cholesterol synthesis by the gastrointestinal tract: local- ization

Journal of Clinical InvestigationVol. 44, No. 8, 1965

Cholesterol Synthesis by the Gastrointestinal Tract: Local-ization and Mechanisms of Control *JOHN M. DIETSCHY t AND MARVIN D. SIPERSTEIN :

(From the Department of Internal Medicine, The University of Texas Southwestern MedicalSchool, Dallas, Texas)

The synthesis of cholesterol has been demon-strated in virtually every tissue of the mammalianbody with the important exception of the maturenervous system (1). Yet studies in this field haveprimarily centered about cholesterogenesis in theliver for at least two important reasons: first, earlyreports suggested that the liver was the sole en-dogenous source of circulating cholesterol (2), andsecondly, the rate of hepatic cholesterol synthesiswas shown to be controlled by a sensitive feedbackmechanism of considerable practical and theoreticalinterest (3, 4). By contrast, little information isavailable concerning either the physiologic im-portance or the means of regulation of cholestero-genesis in the intestine. Yet the recent report ofLindsey and Wilson (5), which unequivocallydemonstrates that the intestine contributes to thecirculating cholesterol pool, emphasizes the needfor a more thorough evaluation of cholesterol syn-thesis by the gastrointestinal tract. The presentstudies, therefore, were undertaken to investigatethree aspects of intestinal sterol synthesis: 1) char-acterization of synthetic rates along the length ofthe gastrointestinal tract, 2) localization of the syn-thetic site within the small bowel wall, and 3) eval-uation of mechanisms operative in the control ofintestinal cholesterogenesis.

* Submitted for publication December 28, 1964; ac-cepted April 22, 1965.This investigation was supported by U. S. Public

Health Service research grants AM 03906 from the Na-tional Institute of Arthritis and Metabolic Diseases andCA 05090 from the National Cancer Institute and grantDRG 747A from the Damon Runyon Memorial Fund forCancer Research.t Postdoctoral fellow, National Institute of Arthritis

and Metabolic Diseases. Presently in the Gastroenterol-ogy-Liver Section, University of Texas SouthwesternMedical School.

i: Recipient of Research Career Award, National HeartInstitute. Address requests for reprints to Dr. MarvinD. Siperstein, Dept of Internal Medicine, University ofTexas Southwestern Medical School, Dallas, Texas 75235.

Methods

Animal preparation. Female Sprague Dawley ratsweighing 180 to 200 g were used in the present study.Preliminary experiments with male rats showed no sig-nificant difference in data obtained from either sex. Allanimals were allowed water and Purina rat chow adlibitum before being killed or operated upon. In someexperiments groups of animals were given a ground ratdiet to each 100 g of which was added 5 g of cholesteroldissolved in 5 g of oleic acid; the control diets for thesestudies contained an equivalent amount of oleic acid butno added cholesterol. Bile fistula animals were preparedunder ether anesthesia by intubation of the common bileduct with a polyethylene catheter (o.d., 0.038 inch) thatwas exteriorized through a dorsal stab wound. Controlanimals were similarly operated upon except that thecommon duct was not entered. Both bile fistula andsham-operated animals were maintained in restrainingcages postoperatively and allowed free access to rat chowdiet and water unless otherwise specified in the Resultssection. There was no significant difference in the netfood intake between the two types of animal.

Bile fistula and sham-operated animals were also pre-pared with indwelling intestinal catheters. Polyethylenetubing (o.d., 0.050 inch) was inserted about 1 cm into theintestinal lumen through a puncture wound in the anti-mesenteric border of the bowel and secured with a purse-string suture. The catheter was also sutured superficiallyto the serosa of the intestine to keep the tip directedcaudally within the lumen, and the other end of the tubewas brought to the outside of the animal through a dorsalstab wound. Perfusion of the intestinal lumen was per-formed through the indwelling catheter by a constantrate infusion pump 1 at a rate of 1.0 ml per hour.

