The Journal of Clinical InvestigationVol. 46, No. 9, 1967

Difference in Hepatic Metabolism of Long- and Medium-Chain Fatty Acids: the Role of Fatty Acid Chain

Length in the Production of the AlcoholicFatty Liver *

CHARLESS. LIEBER,4 ANDRE' LEFEVRE, NORTONSPRITZ,§LAWRENCEFEINMAN,II ANDLEONOREM. DECARLI

(From the Liver Disease and Nutrition Unit, Second (Cornell) Medical Disision, BellevueHospital, and the Department of Medicine, Cornell University Medical College,

New York)

Abstract. Replacement of dietary triglycerides containing long-chain fattyacids (LCFA) by triglycerides containing medium-chain fatty acids (MCFA)markedly reduced the capacity of alcohol to produce fatty liver in rats. After24 days of ethanol and MCFA, the increase in hepatic triglycerides was only 3times that of controls, whereas an 8-fold rise was observed after ethanol andLCFA. The triglyceride fatty acids that accumulated in the liver after feed-ing of ethanol with MCFAcontained only a small percentage of the MCFA;their composition also differed strikingly from that of adipose lipids.

To study the mechanism of the reduction in steatosis, we compared oxida-tion to CO2and incorporation into esterified lipids of 14C-labeled chylomicronsor palmitate-14C (representing LCFA), and of octanoate-14C (as MCFA) inliver slices and isolated perfused livers, in the presence or absence of ethanol.Ethanol depressed the oxidation of all substrates to CO2; MCFA, however,was much more oxidized and reciprocally much less esterified than LCFA,with a 100-fold difference in the ratio of esterified lipid-14C to 14CO2. Fur-thermore, in hepatic microsomal fractions incubated with a-glycerophosphate,octanoate was much less esterified than palmitate. This propensity of MCFAto oxidation rather than esterification represents a likely explanation for thereduction in alcoholic steatosis upon replacement of dietary LCFA by MCFA.

Introduction

WVe have shown previously, in man (2) and inrats (3), that dietary fatty acids represent a largepercentage of the triglyceride fatty acids that ac-

* Submitted for publication 20 March 1967; accepted8 June 1967.

Presented in part at the Annual Meeting of the Ameri-can Gastroenterological Association, Chicago, May 1966and published in abstract form (1). This study wassupported in part by U. S. Public Health Service re-search grants AM09536, AM10893, and AM06284 fromthe National Institute of Arthritis and Metabolic Dis-eases and grant NB03345-04 from the National Instituteof Neurological Diseases and Blindness.

4 Recipient of a U. S. Public Health Service ResearchCareer Development Award (K3-AM-22,590) from theNational Institute of Arthritis and Metabolic Diseases.

cumulate in the liver after prolonged ingestion ofalcohol 1 with a normal diet containing LCT (tri-glycerides comprised of long-chain fatty acids).

§ Established Investigator of the Health Research Coun-cil of the City of New York (I-128). Present address:The Rockefeller University, New York, N. Y.

11 Supported by a Postdoctoral Research Traineeship inGastroenterology (TI AM 5091) and a Research Fel-lowship (F2 AM29096-01) from the National Instituteof Arthritis and Metabolic Diseases, U. S. Public HealthService.

Address requests for reprints to Dr. Charles S. Lieber,Director, Liver Disease and Nutrition Unit, Second(Cornell) Medical Division, Bellevue Hospital, FirstAvenue and 26th Street, New York, N. Y. 10016.

1 In this paper, ethanol and alcohol are used synony-mously.

1451

LIEBER, LEFEVRE, SPRITZ, FEINMAN, AND DECARLI

LCT MCT

0oorCalories

(% of total)80 V

601-

40 _

20

0MEthyl- Linoleote 2 mg/cal

**Including Methionine 1.50 mg/calCholine Chloride 0.25 mg/col

FIG. 1. SCHEMATICREPRESENTATIONOF THE COMPOSITION OF THE FOURISOCALORIC LIQUIDDIETS FED TO RATS FOR 24 DAYS. LCT, triglycerides containing long-chain fatty acids;MCT, triglycerides containing medium-chain fatty acids.

When the dietary LCT were isocalorically re-placed by MCT (triglycerides which consist ofmedium-chain fatty acids) it was noted, in a pre-liminary observation, that hepatic steatosis follow-ing alcohol ingestion was markedly reduced (4).In an attempt to explain this difference, we com-pared the hepatic metabolism of long-chain fattyacids (LCFA) and medium-chain fatty acids(MCFA) in the present study.

