractional turnover rates for the injected label in the two groups was calculated, labeling of liver and carcass lipids 2 min after injection were studied, and the concentrations of liver glycerides, phospholipids, and plasma free fatty acids were measured at this time. I. The fractional turnover rate of plasma free fatty acids was significantly decreased by partial hepatectomy. In spite of this the flux of plasma free fatty acids per min remained essentially unchanged. 2. Liver glyceride and plasma free fatty acid concentrations were significantly increased by partial hepatectomy while liver phospholipid concentration was unaffected. 3. More label was found in liver lipids in the partially hepatectomized group per g wet tissue, and a much higher proportion of this radioactivity was in the nonphospholipid fraction as compared to the laparotomized group. It is suggested that an increased incorporation of plasma free fatty acids into liver triglycerides is the major factor in the liver fat accumulation after partial hepatectomy.

INTRODUCTION

After partial hepatectomy in rats, the residual liver soon becomes infiltrated with fat, mainly triglycerides 1- b. The liver glyceride concentration per g wet tissue reaches maximal levels around 24 h after operation4 and then declines towards normal at 72 h pastoperatively5. The cause of this early fatty liver is not known, but it seems likely that changes in lipid transport rather than in lipid synthesis are the factors responsiblea. After partial hepatectomy a series of changes occurs in liver circulation, the result of which is an increase in blood flow per g wet tissue7-g. Since hepatic uptake of plasma free fatty acids seems to be a function of the plasma free fatty acid * Present address: Department

of Physiological

Chemistry,

University

of Ume%, IJme%, Sweden.

Biochim. Biophys. Acta, 152 (1968) 237-243

concentration

and blood flo~~O_~:~it is likely that, under conditions of plasma free fatty acids is increased.

of increa~ctl Ii\-c,r

blood flow, the liver uptake To investigate fatty

this possibility

acids in the partially

we have studied

hepatectomized

the metabolism

rat. As a suitable

tracer,

d p1~11lafret, :‘H ,oleic acid

was chosen, since this fatty acid is not converted to other fatt!, acids to anv appreciable extent in the rat”. Two experiments were performed on rats \vlrich I~acl been partially hepatectomized IS 11 before. In the first experiment we stud&l tire disappearance from the circulating blood of intravenously injected “Hjoleic acid corn--Ð plexed to 53; bovine serum albumin and in the second experiment tissue distribution of label z min after the injection. MATERIALS

tlie

England)

was

ANI) JIETHOI)S

Pre;haration of labeled iiajectiofz solutiom _g,ro-3H,:01eic acid (The Radiochemical purified

\vc studied

twice by reversed

according

phase

to BORGSTl~ijh116. It was complexedr7

(4G Serva,

Heidelberg,

Germany)

Centre,

Amersham,

cllromatograplly15 (Expt.

and by liquid-liquid partition to either 50/b bovine serum albumin

I) or rat serum (Expt.

II).

A ninaal fwoceduves Male

SpragueeDawley

rats

(AB

Anticimex,

Stockholm,

Sweden)

weighing

200-260 g were used in both experiments. Cp to the time of operation they were fed an ordinary laboratory diet and water ad libitwm. Partial hepatectomy was performed as described by HIKIX ASI) AKDERSSONIC. Control

respective

rats were subjected

surgical procedures

to laparotomy

and handling

of the liver. After the

and up to the time of death the rats were allowed free

access to water but no food. Mean body weights at the time of death are given in text to Fig. z and Table I. Exfit. I. The rats were lightly anaesthetized min complex

was rapidly injected

with ether and the fatty acicl-albu-

into one of the exposed

neck veins. At intervals,

blood samples (0.1~0.2 ml) were withdrawn from the other neck vein, and transferred into pre-weighed tubes containing chloroform-methanol (z : I, v/v). Ex$t. II. The fatty acid-rat serum complex was injected intravenously in the same way as described above. 2 min later the rats were exsanguinated through the abdominal aorta. Exactly 5 ml of blood was taken out, of which I ml was immediately injected into tubes containing chloroform-methanol (2 : I, v/v), The liver was cut out, rinsed in water, blotted dry and homogenized in 20 vol. chloroform~methanol (2: I

v/v). The necrotic

liver tissue around the ligatures in the partially hepatectomized rats was transferred to separate tubes with chloroform~methanol (2: I, v/v). The remains of the rat, the carcass, were transferred to r-1 flasks containing 300 ml of 2096 ethanolic

KOH.

