Effect of Dietary Unsaturated Oils on the Biosynthesisof Cholesterol, and on Biliary and Fecal Excretionof Cholesterol and Bile Acids in Rats1

CHAKKODABYLU S. RAMESHA, RANAJIT PAUL2ANDJAGANNATH GANGULY3

Department of Biochemistry, Indian Institute of Science,Bangalore-560 012, India

ABSTRACT The aim of the present work was to investigate whether thehypocholesterolemic effect of polyunsaturated oils is due to inhibition of cholesterol synthesis or increased excretion of cholesterol and bile acids through thebile and feces of animals. Separate groups of rats were fed diets containing 10%safflower oil, coconut oil or hydrogenated vegetable oils for 30 days, after whichthe hepatic cholesterol and bile acid synthesis and their excretion through thebile and feces were studied. As compared to the rats in the other two groups, thosegiven the diet containing 10% safflower oil showed markedly increased rates ofbile flow and excreted through their bile and feces markedly higher amounts ofcholesterol and bile acids. At the same time incorporation of [l-14C]acetate and[2-14C]mevalonate into the liver cholesterol and conversion of [4-14C]cholesterolinto 14C-bileacids were also higher in the same rats. In the light of these observations it has been discussed that in the animals given polyunsaturated oils, biliaryand fecal loss of cholesterol and bile acids far outweighs the activation of cholesterol synthesis and thereby effectively lowers the serum cholesterol levels. J.Nutr. 110: 2149-2158, 1980.INDEXING KEY WORDS unsaturated fat •cholesterol •bile acidscholic acid •HMG-CoA reducíase •cholesterol 7a-hydroxylase •biliaryexcretion •fecal excretion •neutral sterols

Even though it is well established that excretion of cholesterol is probably thediets rich in polyunsaturated lipids lower most important mechanism of cholesterol-serum cholesterol levels in animals, the lowering effects of polyunsaturatedactual mechanism of such hypocholes- lipids (2).terolemic effects of polyunsaturated We are now showing here that feedinglipids is not properly understood yet. We diets containing a polyunsaturated oil likehave recently shown that in rats, the safflower oil not only causes marked stim-feeding of polyunsaturated lipids in the ulation of bile flow and increased fecalform of safflower oil or soya lecithin excretion of cholesterol in rats, but at thestimulates the rate of bile flow, while same time activates cholesterol and cholicat the same time the concentration of acid synthesis in the liver of such rats. Incholesterol per milliliter of bile shows the light of these findings it is suggestedsignificant increase, so that markedly _higher amounts of cholesterol are ex- Received for publication 29 February 19SO.Creted through the bile of SUch ratS (1). 'Supported by a grant from the Department of Science and

. , i-i Technology, Government of India, New Delhi.In a recent review We have dlSCUSSed ' Recipient of a Senior Research Fellowship, CSIR, New Delhi.

infnrmuHnn nn fViic acrwx^t Present address: Department of Microbiology, University ofiniormation on mis aspect VjrginiaSchoo,ofMedicinechariottesviiie,VA22901.and have pointed out that biliary and fecal •TOwhomreprintrequestsshouldbesent.

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2150 C. S. RAMESHA, R. PAUL AND J. GANGULY

TABLE 1

Fatty acid compositions of the oils fed and the effect of the dietary oils on the fatty acid compositionof the phospholipids and cholesteryl esters of the liver microsomes1

Liver microsomes

FattyacidC10:0C12:0C14:0C16:0C18:0C18:lC18:2SO,——6.03.112.3576.92Oil

fedCO7.1627.5036.6118.802.811.832.54PhospholipidsD——25.6221.3647.84.2SO——10.021.025.842.19CO——17.3223.0335.8624.51D——18.9124.7634.2817.50CholesterylestersSO——21.73—20.2356.46CO__3.058.3433.46—33.1920.11D_—25.087.8043.4722.55

' The animals received the diets containing the different oils for 30 days, after which they were killedand the liver microsomes were isolated according to Shefer et al. (9). The fatty acids of the microsomes wereanalysed as described by Paul and Ganguly (1). Values are expressed as the percentage of the total peakarea, minor peaks (<2.0%) being neglected. SO = Safflower oil; CO = coconut oil; D = Dalda (hydro-genated vegetable oil).

that the increased biliary and fecal excretion of cholesterol outweighs the stimulation of cholesterol synthesis in the ratsgiven highly unsaturated oil.