Methods

In vivo experiments. Charles River CD male rats 2were purchased in groups of four littermates each. Theywere maintained on Purina Laboratory Chow3 and tapwater ad lib. until they reached a weight of 100-150 g;they then were housed in individual wire-bottomed cages,and given liquid diets in Richter drinking tubes as theonly source of food and water. The composition of thebasic ethanol and control diets was as described before(5), except for the oil mixture. The latter consisted,per liter of diet, of 2 g of ethyl linoleate (supplementedwith vitamin A acetate, 2 mg; calciferol, 10 /ig; a-tocopherol acetate, 30 mg), plus either 10.0 g of corn oiland 34.3 g of olive oil given as a source of LCFA tohalf the rats or 49.5 g of MCT4 given alternatively as a

2 Purchased from Charles River Breeding Laborator-ies, Inc., North Wilmington, Mass.

3 Purchased from Ralston Purina Co., St. Louis, Mo.4 MCTwas kindly provided by Drew Chemical Corp.,

Boonton, N. J.

source of MCFAto the other half of the animals. Forthe isocaloric replacement of the olive-corn oil mixtureby MCT, the latter was considered to have a caloricvalue of 8.0 cal/g.5 The animals were studied in groupsof four littermates each. Two of the littermates re-ceived the LCT and MCT control diets respectively,while the other two were given the same diets in whichcarbohydrates were progressively and isocalorically re-placed by ethanol, as described previously (5). A sche-matic representation of the four diets is given in Fig. 1.Each litter of four animals was group-fed daily; thevolume of liquid diet was limited to the intake of themember with the lowest consumption.

After 24 days, the animals were sacrificed by decapi-tation and the liver and epididymal fat pads were quicklyexcised. Total lipid and triglyceride concentrations inthe liver (4) and fatty acid composition of the hepatictriglycerides and of the adipose tissue lipids (3) weredetermined as described previously, except that in orderto avoid loss of MCFA, evaporation of solvents was car-ried out at room temperature. Recovery of MCTaddedto the original lipid extract varied between 85 and 93%.

Liver perfusion experiments. Isolated rat livers wereperfused according to the technique of Miller, Bly, Wat-son, and Bale (6), using heparinized rat blood diluted(2 : 1) with a Ringer 6 solution to which bicarbonatehad been added (final concentration: 30 mEq/liter).Precautions were taken according to Kay and Entenman

5 Drew Chemical Corp., Boonton, N. J. Personalcommunication.

6 Purchased from Abbott Laboratories, North Chicago,Ill.

1452

LONG- AND MEDIUM-CHAIN FATTY ACIDS IN ALCOHOLIC FATTY LIVER

(7) to avoid injection of heparin into the rats fromwhich blood, lymph, or liver was obtained. Glucose wasadded to achieve a final concentration of 150 mg/100 ml.The perfusions were carried out in paired experiments,with or without ethanol; for each member of the pair,equal volumes of the same perfusate, ranging from 100to 135 ml, were used. The gas phase consisted of a mix-ture of 02 (98%o) and CO2 (2%) (v/v).

2-hr before the perfusion, the rats whose livers wereused for the ethanol experiments were given 2-2.4 g/kgethanol, by gastric tube, while the corresponding controlrats received an isocaloric amount of carbohydrate.

At the onset of the perfusion, a loading dose of ethanolwas added to achieve an initial concentration of 200 mg/100 ml. Thereafter, half the initial ethanol dose wasgiven hourly to maintain the alcohol concentrationthroughout the experiment at a level of 50-100 mg/100 mlof perfusate. Three groups of three to four paired per-fusions each were performed with the addition of:

(a) '4C-labeled chylomicrons, obtained as describedpreviously (3), including the avoidance of proceduresapt to alter the chylomicrons, such as chilling. Chylo-micron lipid was 1.07-3.07 mg/ml of final perfusate withapproximately 200,000 dpm/mg of lipid. Approximately2% of the label was in free fatty acids, 2% in phospho-lipids, and the remainder in triglycerides.

(b) "C-labeled LCFA (palmitate-1-"C) ,7 bound to al-bumin (8), added at a concentration of 0.03-0.05 /AEq/ml of perfusate (0.27-0.38 Ac/ml). To equalize the lipidloads, we performed half of the perfusions with ad-dition of nonlabeled chyle in amounts similar to those ofthe experiments in which "C-labeled chylomicrons wereperfused. Unlabeled, albumin-bound palmitic acid (0.38,AEq/ml) was added to the perfusions that did not con-tain chylomicrons.