Tissue preparation. Each animal was killed by decapi-tation and the gastrointestinal tract immediately excisedand placed in cold Krebs bicarbonate buffer (6). Onlythe distal half (glandular portion) of the stomach wasused in these experiments. The entire small bowel wasdivided into 10 equal parts of about 10 to 13 cm in length,and the intestinal contents were removed from each seg-ment by flushing with cold buffer. These segments werenumbered from one to ten, number one being the mostproximal segment. The cecum and large bowel werealso cleansed with buffer; the large bowel was separatedfrom the cecum and divided into three equal segments that

1 B. Braun, Melsungen, West Germany.

1311

JOHN M. DIETSCHY AND MARVIN D. SIPERSTEIN

were numbered from one to three, number one again be-ing the most proximal segment. One-millimeter thickslices were then prepared with a McIlwain tissue slicerfrom each portion of the gastrointestinal tract understudy. Four hundred mg of slices was placed in incuba-tion flasks that contained 5.0 ml of Krebs bicarbonatebuffer (pH 7.4) previously gassed with 95%o oxygen: 5%C02, 1.0 yc of acetate-2-C", and 10 /moles of sodium ace-tate. The flasks were gassed with 95%o oxygen: 5% C02,capped, and placed in a metabolic shaker at 370 C for 2hours.

In order to compare the metabolism of various portionsof the small bowel wall a scraping technique was de-vised that allowed the subdivision of the wall into rela-tively pure preparations of villi, crypts, and smoothmuscle. After flushing a bowel segment with buffer, itwas opened along the mesenteric border, laid with the mu-cosal side up on a glass plate, and kept moistened withcold buffer. Lightly scraping the mucosa with the edgeof a glass microscope slide removed most of the villi.A more forceful scraping removed a second tissue layerthat contained most of the intestinal crypts and left onlythe denuded smooth muscle layer. The villi and cryptswere suspended directly in the buffer mixture while thesmooth muscle layer was first sliced then incubated asdescribed above. The histology of these intestinal layerswas determined on formalin fixed, hematoxylin and eosinstained sections of each preparation.Chemical methods. Methods for determining the in-

corporation of radioactive acetate into digitonin pre-cipitable sterols, long chain fatty acids, and carbon dioxidehave been previously described in detail (3). Brieflythese procedures may be outlined as follows: After incu-bation, the contents of the flask were acidified with 1 NH2SO4, and the C02 evolved was trapped in 1 ml of 1 NNaOH previously placed in the center well of each flask.A sample of this alkali solution was counted in thescintillation fluid described by Bray (7). The flasks'contents were then saponified, made up to a 50% ethanolicsolution, and extracted with petroleum ether to removenonsaponifiable lipids. After acidification, the residue wasnext extracted with pentane to remove acidic lipids.These pentane extracts were backwashed with water, anda sample was counted in a scintillation fluid containing0.3%o diphenyloxazole and 0.015% p-bis-phenyloxazolyl-benzene in toluene (DPO-POPOP solution). Sterolswere precipitated from the petroleum ether extracts asthe digitonide, dehydrated with acetone, washed with di-ethyl ether, and dissolved in methanol. A sample of thissolution was placed in DPO-POPOP scintillation fluidfor C14 assay. All samples were counted in a Packardliquid scintillation counter, series 314E. The data are ex-pressed as the millimicromoles of acetate-2-C4 incorpo-rated into digitonin precipitable sterols (DPS), fattyacids (FA), and carbon dioxide (CO2) per gram wetweight of tissue or, in some instances, as the C" countsper minute found in each of these fractions.For purposes of fractionation of the digitonin precipi-

table sterols by thin layer chromatography (TLC), thedried sterol digitonides were first dissolved in 1.0 ml of

ethanol that had been saturated with a 90%o potassiumhydroxide solution. The free sterol was then extractedfour times with petroleum ether; each ether extract wasfiltered and taken to dryness under nitrogen. The sterolmixture was redissolved in a small volume of chloroformand spotted on 20- X 40-cm plates coated with either silicagel G 2 or a mixture of silica gel G and silver nitrate.Three different solvent systems were employed to de-velop the thin layer chromatograms. 1) Separation oflanosterol, A7- and Y8-methostenol, A7-cholestenol, and anarea containing both cholesterol and cholestanol was car-ried out on plates coated with silica gel G using benzene:ethyl acetate, 5: 1 (8). 2) In instances in which resolu-tion of A7-cholestenol from the cholesterol-cholestanolspot was inadequate, these areas were respotted on silicagel G coated plates and run in benzene: ethyl acetate,20: 1 (8). A pad of filter paper was clamped to thetop of each of these plates to allow the developing fluid toascend continuously for 36 hours. 3) Separation of cho-lestanol from cholesterol was accomplished on silver ni-trate-silica gel G plates run in benzene: hexane, 15: 85(9). Individual spots were scraped from the plates andcounted directly in the DPO-POPOP scintillation fluidusing an internal standard to correct for quenching.Duplicate spots were eluted with ethanol, dried, redis-solved in chloroform, and analyzed by gas-liquid chroma-tography (GLC) using a 6-foot column packed withGaschrom P coated with 0.75%o neopentyl glycol succinateat 2300 C with a gas flow of 100 ml per minute (10).Tentative identification of individual spots was made onthe basis of a comparison of Rf values and retention timeswith standard sterols in both the TLC and GLC systems,respectively.