MATERIALS AND METHODS

Materials. (l-14C)-Acetate (specificradioactivity 49 mCi/mmole) was obtained from the Bhabha Atomic ResearchCentre, Bombay. DL(2-14C) Mevalonicacid lactone (specific radioactivity 13.0mCi/mmole), DL(l-14C)-mevalonic acidlactone (specific radioactivity 6.0 mCi/mmole) and (4-I4C)-cholesterol (specificradioactivity 55.6 mCi/mmole) were obtained commercially (RadiochemicalsCentre, Amersham, England) as was (3-14C)-/3-Hydroxy/3-methylglutarate (HMG,specific radioactivity 7.7 mCi/mmole, NewEngland Nuclear Corporation, Boston,MA). All radioactive compounds wereused after purification by thin-layerchromatography (TLC).

The non-radioactive DL-mevalonic acidlactone, HMG, ATP, CoA, glucose-6-phos-phate, glucose-6-phosphate dehydrogenase,NADP, dithiothreitol (DTT), hexamethyldisilazone and trimethyl chlorosilanewere purchased (Sigma Chemical Co., St.Louis, MO). Sources of other materialsand purification of the solvents were asdescribed by Paul and Ganguly (1).

After suitable dilution of the radioactiveHMG with non-radioactive HMG, it wasconverted into its CoA derivative by themethod of Louw et al. (3). The finalspecific radioactivity of the HMG-CoAused in this work was 275 disintegrationsper minute (dpm/per nanomole).

Treatment of animals. Three types ofoils—safflower oil, coconut oil and Dalda(a commercial preparation of hydro-genated vegetable oil)—were used. Thefatty acid compositions of these oils aregiven in table 1. In order to prevent significant peroxidation during use, the oilswere stored in sealed containers in a refrigerator.

Pellets of a stock diet (M/s HindustanLever Ltd., Bombay) were crushed intosmall pieces and mixed daily with the different oils in the ratio of 1:10 (oihdiet,w/w). Male albino rats of this Institutestrain and weighing about 100-110 g.were allowed to eat ad libitum for 30 daysdiets freshly mixed with the respectiveoil, after which they were used for theexperiments as described in appropriateplaces. At the end of such dietary regimens there was very little difference inthe final body weights and liver weightsof the rats of the different groups; thesevalues were in the region of 160 g and9.0 g, respectively.

Collection and analysis of bile. Bile

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UNSATURATED OIL AND CHOLESTEROL METABOLISM 2151

cannulae were established in the rats afteran intraperitoneal injection of Nembutal(40 mg/kg body weight), as described byPaul and Ganguly (1). The volumes of thebile collected were recorded every hourand the collection was continued for 4hours, after which suitable portions of thesamples collected in 4 hours were analyzed for radioactivity, cholesterol andbile acids as described earlier (1).

Analysis of fecal sterols. Feces of sixrats from each group were separately collected during the last 24 hours of therespective dietary treatment, pooledwithin each group, sun-dried and madeinto powder in a pestle and mortar. Representative samples of 0.5-1.0 g of thepowdered feces were analyzed for neutralsterols essentially according to Grundy etal. (4), and during the process of analysis(4-I4C)-cholesterol of known specificradioactivity was added externally for determining the loss of the sterols duringprocessing.

Trimethylsilyl (TMS) derivatives of theneutral sterols were prepared by themethod of Grundy et al. (4) and wereseparated by gas-liquid chromatography(GLC). The GLC was carried out on a5-foot glass column packed with SE 30:NGF:QF1 on a glass chrome P and withnitrogen as the carrier gas (15 psi at 210°).5a-Cholestane was used as the internalstandard for quantitation of the sterols.