(c) "C-labeled MCFA (octanoate-l-"C) 7 added at afinal concentration of 0.78 ,uEq/ml (0.26 ,uc/ml), withalbumin in an amount equal to that used with palmitate-4C. The fatty acid load (in milligrams) of these ex-

periments was comparable to that of the perfusions withpalmitate-"C (supplemented with nonlabeled palmiticacid).

In all instances, the labeled lipid material was addedafter a control period of 30 min during which the ade-quacy of the perfusion was judged by the continuous ex-cretion of bile and by the blood flow, which averaged1.79 + 0.17 ml/g of liver per min.

CO2 was collected in a 100 ml trap of 2 N sodium hy-droxide solution, renewed every 30 min. Aliquots weresuspended with a thixotropic gel powder (Cab-O-Sil)8(9) and 14CO2 radioactivity was determined in a liquidscintillation counter (10). In three pairs of perfusions(two with chylomicron-"C, one with palmitate-"C) totalC02 was measured repeatedly by the procedure of VanSlyke (11) in the in- and outflow blood of the liver andthe total C02 produced was calculated from the difference

7Purchased from New England Nuclear Corp., Boston,Mass.

8 Purchased from Packard Instrument Co., Inc.,Downers Grove, Ill.

in C02 concentrations and the blood flow rates. The dura-tion of the perfusion varied between 2 and 4 hr. At theend of the perfusion, livers were flushed with saline andweighed. Total lipids were extracted from the liver asdescribed for the in vivo studies and phospholipids wereseparated from the neutral lipids by silicic acid columnchromatography as described previously (3).

Aliquots of the liver were also taken for protein de-termination (12) in order to express the results as afunction of the protein content.

Ethanol concentration in the perfusion medium was de-termined by the procedure of Bonnichsen (13).

Liver slice experiments. These studies were conductedin five series of four incubation flasks each. Liver slices,obtained as described previously (10), were randomlydistributed among the four flasks at approximately 0.5 gper flask. The incubation medium was as describedpreviously (10), with added albumin (4 g/100 ml). Totwo of the flasks, palmitate-1-"C, bound to albumin (8),was added at a concentration of 0.2 ,umole/ml (0.4 uc/ml); the two other flasks received, instead of palmitate-1-"C, octanoate-1-"C, at a final concentration of 0.4,umole/ml (0.4 ,uc/ml). To half the flasks, ethanol wasadded at a final concentration of 10 umoles/ml, while theother flasks received, as control substrate, 10 ,moles/mlof acetate.

After a 3 hr incubation period, "CO2 produced wascollected and counted as described previously (10).The liver slices were washed with saline twice; totallipids were extracted and neutral lipids and phospho-lipids separated as described above in the perfusionexperiments.

Labeling of the lipid fractions was determined in aliquid scintillation counter as described previously (10).

Experiments with hepatic tnicrosomes. The microsomalfraction was obtained by ultracentrifugation accordingto Stein and Shapiro (14). It was then incubated at37°C in a shaking water bath with palmitate or octano-ate (0.2-1.2 ,umoles/3 ml), glycerophosphate-"C (10,umoles, 0.5 ,uc/3 ml) ,9 and the necessary cofactors foresterification, in the medium described by Stein andShapiro (14); the fatty acid concentration range wasextended to the limit of solubility of palmitate. Simul-taneously, blank incubations were performed in the ab-sence of microsomes. At the end of 30 min, total lipidswere extracted (15) and the amount of esterifed a-glyc-erophosphate was assessed by the radioactivity of thelipid extract corrected for the blank value and expressedper milligram of microsomal protein. Protein was mea-sured according to Lowry, Rosebrough, Farr and Ran-dall (16) and radioactivity was determined as describedabove.

In all experiments, each individual result was com-pared to its corresponding control, and the mean of theindividual differences was tested by the Student t test(17). The variance of the means was estimated by thestandard error.

9 Purchased as 1-a-glycerol-U-"C phosphate from In-ternational Chemical and Nuclear Corporation, City ofIndustry, Calif.

1453

LIEBER, LEFEVRE, SPRITZ, FEINMAN, AND DECARLI

Results

In vivo experiments. As indicated in Fig. 2,both groups of rats given ethanol had a significantincrease of hepatic triglycerides, in comparisonwith their respective controls. This increase, how-ever, was much less pronounced after ethanol-MCT (3-fold triglyceride rise; P < 0.02) thanethanol-LCT (8-fold rise; P < 0.001), with a sig-nificant difference between the groups (P < 0.01).The total lipids had similar changes: 48.1 ± 4.2mg/g in the LCT control, 118.8 ± 13.1 mg/g inthe LCT-ethanol, 54.0 ± 2.9 mg/g in the MCTcontrol, and 72.0 ± 7.4 mg/g in the MCT-ethanolgroups. These results indicate that the bulk ofthe total lipid changes can be accounted for by theaccumulation of triglyceride.