Expt. I. Blood lipids were extracted with chloroform-methanol (z:I, v/v) washed with 0.4 vol. of 27& KH,PO, and the chloroform extract filtered directly into counting vials. Expt. II. The liver homogenate was filtered into a separatory funnel and 0.4 Biochiwz.

Bio$hys.

Acfa,

I_~L (1968)

237-243

FREE FATTY ACID METABOLISM AFTER PARTIAL HEPATECTOMY

239

~01. 2% KH,PO, was added. After separation of the phases the chloroform extract was dried over anhydrous Na,SO,. Non-phospholipids were separated from phospholipids on silicic acid columns 19. The lipids of the necrotic liver tissue around the ligatures were extracted in the same way and aliquots of the extracts were transferred to counting vials and counted. Only negligible amounts of radioactivity were found. The carcass was digested in 20% ethanolic KOH, refluxed on a boiling-water bath overnight and the fatty acids extracted from an aliquot of the digest20. The radioactivity of the blood samples of Expts. I and II as well as that of aliquots of total liver lipid extract, of non-phospholipid and pllospholipid fractions from the columns, and of the KOH digest of the carcass were counted in a Packard Tri-Carb liquid scintillation spectrometer, Quenching was corrected for by addition of an internal standard. Plasma from the 4-ml portion of blood in Expt. II was obtained by centrifugation. I ml of plasma was pipetted into 4 ml of isopropanol-1.5 M H,SO, (40: I, v/v). Two ml of distilled water and 5 ml of heptane were added and the tubes shaken. After separation of the phases, 4 ml of the heptane layer were transferred to new tubes containing 4 ml of lower phase from a blank extraction and the tubes shaken again. 3 ml of the heptane layer were then titrated using Nile blue as indicator. This double partitioning against lower phase was done to minimize the influence of metabolic acids. Nlodel experiments showed the recovery of added or-l~C]palmitic acid in the first extraction step to be better than 97%, only negligible amounts of which were lost in the second partitioning. Glyceride glycerol was determined by the method of VAN HANDEL AND ZILVERSMITH* and lipid phosphorus was determined according to the procedure of KINGLY. RESULTS

Fig. I shows the disappearance of the intravenously injected [SH]oleic acidalbumin complex from the circulating blood during the first z min after injection. The disappearance was assumed to follow a first-order exponential relationship: C = C,e+t

Fig. I. Disappearance from the circulating blood of intravenously injected [Wloleic acid complexed to 5% bovine serum albumin. The rats were either partially hepatectomized or laparotomized 18 h before. Disappearance curves for 7 individual rats in each group are plotted. Values are given as percentage of injected radioactivity present in I ml of whole blood at the respective times. o---.0, partially hepatectomized rats; O----O, laparotomized rats. Bid&n.

Biophys.

Acta,

1.52 (1968) 237-243

c;. r:lis, ‘I OI.I\~I~I~OS.\

240

during the first 90 sec. Only the values up to this time were used since, as judged from the curves, the disappearance did not follow at longer times. Using the method of least squares the intercepts, C, the rates, k, and their respective standard deviations were calculated giving the following equations. Hepatectomized rats C = Q,6.e-(i.2@ j- 0.07). t (9~:

for the calculations the above rcluation fractional turnover for tlie two groups,

7)

Controls c = 7,7.e&1.84 + 0.08) t (K _ 7) The fractional turnover rates correspond to half-lives of 0.55 and 0.38 min, respcctively, and differ significantly (P < 0.01). The difference between the intercepts is not statistically significant. This suggests that the blood volume wherein the isotope was initially mixed was of a similar magnitude in the two groups. Table I gives the chemical amounts of liver triglycerides, liver phospholipids, and plasma free fatty acids. The triglyceride concentration per g wet liver tissue was significantly higher (P < 0.01) in the partially hepatectomized group and is in good TABLE

I

LIVER

TRIGLYCERIDE,

AFTER

PARTIAL

LIVER

HEPATECTOMY

PHOSPHOLIFID, OR

LhPAROTO.\IY

r\ND

PLASM.4

(EXFT.