Assay of cholesterol-Ta-hydroxylase.The activity of this enzyme was assayedin the liver microsomes essentially according to Mitropoulos and Balasubra-maniam (5), with the following modifications. The standard incubation mixturescontained in a total volume of 1.8 ml,potassium phosphate buffer (0.1 M, pH7.4), nicotinamide (30 ITIM),EDTA (2 ITIM),MgCl2 (5 HIM),NADP (1 mM), glucose-6-phosphate (10 ITIM),glucose-6-phosphatedehydrogenase (1 unit) and 8-10 mgmicrosomal protein. The mixture was pre-incubated at 37°for 10 minutes and thereaction was started by adding 6.1 x IO4dpm of the (4-'4C)-cholesterol suspendedin Tween 80. The incubation was continued for 30 minutes at 37°with constantshaking.

At the end of 30 minutes the reaction

was stopped by the addition of 10 mlchloroform: methanol (2:1, v/v) and 1 ml0.25 M H2SO4.The precipitated proteinswere centrifuged off and the total lipidswere extracted from the supernatant bythe Bligh and Dyer (6) procedure. Theextracted lipids were spotted on silica gelC plates and the chromatograms weredeveloped at 4°with diethyl ether, afterwhich the plates were exposed to iodinevapor and the spots corresponding tothose of standard 7a-hydroxycholesterolwere scraped into scintillation vials fordetermination of the radioactivity.

Each reaction was run in duplicate andan average of the two values was taken.A blank reaction containing boiled micro-somes was also run in order to determineauto-oxidation and the blank counts weresubtracted from those of the experimentalsamples. In this procedure the counts dueto auto-oxidation were about 5-7% of thetotal counts in the 7a-hydroxycholesterol.

Incorporation of(l-i4C)-acetate and(2-t4C)-mevalonate into liver cholesterol

Acetate and mevalonate in vivo. Thirtyminutes after an intraperitoneal injectionof the labeled acetate or mevalonate,the rats were killed by cervical dislocation, after which the livers were homogenized in 80% aqueous ethanol (v/v), andthe homogenate was saponified in 10 NNaOH (1 ml/g liver tissue). The non-saponifiable lipids were extracted withlight petroleum ether (boiling point 40-60°)and the solvent was evaporated todryness in vacuo, after which the residuewas redissolved in known amounts ofchloroform. Small portions of the chloroform solution were spotted on silica gelC plates and the chromatogram wasdeveloped by using the solvent system,n-hexane:diethyl ethenacetic acid (30:6:0.5, v/v), after which the plates wereexposed to iodine vapor and the spotsdue to cholesterol were scraped intoscintillation vials for determination of theradioactivity.

In those experiments where the (1-14C)-acetate was injected, after extraction ofthe non-saponifiable lipids, the aqueousphase was acidified to pH 2 with 12 N HC1

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2152 C. S. RAMESHA, R. PAUL AND J. GANGULY

and the free fatty acids were extractedwith light petroleum. The rest of theprocedure for the separation of the freefatty acids on silica gel C plates and forthe measurement of the radioactivityin the separated free fatty acids was thesame as described above for cholesterol.

Mevalonate in vitro. These experiments were carried out according to theprocedure described by Shama Bhat andRamasarma (7) by using the post-mito-chondrial supernatant of the liver homog-enate as the source of the enzymes. Ina total volume of 2 ml, the reaction mixture consisted of phosphate buffer (pH7.4, 250 mM), nicotinamide (60 /Limóles),ATP (5 /¿moles), MgCl2 (20 Amóles),DL(2-14C)-mevalonicacid(1.8 x 105dpm/300 nmoles) and the post-mitochondrialsupernatant (0.5-1.0 ml). The reactionwas carried out at 37°in a water bath with

constant shaking and at the end of 30minutes it was stopped by the additionof 25 ml of 80% ethanol (v/v). Subsequentextraction of the lipids, separation of thecholesterol by TLC and measurement ofthe radioactivity in the sterol were as described above for the in vivo experiments.

Determination of the rate, of releaseof !4CO2/rom (l-l4C)-mevalonate

in vitro and assay of themevalonatepyrophosphatedecarboxylase activity in the liverhomogenate

The rate of 14CO2release was determinedby following the procedure described byShama Bhat and Ramasarma (8). Thereaction mixture was essentially the sameas used for the study of (2-14C)-mevalonate

incorporation into the liver cholesterolin vitro, except that nicotinamide wasomitted here, while (l-HC)-mevalonicacid (1.4 x IO5 dpm/300 nmoles) wasused and the total volume was made up to1.7 -ml. The reaction was carried out instoppered Warburg flasks at 37°without

shaking. The side arm contained 0.5 ml ofl M H2SO4, while the central well contained 0.2 ml of l M KOH. At the end of20 minutes the reaction was stopped bytipping the contents of the side arm intothe reaction mixture and the flasks were

left at the room temperature for 1 to 2hours, after which small samples weretaken from the central well for the determination of the radioactivity.