In Fig. 3, we have represented the compositionof the major fatty acids of the hepatic triglycerides,which accumulated in the rats given MCT-ethanol,together with that of the corresponding adiposetissue. The fatty acids of the hepatic triglyceridescontained only a small percentage of MCFA, butcomprised instead a large amount of endogenouslysynthesized LCFA such as palmitate and oleate.The fatty acid composition of the hepatic tri-

LCT

80jmg perg Liver

(wet weight)

60 [

501F40-

30 -

201-

10 1

01SUCROSE ETHANOL(Control) (36% of calories)

glycerides differed also from that of adipose tissuein two respects: adipose lipids had a greater pro-portion of MCFA than liver triglycerides; fur-thermore the linoleate concentration of adiposelipids (15.6 + 1.5% of total fatty acids) was muchhigher than that of liver triglycerides (5.6 ±1.5%). In the rats given the MCTcontrol diet,similar findings were observed, except for a lowerhepatic triglyceride concentration.

Liver perfusion experiments. In the three ex-periments in which alcohol was added to the 14C-labeled chylomicrons in the perfusion media, theproduction of total 14CO2 was markedly depressedfrom average control values of 40,500 dpm (per100 mgof liver protein) to 5060 dpm after ethanol.This is illustrated for one pair of studies in Fig. 4.As indicated in Fig. 5, a comparable depression of14CO2 production was observed from free fattyacids, both long-chain (palmitate-14C) and me-dium-chain (octanoate-'4C). In the livers per-fused with palmitate-14C and unlabeled chylomi-crons (Fig. 5, center panel, solid lines), the 14CO2produced was depressed from an average 722,600dpm/100 mg of liver protein in the controls to94,000 dpm in the ethanol studies, while the cor-responding values in the perfusions with palmi-

MCT

SUCROSE ETHANOL(Control) (36% of calories)

FIG. 2. EFFECT OF ISOCALORIC SUBSTITUTION OF LCT BY MCT ONHEPATIC TRIGLYCERIDE ACCUMULATION. The rats were fed isocaloricliquid diets for 24 days, with 41 % of calories as long-chain triglycerides(LCT, olive-corn oil mixture), or medium-chain triglycerides (MCT).

1454

LONG- AND MEDIUM-CHAIN FATTY ACIDS IN ALCOHOLICFATTY LIVER

40

30

20

101

oL 8:0 + 10:0 16:0 18:1(octanoate + (palmitate) (oleate)decanoate )

FIG. 3. COMPARISONOF FATTY ACID COMPOSITION OF ADIPOSE LIPIDS

(DOTTED BARS) AND LIVER TRIGLYCERIDES (CROSS-HATCHED BARS) INRATS FED ETHANOL AND MCT-CONTAINING DIETS OVER 24 DAYS. Asimilar fatty acid composition was observed in the control animals;their hepatic triglyceride concentrations, however, were lower (seeFig. 2).

tate-14C and unlabeled palmitic acid (Fig. 5, cen-ter panel, dashed lines) were 213,600 and 45,100dpm, respectively. In the perfusions with octano-ate, the corresponding values were 750,700 and283,700 dpm, respectively. That this reduction in14CO0 produced from various substrates upon ad-dition of ethanol is not solely due to isotopic dilu-tion by metabolites of alcohol is indicated by theobservation that, when measured, total CO2 pro-duction was also found to be depressed, from 0.65+ 0.15 Mmole/min (per 100 mg of liver protein)

in the control perfusions to 0.20 + 0.03 in theethanol experiments.

The per cent recovery (for the total duration ofthe control experiments) in 14CO2 of the lipid la-bel administered was much lower when 14C-la-beled chylomicrons were used (0.3-2.3%o), thanwhen palmitate-"C (3-10%) or octanoate-"C(10-12%) was perfused.