Results

Sterol synthesis along the length of the gastro-intestinal tract. Initial studies were carried out toevaluate variations in the sterol synthetic activityat different levels of the gastrointestinal tract.Whole-wall-thickness slices were made from theesophagus, glandular stomach, and small and largebowel, and the ability of each of these tissues to in-corporate acetate-2-C4 into digitonin precipitablesterols per gram wet weight of tissue was deter-mined. As demonstrated in Figure 1, all portionsof the gastrointestinal tract from the esophagus tothe terminal large bowel were capable of incor-porating acetate-2-C'4 into digitonin precipitablesterols. The glandular stomach was approxi-mately 2.5 times more active than the esophagus.Small bowel synthetic activity typically variedmarkedly at different levels along its length.The proximal half of the small intestine was con-sistently the least active area in the entire gut,

2 Brinkmnan Instruments Inc., Westbury, N. Y.

1312

CHOLESTEROL SYNTHESIS BY THE GASTROINTESTINAL TRACT

1 2 3 4 5 6 7 8 9 10

Small Bowel Segments

E 1 2 3w Colon

c-' Segments

FIG. 1. CHOLESTEROL SYNTHESIS AT DIFFERENT LEVELS OF THIE GASTROINTESTINAL TRACT.This diagram compares the ability of tissue slices prepared from different levels of the gastro-intestinal tract to incorporate acetate-2-C"4 into digitonin precipitable sterols (DPS) per gramwet weight of tissue during a 2-hour incubation period. The small and large bowels have beensubdivided into 10 and 3 segments of equal length, respectively.

converting only 10 to 35 mbtmoles of acetate-2-C14to digitonin precipitable sterols per gram of tis-sue. Activity rapidly increased, however, in moredistal segments, and maximal incorporation oc-curred in the terminal three ileal segments where90 to 130 mptmoles of acetate-2-C14 was convertedto digitonin precipitable sterols per gram of tis-sue. The cecum was relatively inactive, but, again,the rate of incorporation increased in more distalsegments of the colon.

Sterol synthesis in different layers of the smallbowel wall. In order to obtain tissue preparationsof a more uniform cell type the intestinal wall wasdivided into several functionally distinct layers,and synthetic rates were determined in each ofthese different preparations. The data presentedin Table I show the mean values of two experi-ments. Three contiguous six-cm long segmentsof mid-jejunum were cut from the intestine of anormal animal. One segment was sliced in totoand placed in an incubation flask (designated

"whole wall" in Table I). A second segment wasopened along its mesentary border, and the villiwere separated from the crypts and muscle; thesetwo preparations were placed in incubation flasksand are designated as "villi" and "crypts and

TABLE I

Incorporation of acetate-2-C'4 into carbon dioxide (CO2),digitonin precipitable sterols (DPS), and fatty acids

(FA) by different tissue fractions of the jejunal andileal wall*

Acetate-2-C'4 incorporated into C02, DPS,and FA per 2-hour incubation

Jejunum IleurnTissuelayer C02 DPS FA C02 DPS FA

cpm X 10-3Whole wvall 684 22 27 554 40 29Villi 92 0.03 0.04 36 < .01 0.25Crvpts and

muscle 423 16 14 370 29 25Muscle 56 0.12 0.36 69 0.04 0.23

* Each fraction represents the amount of tissue obtainedfrom a 6-cm length of intestine.