The decarboxylase activity was measuredas described by Shama Bhat and Ramasarma (8) by using 38,000 x g supernatantof the liver homogenate as the source ofthe enzyme. In a final volume of 1 ml, theassay system consisted of Tris-HCl buffer(pH 7.4, 0.05 M), ATP (10 mM), MgCl2(12.5 HIM), 2-mercaptoethanol (12.5 mM)and 0.6 ml of the 38,000 x g supernatant.The incubation mixture was taken in theouter well of a Warburg flask, while thecentral well and the side arm contained0.2 ml of l MKOH and 0.5 ml of 2 N H2SO4,respectively. The reaction was started byadding (l-14C)-mevalonic acid pyrophos-phate (0.05 mM, specific radioactivity 500dpm/nmole), after which the flasks werestoppered and incubated at 37°for 20

minutes. The rest of the procedure wassame as described for the study of therate of release of 14CO2from (l-14C)-meva-

lonate in vitro.Assay of HMG-CoA reducíase activity.

The rats were killed by cervical dislocation between 0900 and 1000 hoursand the livers were immediately transferred into chilled solutions containing30 mM EDTA, 70 mM NaCl, 10 mM DTTand 50 mM potassium phosphate buffer(pH 7.4), after which the livers were cutinto small pieces with a pair of scissorsand homogenized in a Potter-Elvehjemtype of homogenizer. The microsomalpellets obtained from the homogenate bythe method of Shefer et al. (9) were suspended in the same buffer and the suspension was used as the source of theenzyme. The reactions were carriedout according to the procedures describedby Shapiro et al. (IO). In a final volumeof 100 fu, the reaction mixture consistedof microsomal protein (200-300 ¿ig),glucose-6-phosphate (5 Amóles), NADP(450 nmoles), glucose-6-phosphate de-hydrogenase (0.5 unit) and phosphatebuffer (pH 7.4, 0.05 M).

The mixture was preincubated for 5minutes at 37°,after which the reaction

was started by the addition of 50 nmolesof (3-14C)-HMG-CoA (specific radioactivity

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UNSATURATED OIL AND CHOLESTEROL METABOLISM 2153

275 dpm/nmole). The reaction was carriedout at 37°for 15 minutes with constantshaking and was terminated by the addition of 10 ¡uof 4 N NaOH (which alsohydrolysed the remaining thioesters).After adding 25 /¿Iof 10 MHC1 and smallamounts of unlabeled mevalonic acid, themixture was further incubated for anadditional 30 minutes for complete lacto-nization of the mevalonic acid. The precipitated proteins were centrifuged offand the product mevalonolactone wasseparated by TLC on silica gel C platesby using the solvent system, acetone:benzene (3:2, v/v), after which the regioncorresponding to that of standard mevalonolactone was scraped into scintillationvials for determination of the radioactivity.

Other methods. Extraction of the lipidsfrom the tissues and homogenates, separation of the neutral lipids, phospholipidsand bile acids, estimation of cholesterol,phospholipids, bile acids and proteins,analysis of the free fatty acids by GLCand counting of the radioactivity were asdescribed earlier (1).

RESULTS

Effect of the dietary oils on the fatty acidcomposition of the phospholipids andcholesteryl esters of the liver microsomes

Table 1 shows that the nature of thedietary oils markedly influenced the fattyacid composition of the liver microsomallipids of the rats. Thus safflower oil feeding led to a marked increase in the lino-leate values of both phospholipids andcholesteryl esters, which was mostly atthe expense of their oleate and palmitatecontents.