No consistent difference was found between thealcohol and control perfusions in the absoluteamount of label recovered in the neutral lipid orphospholipid fractions. The average absolutevalues of radioactivity recovered in the hepaticneutral lipids and phospholipids were 171,400and 93,200 dpm (per 100 mg of liver protein) inthe perfusion with ethanol and "C-labeled chylo-microns; the corresponding controls were 198,500and 124,400 dpm. In the perfusions with ethanol,

palmitate-1"C, and unlabeled chylomicrons, theradioactivity in hepatic neutral lipids and phos-pholipids was 1,811,100 and 2,673,100 dpm (per100 mg of liver protein), respectively; the corre-sponding controls were 1,640,700 and 2,191,100dpm. In the perfusions with ethanol, palmitate-

DPM

0Minutes

FIG. 4. EFFECT OF ALCOHOLONTOTAL 'CO2 PRODUCTIONFROM"C-LABELED CHYLOMICRONSIN A PAIR OF ISOLATEDPERFUSEDRAT LIVERS.

%of totalfatty acids(mean ±SE)

_ _

i_18:2

(linoleate)

1455

LIEBER, LEFEVRE, SPRITZ, FEINMAN, AND DECARLI

CHYLOMICRONS-4C

100

%ofcontrol

50

PALM TATE-14 C OCTANOATE-14C

FIG. 5. COMPARISONOF THE EFFECT OF ETHANOL ON "CO2 PRODUCTION FROM EITHER 1C-LABELED CHYLOMICRONS,PALMITATE, OR OCTANOATE. In the palmitate-"C experiments indicatedby a solid line, unlabeled chylomicrons (in amounts comparable to those used in the studieswith "C-labeled chylomicrons) were added to the perfusate; in the palmitate-"C experimentsindicated by a dashed line, unlabeled palmitic acid (0.38 t&Eq/ml) was added.

14C, and unlabeled palmitic acid; the radioactivityin hepatic neutral lipids and phospholipids was1,260,400 and 796,000 dpm, respectively (controls:982,200 and 1,151,200 dpm); the corresponding

CHYLOMICRONS-"4C

100l

50 -

0

values in the perfusions with octanoate-14C were122,100 and 45,600 dpm with ethanol and 110,600and 57,900 dpm in the controls.

The above results of incorporation of 14C in

PALMITATE-" C OCTANOATE-"4C

FIG. 6. COMPARISONOF THE INCORPORATION INTO HEPATIC NEU-TRAL LIPIDS (STRIATED AREAS), PHOSPHOLIPIDS (SOLID AREAS), ANDCO2 (CROSS-HATCHEDAREAS) OF THE "C-LABEL DERIVED FROMISO-LATED LIVERS PERFUSED WITH CHYLOMICRONS-"C, PALMITATE-"C,ANDOCTANOATE-"C. The radioactivity of each fraction is expressedas percentage of the total labeling recovered in these three majorfractions.

1456

L'ue t u

LONG- AND MEDIUM-CHAIN FATTY ACIDS IN ALCOHOLICFATTY LIVER

PALMITATE-14C (OApc:02pmole /ml.

5,000,000

4,500,000

le1:SE)T

1,000,000

500,000

0L- CONTROL ETHANOL(acetate 10imoles I ml) (lOpmoles / ml)

) OCTANOATE-'4 C (0.4pc; 0.4pmole /ml)

CONTROL ETHANOL(acetate 1OgmolesJ ml) (10pmoles /lml)

FIG. 7. COMPARISONOF THE INCORPORATION INTO HEPATIC TOTAL LIPIDS-1C(STRIATED BARS) AND CO2 (CROSS-HATCHEDBARS) OF THE LABEL DERIVED FROMPALMITATE-14C AND OCTANOATE-14C, IN LIVER SLICES INCUBATED FOR 3 HR WITH

ETHANOLOR ACETATE.

CO2 and lipids reveal the striking find that therelative distribution of the label between esterifiedlipids and 14CO1 was very different depending on

the chain length of the fatty acids used. With"4C-labeled chylomicron and palmitate, much more

of the label was present in the esterified lipidsthan in 14CO2 (in the control experiments as wellas in those with addition of ethanol), whereas thereverse was true in the studies with octanoate-14C(Fig. 6). As a result, after ethanol, the ratio oflabeled esterified lipids to 14CO2 was approximatelya hundred times lower in experiments with la-beled MCFA (0.59 + 0.17) than with labeledLCFA (palmitate: 47.0 7.2; chylomicron:52.0 + 11.5).