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1313


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MUSCLEAl

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~~$)t~~~~~~:4V~~~~~t.% .. .Y?9.. -L~~~~~~~~~~~~2d

FIG. 2. PHOTOMICROGRAPHS OF TISSUE LAYER PREPARATIONS OF THE SMALL BOWEL WALL. These photomicrographsillustrate the histology of each of the tissue fractions prepared from the wall of both the jejunum and ileum as de-scribed in the text. The relative synthetic activity of each of these fractions is given in Table I.

ILEUM

1314


TABLE II

Incorporation of acetate-2-C14 into digitonsn precipitablesterols by jejunal and ileal villi, crypts, and smooth

muscle*

Acetate-2-CU incorporation into DPS

Experi- Experi-ment 1 ment 2

cpmJejunum Villi 67 110

Crypts 632 666Muscle 34 57

Ileum Villi 158 24Crypts 4,640 4,550Muscle 22 41

* Each fraction represents the amount of tissue obtainedfrom a 5-cm length of intestine.

muscle," respectively, in Table I. The third seg-ment was similarly opened and vigorously scrapedto remove all tissue except the smooth muscle.This was sliced for incubation and is designated as"muscle" in Table I. Similar preparations weremade from three 6-cm segments of mid-ileum. Asmall portion of each tissue fraction was used forhistologic examination (Figure 2), and the re-mainder was evaluated for its ability to incorporateacetate-2-C'4 into digitonin precipitable sterols,carbon dioxide, and fatty acids.As is evident from the data in Table I, all tissue

fractions oxidized significant amounts of acetateto carbon dioxide, yet these same tissue layerswere not all capable of active lipid synthesis. Thevilli preparations, which histologically containedvirtually no extraneous tissue (Figure 2), did notincorporate acetate into either digitonin precipi-table sterols or fatty acids to any significant degree.In contrast, crypt-muscle preparations activelysynthesized labeled digitonin precipitable sterolsand fatty acids, and, indeed, synthesis of these lip-ids approached the rates observed in the wholewall preparations. Histologically (Figure 2)roughly half of the tissue bulk of the crypt-musclelayer was made up of intestinal crypts while halfwas smooth muscle. That the intestinal smoothmuscle alone incorporated little acetate into digi-tonin precipitable sterols or fatty acids is apparentfrom the data in the last line of Table I. Theseexperiments strongly implied that the cells of thecrypt layer of the intestinal wall were the site ofincorporation of the major portion of acetate-2-C14into digitonin precipitable sterols and fatty acids.

It was obviously desirable, therefore, to studysterol synthesis in isolated crypts. Table II showsthe results of two experiments in which five-cmlong pieces of jejunum and ileum were fractionatedinto villi, crypts, and smooth muscde and evaluatedas to their synthetic ability. Of the three intestinallayers, the isolated crypts showed by far the high-est rates of incorporation of acetate-2-C14 into digi-tonin precipitable sterols. In the case of the ileum,sterol synthesis in the crypt cells was at least 30times that in the villi or the muscle layers. Theresults of this and the preceding experiments,therefore, dearly demonstrate that the major siteof intestinal sterol synthesis lies in the crypt celllayer of the gut.

Influence of prolonged cholesterol feeding andfasting upon sterol synthesis by the bowel. Bothfasting (11) and cholesterol feeding (12-15) areknown to produce profound depressions of hepaticcholesterogenesis, effects that imply importantphysiological mechanisms for over-all sterol bal-ance within the intact organism. The effect ofboth of these dietary manipulations upon choles-terol synthesis in the intestine was therefore evalu-ated. Four animals were placed on a 5%o choles-terol diet for 6 weeks while four other animalswere placed on a control, cholesterol-free diet. Athird group of four animals was deprived of foodbut allowed water for 48 hours before being killed.Representative sections were taken from each ani-mal's duodenum, mid-jejunum, mid-ileum, trans-verse colon, and liver and tested for their ability toincorporate acetate-2-CG4 into digitonin precipitablesterols. The mean results (± 1 SE of the mean)of these studies are shown in Table III. As ex-pected, hepatic sterol synthesis was markedly de-

TABLE III

Effect of cholesterol feeding and of fasting upon digitoninprecipitable sterol synthesis by different portions of the

gastrointestinal tract

Acetate-2-C4 converted to DPS per gramtissue (mean 4 animals ± 1 SE)

Cholesterol-fed FastedTissue Control 6 weeks 48 hrs

mpmoiesDuodenum 17 i 4 16 ± 4 17 ± 3Jejunum 15 i 10 8 + 5 7 ± 2Ileum 119 ± 38 89 ± 17 69 i 9Colon 38 ± 9 60 4 4 33 ± 6

Liver 232 ± 66 1.2 ± 0.3 15 ±i 3

1315


pressed by both cholesterol feeding (to 0.5% ofcontrol) and by fasting (to 6.5% of control); bycontrast, in no instance was sterol synthesis byany portion of the bowel depressed to a similar de-gree by these dietary manipulations.