Bile flow and biliary excretion of cholesterol and bile acids. Table 2 showsthat the rate of bile flow was appreciablyhigher in the rats receiving the diet containing safflower oil and this is in generalagreement with our earlier findings (1).It can also be seen from the same tablethat excretion of cholesterol per milliliterof bile was markedly higher in the samerats, so that the total excretion of cholesterol and of the radioactivity in cholesterol over a period of 4 hours was also

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2154 C. S. RAMESHA, R. PAUL AND J. GANGULY

TABLE 3

Fecal excretion of neutral sterols

OilfedSOCODExpt. No.(i)

(¡i)(i)

(u)(0(ii)Neutral

sterols1mg/rat/day17.6

14.910.87

7.39.36

8.83

1Each figure is from pooled feces from six rats andthe results of two separate sets of experiments aregiven here.

markedly higher in these rats. Thus, while273.01 fig of cholesterol was found permilliliter of bile in the safflower oil-fedrats, the corresponding values were 118.23pig and 106.70 pig in the coconut oil-fedand Dalda-fed rats, respectively. Similarly,while 590.99 fig of cholesterol was excretedin 4 hours by the safflower oil-fed rats,the corresponding values were 221.18fig and 188.29 /tig in the coconut oil-fedand Dalda-fed rats, respectively.

The excretion of the total bile acids andof the radioactivity in the bile acids wasalso significantly higher in the saffloweroil-fed rats (table 2). It should, however,be noted that the difference in the bileacid values in the safflower oil-fed ratsand in the other rats was less marked thanin the case of cholesterol and the radioactivity in cholesterol.

Since cholic acid is the major bile acidof rat bile, the effect of feeding diets containing the three types of oils on the cholicacid and chenodeoxycholic acid contentsof the bile was investigated; the sametable (table 2) shows that the saffloweroil-fed rats had higher cholic acid permilliliter of bile, as a consequence ofwhich these rats excreted markedly higheramounts of cholic acid in 4 hours. Thus,while these rats excreted 9.77 mg ofcholic acid in 4 hours, the correspondingvalues for the coconut oil-fed and Dalda-fed rats were 5.77 mg and 5.62 mg., respectively. It can be further seen fromtable 2 that the conversion of the injectedradioactive cholesterol into cholic acid

was also significantly higher in the ratsgiven safflower oil. In sharp contrast, therewas no significant difference in the chenodeoxycholic acid values per milliliterof bile of the three groups of rats.

Fecal excretion of the neutral sterols inthe rats fed diets containing different oils

Table 3 shows that such marked difference in the excretion of cholesterolthrough the bile of the three groups ofrats was fully reflected in the neutralsterol contents of their feces. Thus, whilethe safflower oil-fed rats excreted 17.6 mgand 14.9 mg of neutral sterols per rat perday in the two sets of experiments, thecorresponding values for the coconut oil-fed and Dalda-fed rats were 10.87 mg,7.3 mg, 9.36 mg and 8.83 mg, respectively.

Cholesterol 7a-hydroxylase activity ofthe liver microsomes. The results presented so far have demonstrated thatfeeding safflower oil not only stimulatesbile flow and excretion of cholesterol andcholic acid through the bile, but alsoactivates conversion of cholesterol intocholic acid. Therefore, the activity of theenzyme cholesterol 7a-hydroxylase, whichis a rate-limiting enzyme in the pathwayof conversion of cholesterol into cholicacid, was examined in the liver micro-somes of these rats and the results presented in table 4 demonstrate that itsactivity was markedly higher in the safflower oil-fed rats, as compared to theother two groups.

TABLE 4

ChoIesterol-7a-hydroxi/I(i(ie activity'

Oil fed Enzyme activity

nmoles of 7a-hydroxycholesterolformedlmg protein/hr

SOCODP

valuesSOvsCOSOvs

DCOvs D2.92

±0.321.72±0.111.68

±0.31<0.01<0.01N.S.

' Values are mean ±SEM from six rats in eachgroup.

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UNSATURATED OIL AND CHOLESTEROL METABOLISM 2155

Incorporation of(l-liC)-acetate and(2-l4C)-mevalonate into the liver lipids

(l-ì4C)-Acetatein vivo. Although theseexperiments showed that the dietary saf-flower oil causes marked increase in thebiliary and fecal excretion of cholesteroland bile acids as well as increased conversion of cholesterol into cholic acid, itwas also possible that feeding of the un-saturated oil may inhibit cholesterol synthesis. But table 5 demonstrates that therate of incorporation of the injected 14C-acetate into the liver cholesterol wassignificantly higher in the safflower-oilfed rats, while its incorporation into thetotal fatty acids was more or less comparable in the three groups.