FREE

FATTY

ACID

COP;CENTRATION

18 h

11)

All values are mean 1 S.D. of 5 rats. Mean body and liver weights for the partially hepatectomized rats mere 207 g (range 200-22 I g) and 3.1 g (2.8-3.5 9) and for the laparotomized rats 2 TT i: (203-222 00)and 7.7 g (6.9-X.9 g).

Treatmmt Phospholifiids (pm&s/g w’et tissue) Partial hepatectomy Laparotomy

58.7 53.5

‘~ 8.8

q.8

agreement with the results of JOHSSOK AKD ALBERTA. The concentration of phospl~olipids was similar in the two groups. Plasma free fatty acid concentration was significantly higher (P < 0.01) in the partially hepatectomized group. From the data on plasma free fatty acid concentrations and the assumption that the plasma volume was 8 ml in both groups, the flux of plasma free fatty acids per min was calculated as follows: Fractional turnover rate x plasnia free fatty acid concentration x S. TIE calculated fluxes were 8.29 and 8.27 pequiv/min in the laparotomized and the partially hepatectomized groups, respectively. Table II shows the percentage of label in liver lipids and liver lipid fractions per g wet liver tissue 2 min after injection, There was significantly more label in the non-phospholipid and significantly less in the phospholipid fraction in the partially hepatectomized group. (In both cases P < 0.01.) The distribution of label between lipid classes was different in the two groups. In the partially hepatectomized group more label was in the non-phospholipid fraction, and less in the phospholipid fraction if compared with the corresponding fractions Biochim. Biophys. Acta, 152 (1968) zj7-qj

FREE FATTY ACID METABOLISM

TABLE

241

AFTER PARTIAL HEPATECTOMY

II

RADIOACTIWTYIN LWER LIPIDFRACTIONS 2 min AFTER INJECTION OF [~H]OLEIC ACID COMPLEXED TO RAT SERUM (EXPT. II) All values are mean * legend to Table I.

S.D. of 5 rats. Mean

and range of body

and liver weights

Radioactivity (% of injected dose per g liver

Treatment

Total Partial hepatectomy Laparotomy

~~

2.9 i 0.7 2.0 :k 0.2

Non-phospholipid

are given in

wet wt.) ._~

Phospholipid

2.4 t 0.6

0.48 &

1.4 *

0.55 -t 0.01

0.2

0.01

of the laparotomized group. The non-phospholipid radioactivity to phospholipid radioactivity ratio was 5.1 and 2.6 in the partially hepatectomized and laparotomized group, respectively. Table III shows the tissue distribution of label in Expt. II. There was considerably less label in the whole liver of the partially hepatectomized rats, but, as shown in Table II, there was more label per g of liver in this group. Carcass radioTABLE

III

RADIOACTIVITY IN THE WHOLE LIVER AND CARCASS LrFrns 2 min AFTER THE INTRAVENOUS INJRCTION OF [3H]~~~~~ ACID COMPLEXED TORAT SERUMINTO RATS 18 h AFTER PARTIALHEPATECTOMY OR

LAPAROTOMY

Liver radioactivity is given as percent of injected dose per whole liver. Carcass radioactivity was corrected for radioactivity present in the blood as described in the text. All values are mean + S.D. of 5 animals. (Mean and range of body and liver weights are given in the legend to Table I.)