However, in the preceding experiment acetate-2-C14 incorporation was measured only in the totaldigitonin precipitable sterols; it was possible,therefore, that the specific synthesis of cholesterolwas inhibited but that this effect was masked bythe simultaneous accumulation or overproductionof another labeled 3-,8-OH sterol. For this reason

thin layer chromatography was used to determinethe relative labeling of specific sterols in the digi-tonin precipitable fractions in each of the experi-mental groups. These data are reported in ex-

periment 1 of Table IV. The incorporation of ace-

tate-2-C14 into specific sterols was similar in thethree groups of animals, and most importantly,cholesterol synthesis in the intestines of the choles-terol-fed and fasted rats was not significantly dif-ferent from that in the intestine of the control ani-mal. Thus, cholesterol synthesis by the intestine,in striking contrast to the situation in the liver, ap-

pears to be virtually insensitive to both prolongedcholesterol feeding and prolonged fasting.

Effects of bile upon intestinal sterol synthesis.During the course of studies on the feedback con-

trol of cholesterol synthesis it was noted that in-testinal slices from animals with bile fistula were

approximately 10 times more active in incorpo-rating acetate-2-C14 in the digitonin precipitablesterols than were slices taken from animals withintact biliary systems. This finding was extendedby comparing the mean rates of incorporation ofacetate-2-C14 into digitonin precipitable sterols,

fatty acids, and carbon dioxide by intestinal slicesfrom four sham-operated control animals and fromfour animals with biliary diversion of 48 hoursduration. These data are shown in Figure 3;the striking stimulation of small bowel sterol syn-

thesis produced by biliary diversion is apparent inevery segment and amounted to as much as 13-foldin the middle segments. By contrast, neither thestomach nor colonic segments showed this effect.The increase of sterol synthesis produced by di-version of bile is clearly not due to a nonspecificstimulation of small bowel mucosal metabolismsince, as shown in the lower two panels of Figure3, fatty acid synthesis was actually somewhat de-pressed in the small intestine of the fistula animals,whereas the oxidation of acetate to carbon dioxidewas nearly identical at all levels of the gastroin-testinal tract in both experimental groups. Whenthe digitonin precipitable sterols synthesized bythe intestine of an animal with a bile fistula were

fractionated into specific sterols by thin layer chro-matography, it could be shown that the synthesisof all sterols examined was increased by bile di-version; however, the marked increase in choles-terol synthesis was clearly the major cause for theenhanced sterol synthetic activity (experiment 2,Table IV).

Localization of the bile effect upon intestinalcholesterogenesis. Although the experiments justdescribed clearly imply that bile contained a po-

tent inhibitor of intestinal cholesterogenesis, theydid not distinguish whether this inhibitor actslocally upon the mucosal cells or whether it under-goes an enterohepatic circulation and so suppressesintestinal sterol synthesis by virtue of its presence

in the blood. These two possibilities were exam-

TABLE IV

Incorporation of acetate-2- C'4 into specific sterols by intestinal slices obtainedfrom the ileum (segment 8) ofa control, cholesterol-fed, and fasted animal (experiment 1) as well as from an animal with biliary diversion (experiment 2)

Acetate-2-C14 converted to specific sterols per gram tissue

MethostenolExp. Animal preparation Cholesterol (W7 and As) Cholestanol A7-Cholestenol Lanosterol

mumolesControl 73 3.6 0.9 1.8 3.6

1 Cholesterol-fed, 90 2.5 1.2 1.2 3.74 weeks

Fasted, 48 hrs 62 1.6 0.8 0.8 3.3

2 Bile fistula, 220.1 24.7 13.7 16.548 hrs

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1 ' 2 ! 3 4 5 6 ' 7 8 9110 'r 1* 2 3Smal I Bowel Segments ' ColonE Segments

FIG. 3. EFFECT OF BILE UPON GASTROINTESTINAL STEROL AND FATTY ACID SYNTHESIS.The data in this diagram compare the ability of gastric and intestinal slices obtained fromsham-operated rats and similar slices taken from animals that had biliary diversion for 48hours to incorporate acetate-2-C" into digitonin precipitable sterols (DPS), fatty acids(FA), and carbon dioxide (COs) per gram of tissue during a 2-hour incubation period.