(2-l4C)-Mevalonate in vivo and in vitro.Table 6 shows that the incorporation ofthe injected radioactive mevalonate wassignificantly higher in the liver cholesterolof the safflower oil-fed rats, as comparedto the other two groups. Similarly, whenincubated with the liver homogenate, theincorporation of the radioactivity into thecholesterol was again markedly higher inthe same rats.

I4CO2 release from (l-i4C)-mevalonate

and mevalonatepyrophosphatedecarboxylase activity

Table 7 shows that both the rate of14CO,release from the radioactive meva-

TABLE 5

Incorporation, in vivo, of >4C-acetate into theliver cholesterol and fatty acids'

OilfedSO

CODP

valuesSOvsCOSOvs

DCOvs DRadioactivity

incholesteroldpm/g

liver3,411

±3881,689 ±1311,767

±151<0.02<0.01N.S.Radioactivity

intotal fattyacidsdpmlg

liver7,952

±1,4139,024 ±2,3047,611

±1,956N.S.N.S.N.S.

1Five microcuries of (l-l4C)-acetate was injectedintraperitoneally into each rat and the rest of theprocedure has been given in Materials and Methods.Values are mean ±SEMfrom 9-12 rats in each group.

TABLE 6

Incorporation, in vivo, and in vitro of (2-l4C)mevalonate into liver cholesterol*

Oil fed In vivo In vitro

dpm/g liver nmoles mevalonateincorporated per

mg proteinlhr

SOCODP

valuesSOvsCOSOvs

DCOvs D4,703

±2862,997±3473,175±412<0.02<0.02N.S.0.24

±0.010.10±0.010.13±0.01<0.001<0.001N.S.

1In vivo: 0.2 /tCi of (2-14C)-mevalonate wasinjected intraperitoneally into each rat and the restof the procedure is given in Materials and Methods.In vitro: The conditions of these experiments aregiven in Materials and Methods. Values are mean±SEMfrom 9-12 rats in each group.

lonate and the mevalonatepyrophosphatedecarboxylase activity were markedlyhigher in the safflower oil-fed rats. Buthere, while the effect on 14CO2releasewas very striking, that on the decarboxylase activity was less marked.

HMG-CoA reductase activity. Table 8shows that the activity of this enzymealso was strikingly higher in the ratsgiven the diet containing safflower oil.

DISCUSSION

We had earlier reported that feedingof highly unsaturated lipids in the formof oil or phopholipids causes markedincrease in bile flow as well as a higherconcentration of cholesterol in the bile ofrats; the results presented here not onlyagree with our earlier observations buthave further shown that the rate of bileflow and biliary excretion of cholesterolwere much higher here than the valuesreported by us earlier (1). It is possiblethat such differences might be due to thedifference in the mode of administrationof the oils in our earlier work and inthe present work. Thus, while in the ourprevious studies the oils were giventhrough a stomach tube, in the presentwork the oils were mixed with the dietand the rats were allowed to eat suchdiets ad libitum.

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2156 C. S. RAMESHA, R. PAUL AND J. GANGULY

TABLE 7

Rate of "COt release and mevalonatepyrophosphatedecarboxylase activity1

Mevalonate-pyrophosphatedecarboxylase

Oil fed UCO2 release activity

nmoles mevalonate nmoles MCO2

converted per released permg protein/hr mg proteinlhr

SOCODP

valuesSOvsCOSOvs

DCOvsD12.41

±1.186.23±1.547.44±0.76<0.05<0.02N.S.17.80

±0.9914.19±2.6014.13±0.90<0.02<0.05N.S.

1Values are mean ±SEM from five to six rats ineach group.

It has also been shown here that, notonly biliary excretion of cholesterol washigher in the safflower oil-fed rats, butsuch rats excreted higher amounts of bileacids also, particularly cholic acid, whichwas reflected in the results of the experiments where biliary excretion of theradioactive bile acids was investigatedin the animals given intraportal injections of the 14C-labeled cholesterol. The

present results have further shown thatthe activity of the rate-limiting enzyme inthe biogenesis of bile acids—cholesterol-7a-hydroxylase—is higher in the ratsgiven dietary safflower oil.