Liver slice experiments. As illustrated in Fig.7, ethanol lowered 14CO0 production from bothlabeled LCFA and MCFA. Furthermore, in ab-solute amounts, there was a 25-fold greater oxida-tion to 14CO2 from the MCFA (octanoate-'4C)than from the LCFA (palmitate-14C) (P <0.001). In contrast, incorporation into hepaticneutral lipids and phospholipids was about 4 timesless with octanoate than with palmitate. The neu-

tral lipid labeling was 901,600 168,700 and4,310,800 + 138,900 dpm/g of liver in the slicesincubated with ethanol and octanoate or palmitate,

respectively (P < 0.001); the corresponding val-ues for phospholipids were 123,700 + 66,300 and413,000 ± 28,600, respectively (P < 0.001). In-corporation of palmitate and octanoate in the he-patic lipids appeared increased by ethanol (Fig.7), but because of variability in the results, the dif-ferences were not statistically significant.

Microsome experiments. As shown in Table I,despite marked variations in activity between dif-ferent microsomal preparations, more radioactivity

TABLE I

Radioactivity in esterifled lipids after incubation of a-glyc-erophosphate-14C with either octanoic acid or palmitic acid*

Micro. Fattysomal acidprepa- concen- Octanoic Palmiticration tration acid acid

pmole/mlA 0.07 658 3240

B 0.20 776 3697

C 0.07 607 27820.20 622 4534

D 0.07 596 10720.20 564 16130.40 500 3198

* dpm/mg of protein; 30Stein and Shapiro (14).

min incubation according to

dpmlgm(mean +

1457

15LIEBER, LEFEVRE, SPRITZ, FEINMAN, AND DECARLI

appeared in esterified lipids when a-glycerophos-phate-14C was incubated with palmitate than withoctanoate, in any given microsomal fraction for allconcentrations tested. For the same microsomalpreparation, even when the octanoate concentrationwas 6-fold that of palmitate, esterification was stillmarkedly lower (Table I).

Discussion

Our study shows that when dietary LCT are

replaced by MCT, fatty liver produced by alcoholis markedly decreased (Fig. 2) and the hepatictriglycerides have a negligible content of dietaryMCFA (Fig. 3). This contrasts with the fattyliver resulting from the feeding of ethanol andLCT, which was found to contain triglycerideswith a high percentage of dietary fatty acids (2, 3).The triglycerides of the ethanol-MCT fatty liveralso had a fatty acid composition markedly differ-ent from that of adipose tissue, with much lesslinoleate and significantly less octanoate anddecanoate (Fig. 3). These findings suggest thatthe triglyceride fatty acids that accumulate in theliver after ethanol-MCT diets do not derive pri-marily from either adipose or dietary lipids, butmay represent endogenously synthesized fattyacids. The observed composition is similar to thatof hepatic triglycerides in fatty liver produced byethanol and low fat, high carbohydrate diets (2,3). The possibility has not been ruled out, how-ever, that in the present study, some of the ac-

cumulation of LCFA in the liver may have re-

sulted from elongation of MCFA. Quantitatively,however, this possibility is probably of minor sig-nificance, in view of the observation that elonga-tion of octanoate occurs only to a minimal degreein the liver (18).

As a possible explanation for the difference inhepatic accumulation of MCFAand LCFA, con-

sideration must be given first to differences intheir metabolic pathways. In contrast withMCFA, which, after intestinal absorption, are car-

ried primarily as free carboxylic acids by the portalroute to the liver (19, 20), LCFA are absorbedas chylomicrons via the lymphatic channels (19,20). The difference in the hepatic fat accumula-tion after ethanol-LCT and ethanol-MCT, how-ever, does not appear to result from a specific ef-fect of ethanol on chylomicron metabolism: a com-

parable depression of oxidation of the lipids in theliver was observed with either chylomicrons orfree fatty acids (Fig. 5). Furthermore, we foundpreviously that ethanol does not alter hepaticuptake of chylomicron (3).' Moreover, whetherunder normal circumstances the liver metabolizescirculating chylomicrons per se or whether they arefirst hydrolyzed in the vascular space or in periph-eral tissues is the subject of controversy (21-24).Consistent with the latter possibility is our ob-servation of a low rate of oxidation of 14C-labeledchylomicrons to 14CO2, compared to that of theoxidation of 14C-labeled fatty acids. The amountof 14CO2 and 14C-labeled phospholipids recoveredin the livers perfused with 14C-labeled chylomi-crons was too high, however, to be accounted forentirely by the small percentages of labeled freefatty acids and phospholipids initially present inthe chylomicron preparation. This means, as wasalso concluded recently by other investigators(23, 24), that some hydrolysis of chylomicrontriglyceride must have taken place, but does notindicate whether this reaction occurred at in-tra- or extracellular sites. Although lipoproteinlipase has been demonstrated in the dog liver(25), whether or not lipoprotein lipase exists inthe rat liver and contributes to chylomicron lipoly-sis is the subject of controversy. According tomost authors, the rat liver does not contain anylipoprotein lipase (26-28), while Mayes and Felts(29) describe some activity in response to largedoses of heparin. Since heparin was used in theliver perfusions of the present study, it conceiv-ably could have played some role in the break-down of chylomicrons to free fatty acids.