Treatment

Partial hepatectomy Laparotomy

Radioactivity ( yO of injected dose) Liver

Carcass

Recovery

9.1 i- 1.7 15.7 Ii 3.4

64.5 zk 3.1 46.5 + 5.0

79.2 i 04.3 *

3.8 4.3

activity was corrected for fatty acid radioactivity remaining in the blood vessels as follows: Corrected carcass radioactivity=carcass radioactivity found-(whole blood volume radioactivity-radioactivity in blood removed for sampling). The blood volume was taken as 7.5 o/oof the body weight. Corrected carcass radioactivity was significantly higher in the partially hepatectomized group (P < 0.01). DISCUSSION

In the present paper we have assumed that the disappearance of the injected fatty acid-albumin complex follows a first-order exponential relationship and that the plasma volume was approximately the same in the two groups of rats. To be able to extend the interpretation of the data to all long chain fatty acids in the plasma free fatty acid pool, we have assumed that oleic acid represents an average fatty acid. We are well aware that these assumptions are open to criticism. The liver normally takes up about one-third of the plasma free fatty acid flux. The reduction in the fractional turnover rate of plasma free fatty acids caused by partial hepatectomy is of similar magnitude. If the partially resected liver does not take up plasma free fatty acids at all this would account for the reduction in fractional turnover rate. Our data show that the partially resected liver takes up a considerable Biochim. Biophys, Acta, 152 (1968) 237-243

212

(i.

Fir.\;,

I.

Ol.I\‘lb(

ROS.\

amount of plasma free fatty acids. Thus the decreased fractional turnover r;ttti c‘;ti, not be explained only by a decreased uptake by the liver. High levels of $asiri;i fm(% fatty acids are associated with lower fractional turnover rates than are 10\v frtac faft!acid IevelP.

Since

the partially

hepatectomized

concentrations than the laparotomized in fractional turnover rate. In spite of the different

rats,

fractional

rats

leave higlier

this inav contribute

turnover

free fatt\. a<,id to tlrcx reduction

rates for plasma

free fatt!-

acids,

the calculated flux of fatty acids through this pool was similar in the two groups. This means that similar amounts of fatty acid enter and leave the plasma free fatty a($(1 pool per unit time in the two groups. The proportion

of the plasma free fatty

acid flux taken up by the liver in t.Ile

two groups is not shown directly by our data. Liver blood flow per g wet tissue weight is increased after partial trel~atecton~~~7~9 and it is tfrus reasonable to assume that the uptake of free fatty acids per g tissue is also increased in the ll~~ate~t(~I~~ize~lrats. The data in Table

II indicate

per g liver was significantly in the fraction

of fatt?,

that this was also the case, since the amount

higher in the partially

acids that

liepatectomized

was immediately

oxidized

liver may, however, have contributed. Since the plasma free fatty acid concentrations groups the specific activity

of the plasma

free fatty

of label

group. A decrease

upon uptake

were different

into the

in the

two

acid pool must also have been

different

after the initial mixing of the injected

activity Because

must have been lower in the hepatectomized rats than in the controls. of this difference, it seems likely that the different amounts of label present

labeled oleic acid. The specific radio-

in the liver phospholipids in the two groups (Table II) reflect the different specific radioactivity of the plasma free fatty acids taken up by the liver, rather than differences in the amount of fatty acids taken up into this fraction. In the liver non-phospholipid fraction, which contains mainly

triglycerides,

a

considerably higher incorporation of label per g liver weight occurred in the partially hepatectomized rats than in the controls. In view of the differences in specific radioactivitv of the plasma free fatty acids discussed above, it seems safe to assume that the chemical amount of fatty acids taken up into this fraction differed even more between poration

the two groups. These data thus show that a considerably increased incorof plasma free fatty acids into liver triglycerides occurs after partial hepa-

tectomy. According to the data this incorporation is 2.4 and 1.4”;, of the plasma free fatty acid flux in the two groups, per g liver. The fluxes of plasma free fatty acids were calculated to be about 8.3 yequiv,/min in both groups. If these figures are assumed to be constant dusing the 18 h after lie~atectollly, the difference in uptake of free fatty acids into the liver glycerides corresponds to about 29 ,umoles triglyceride per g liver. The difference in glyceride content of the liver was found to be 23 ~~moles. Since these figures are of the same order of magnitude it is probable that the increased uptake of free fatty acids into the liver glycerides is a ma.jor factor in the liver fat accumulation

that occurs after partial

2 R. .T). HARKNESS, Biochim.

&OphyS.

J. Ph~siol.

ACta,

London,

1.52 (1968)

hepatectomy.

117 (1952)

2.37-243

267.

FREE FATTY ACID METABOLISM

AFTER PARTIAL BEPATECTOMY

243

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