These findings agree with the previousobservations of McGovern and Quacken-bush (11), who had reported increasedconversion of intraportally injected UC-cholesterol into bile acids in the rats fedsafflower oil, as compared to those fedbeef tallow. But in contrast to the presentfindings, these workers observed no appreciable difference in the total bileacid contents (cholic acid plus cheno-deoxycholic acid) in the bile of the twogroups of rats. Moreover, while McGovernand Quackenbush (12) had found loweramounts of chenodeoxycholic acid in thebile of the safflower oil-fed rats, we haveshown here that the chenodeoxycholicacid values were virtually comparablein the bile of the three groups of rats.

Such differences between our observations and those of McGovern and Quackenbush (12) could be due to the differencein the diets used. Thus, while they hadused high-cholesterol diets, in our workno cholesterol was added to the diets.It may be mentioned in this context thatearlier turnover studies of injected radioactive bile acids in humans (13, 14) and inrats (12) had also shown higher rates ofsynthesis of cholic acid on unsaturatedfat feeding.

The results reported here have furthershown that feeding of the highly unsaturated oil effected a marked increasein the fecal excretion of neutral sterols.It should be recalled in this context thatNestel et al. (15, 16) previously demonstrated that intake of meals containinghighly unsaturated fat leads to significantly higher fecal excretion of neutralsterols in humans. These aspects havebeen extensively discussed by us in arecent review, where we have pointed outthat markedly increased biliary and fecalexcretion of cholesterol should explainthe cholesterol-lowering effects of poly-unsaturated lipids (2).

On the other hand, an alternative mechanism could be inhibition of cholesterolsynthesis in animals given polyunsaturatedfats. In fact, in the past, several groupsof workers had studied the effect ofdifferent types of fats on cholesterolsynthesis in animals. Thus, Avigan andSteinberg (17) had shown higher incorporation of I4C-acetate or T2O into the liver

TABLE 8

HMG-CoA redactase activity1

Oil fed Enzyme activity

nmoles mevalonate formedper mg protein/minute

SOCODP

valuesSOvsCOSOvsDCOvsD319.83

±21.88169.17±21.16142.67±22.14<0.001<0.001N.S.

1Values are mean ±SEM from six to seven ratsin each group.

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UNSATURATED OIL AND CHOLESTEROL METABOLISM 2157

cholesterol of rats given diets containing20% corn oil, while Mukherjee and Alfin-Slater (18) had claimed similar increasedincorporation of 14C-acetate into the livercholesterol of rats receiving 15% cottonseed oil in their diet. Later work of Dupont (19) with rats given 42% corn oiland of Serdarvich and Carroll (20) withrats given 15% corn oil had also shownsimilar activation of cholesterol synthesisin the liver of such rats. In contrast tosuch observations there have been someclaims that feeding of unsaturated fatsinhibits cholesterol synthesis (21-23).But the animals used and the experimental conditions were rather differentin these cases.

Against this background, we demonstrated here that incorporation of (1-14C)-acetate and (2-14C)-mevalonate into theliver cholesterol, as also the rate of 14CO2release from (l-14C)-mevalonate and theactivities of HMG-CoA reducíase andmevalonatepyrophosphate decarboxylasewere all consistently and markedly higherin the livers of the rats receiving saffloweroil. But such activation of cholesterolsynthesis in the rats fed diets containingpolyunsaturated oils is probably to beexpected because, it is well establishedthat feeding of cholesterol or bile acidscauses feedback inhibition of cholesterolsynthesis in the liver of rats (24-26).Since polyunsaturated oil feeding drainsout significant amounts of cholesterol andbile acids through bile and eventuallythrough the feces, it follows that suchconstant removal of cholesterol and bileacids from the liver should lead to activation of cholesterol synthesis in such rats.Therefore, it is clear that in spite of activation of cholesterol synthesis, feeding ofpolyunsaturated oils effectively lowersserum cholesterol levels, which wouldobviously mean that the markedly increased biliary and fecal excretion ofsterols far outweighs the activation ofcholesterol synthesis in these rats.

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