It has been shown that ethanol oxidation in theliver results in the production of nearly equimolaramounts of acetate (30). This could result in adepression of 14CO2 production through isotopicdilution of an acetylCoA-14C intermediate producedduring the metabolism of the 14C-labeled lipidprecursors. The possibility, however, that thisaccounts solely for the 14CO2 reduction has beenruled out in the liver perfusion experiments inwhich both 14CO2 and total CO2 production werefound to be depressed by ethanol. The depressionof 14CO2 and CO2 production suggests that ethanolmay reduce the activity of the citric acid cycle(31); this reduced activity may, in turn, contribute

1458

LONG- AND MEDIUM-CHAIN FATTY ACIDS IN ALCOHOLIC FATTY LIVER

to the hepatic accumulation of LCFA observedafter ethanol (2, 3).

In liver perfusions as well as in liver slices,whether or not alcohol was added, the relativedistribution of the label between esterified lipidsand '4C02 was greatly affected by the chain lengthof the available fatty acid (Figs. 6 and 7). Muchmore of the label from palmitate-14C was re-covered in neutral lipids or phospholipids than inC02, whereas the reverse was true for octanoate-14C. Although ethanol depressed 4CO2productionin both instances, it did not significantly affectthe basic difference in behavior of the fatty acidsof different chain length.

It has been shown that the hepatic enzyme sys-tem responsible for esterification of fatty acids tomore complex lipids (i.e., triglycerides and phos-pholipids) has a much greater affinity for 16 and18 carbon fatty acids than for shorter, 12 and 14,carbon chains (32). The present study indicatesthat this can be extrapolated to octanoate, an eightcarbon MCFA. A similar difference in esterifica-tion of palmitate and octanoate has been describedpreviously in intestinal tissue (33). The differ-ence in the hepatic oxidation of LCFA andMCFAwhich we observed in the present studymay actually result from the reciprocal differencein esterification. The high rate of hepatic oxi-dation of MCFAcould, in turn, be responsible forthe relative lack of their accumulation in hepatictriglycerides, even when administered withethanol.

In conclusion, the present study demonstrates afundamental difference in hepatic metabolism offatty acids depending on their chain length:MCFA (even in the presence of alcohol) wereobserved to be predominantly oxidized and mini-mally esterified, whereas the reverse was foundfor LCFA. This observation probably explainswhy dietary LCFA, but not MCFA, accumulatein hepatic triglycerides, and why much less hepaticsteatosis develops after feeding of alcohol withMCFAthan with LCFA.

AcknowledgmentWeare grateful to Dr. T. P. Almy for his continuous

interest and support.

References

1. Lieber, C. S., A. Lefevre, L. Feinman, and N. Spritz.1966. Importance of dietary long-chain fatty acidsin the pathogenesis of the alcoholic fatty liver.Gastroenterology. 50: 856. (Abstr.)

2. Lieber, C. S., and N. Spritz. 1966. Effects of pro-longed ethanol intake in man: role of dietary, adi-pose, and endogenously synthesized fatty acids inthe pathogenesis of the alcoholic fatty liver. J.Clin. Invest. 45: 1400.

3. Lieber, C. S., N. Spritz, and L. M. DeCarli. 1966.Role of dietary, adipose, and endogenously syn-thesized fatty acids in the pathogenesis of the al-coholic fatty liver. J. Clin. Invest. 45: 51.

4. Lieber, C. S., and L. M. DeCarli. 1966. Study ofagents for the prevention of the fatty liver pro-duced by prolonged alcohol intake. Gastroenter-ology. 50: 316.

5. Lieber, C. S., D. P. Jones, and L. M. DeCarli. 1965.Effects of prolonged ethanol intake: productionof fatty liver despite adequate diets. J. Clin. In-vest. 44: 1009.

6. Miller, L. L., C. G. Bly, M. L. Watson, and W. F.Bale. 1951. The dominant role of the liver inplasma protein synthesis. J. Exptl. Med. 94: 431.

7. Kay, R. E., and C. Entenman. 1961. The synthe-sis of "chylomicron-like" bodies and maintenanceof normal blood sugar levels by the isolated, per-fused rat liver. J. Biol. Chem. 236: 1006.

8. Fredrickson, D. S., D. L. McCollester, and K. Ono.1958. The role of unesterified fatty acid transportin chylomicron metabolism. J. Clin. Invest. 37:1333.

9. Harlan, J. W. 1963. Liquid scintillation countingof C"402 in aqueous carbonate solutions. Advan.Tracer Methodoly. 1: 115.

10. Lieber, C. S., and R. Schmid. 1961. The effect ofethanol on fatty acid metabolism. Stimulation ofhepatic fatty acid synthesis in vitro. J. CGn. Invest.40: 394.

11. Van Slyke, D. D. 1917. Studies of acidosis. II.A method for the determination of carbon di-oxide and carbonates in solution. J. Biol. Chem.30: 347.

12. Gornall, A. G., C. J. Bardawill, and M. M. David.1949. Determination of serum proteins by meansof the biuret reaction. J. Biol. Chem. 177: 751.

13. Bonnichsen, R. 1963. Ethanol-determination withalcohol dehydrogenase and DPN. In Methods ofEnzymatic Analysis. Hans-Ulrich Bergmeyer, ed-itor. Academic Press Inc., New York. 285.

14. Stein, Y., and B. Shapiro. 1958. Glyceride synthe-sis by microsome fractions of rat liver. Biochim.Biophys. Acta. 30: 271.

15. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957.A simple method for the isolation and purificationof total lipids from animal tissues. J. Biol. Chem.226: 497.

1459

LIEBER, LEFEVRE, SPRITZ, FEINMAN, AND DECARLI

16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, andR. J. Randall. 1951. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193: 265.

17. Snedecor, G. W. 1956. Statistical Methods. IowaState College Press, Ames, Iowa. 5th edition.534.

18. Brady, R. O., and S. Gurin. 1950. The biosynthesisof radioactive fatty acids and cholesterol. J. Biol.Chem. 186: 461.

19. Bloom, B., I. L. Chaikoff, and W. 0. Reinhardt.1951. Intestinal lymph as pathway for transportof absorbed fatty acids of different chain lengths.Am. J. Physiol. 166: 451.

20. Borgstrom, B. 1955. Transport form of "C-decanoicacid in porta and inferior vena cava blood duringabsorption in the rat. Acta Physiol. Scand. 34: 71.

21. Morris, B. 1963. The metabolism of free fattyacids and chylomicron triglycerides by the iso-lated perfused liver of the rat. J. Physiol (Lon-don). 168: 564.

22. Felts, J. M., and P. A. Mayes. 1965. Lack of up-take and oxidation of chylomicron triglyceride tocarbon dioxide and ketone bodies by the perfusedrat liver. Nature. 206: 195.

23. Schotz, M. C., B. Arnesjb, and T. Olivecrona. 1966.The role of the liver in the uptake of plasma andchyle triglycerides in the rat. Biochim. Biophys.Acta. 125: 485.

24. Ontko, J. A., and D. B. Zilversmit. 1967. Metab-olism of chylomicrons by the isolated rat liver.J. Lipid Res. 8: 90.

25. Boberg, J., L. A. Carlson, and L. Normell. 1964.Production of lipolytic activity by the isolated, per-fused dog liver in response to heparin. Life Sci.3: 1011.

26. Jeffries, G. H. 1954. The sites at which plasmaclearing activity is produced and destroyed in therat. Quart. J. Exptl. Physiol. 39: 261.

27. Spitzer, J. A., and J. J. Spitzer. 1956. Effect ofliver on lipolysis by normal and postheparin serain the rat. Am. J. Physiol. 185: 18.

28. Morris, B., and J. E. French. 1958. The uptakeand metabolism of C" labelled chylomicron fat bythe isolated perfused liver of the rat. Quart. J.Exptl. Physiol. 43: 180.

29. Mayes, P. A., and J. M. Felts. 1966. Demonstrationof lipoprotein lipase in rat liver. Biochem. J. 99:43P.

30. Lundquist, F., N. Tygstrup, K. Winkler, K. Mellem-gaard, and S. Munck-Petersen. 1962. Ethanolmetabolism and production of free acetate in thehuman liver. J. Clin. Invest. 41: 955.

31. Lieber, C. S. 1966. Hepatic and metabolic effectsof alcohol. Gastroenterology. 50: 119.

32. Kornberg, A., and W. E. Pricer, Jr. 1953. Enzy-matic esterification of a-glycerophosphate by longchain fatty acids. J. Biol. Chem. 204: 345.

33. Dawson, A. M., and K. J. Isselbacher. 1960. Stud-ies on lipid metabolism in the small intestine withobservations on the role of bile salts. J. Clin.Invest. 39: 730.

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