1317


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A. Control B Bi leF i stu a

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D. Bile E Bile DuctInfusion Ligation

I I I I I I I1 I I 1_1 15 6 7 B 9 10 5 6 7 B 9 10 5 6 7 8 9 10 5 6 7 B 9 10

Small Bowel Segment by Number

FIG. 4. LOCALIZATION OF TIHE INII IBITORY EFFECT OF BILE UPON SAMALL 13BOWEL DI(ITONIX PRE-CIPITABLE STEROL SYNTIHESIS. These data show a comparison of the digitonin precipitable sterol(DPS) synthetic activity in the six terminal small bowel segments of animals in five differentexperimental situations as described in the text.indwelling intestinal catheter at the junction ofarrows.

mned in the experiments designated A throughl E inFigure 4. For purposes of comparison experimentA demonstrates the level of incorporation of ace-tate-2-C14 into digitonin precipitable sterols in theterminal six segments of the small bowel of a sham-operated control rat with an intact biliary system.In this and the following experiments only the dis-tal six segments of the small intestine were used,since in the animal with a bile fistula synthetic ac-tivity was maximal and almost uniform in thesesegments (segments 5 to 10). The animal in ex-periment B had a biliary fistula for 48 hours be-fore being killed and demonstrates the expectedincrease in synthetic activity. Animals C and Dboth had biliary fistulae but, in addition, indwellingintestinal catheters were placed so as to enter thebowel at the junction of small bowel segments 7and S. By means of these catheters, isotonic sa-line (in C) and rat whole bile (in D) were infnsedinto the lumen of the terminal three segments ofthe small bowel, leaving the proximal segments(5 to 7) free of bile. The rate of infusion was 1.0

In experiments C and D each animal had ansegments seven an(l eight as indicated by the

nml per hour, and each infusion was continued for48 hours before sacrificing the animal. The incor-poration of acetate-2-C14 into digitonin precipitablesterols by the small bowel of animal C was at theusual high level observed in bile fistula animals;this experiment demonstrated that the trauma ofan intestinal catheter and saline infusion in no wayinfluenced the enhanced synthesis of the terminalthree segments. In contrast, however, the infu-sion of whole bile into the bowel in experiment Dresulted in a marked suppression of synthetic ac-tivity in the terminal three segments (segments 8,9, and 10), whereas the three segments proximalto the point of infusion (segments 5, 6, and 7) in-corporated acetate-2-C14 into digitonin precipitablesterols at the uninhibited rate. These results sug-gested that the inhibitory effect of bile involves adirect action npon the cells of the mncosa whichsynthesize the digitonin precipitable sterols.

However, to evaluate further a possible systemiceffect of the biliary inhibitor of sterol synthesis, afifth animal had a simple bile duct ligation 5 days

1318


before being sacrificed (experiment E). All tis-sues of this animal including the gastrointestinaltract were grossly bile stained, yet the terminal sixsegments of small bowel incorporated approxi-mately 350 mnmoles of acetate-2-CG4 into digitoninprecipitable sterols per gram of tissue, a rate equalto that found in the intestine of an animal with ex-ternal biliarv drainage. These data strongly sup-ported the concept that the inhibitor in bile hasno systemic action, but is effective only when di-rectly in contact with the onucosal surface of thesmall bowel.

Inl animals with biliary fistula, inhibition ofsterol synthesis at any level of the small bowelcould be produced ly the infusion of whole bilefor 48 hours. Three such experiments are shownin Figure 5. In animals A and C whole bile wasinfused through indwelling intestinal catheters atthe junction of small bowsel segments 1 and 2 and

7 and 8, respectively. In experiment B a shortpolyethylene catheter drained bile from the ani-mal's common duct into its small bowel at thejunction of segments 5 and 6, thus leaving seg-ments 1 to 4 free of bile. The results in all threeexperiments were identical; proximal to the pointof infusion there was enhanced sterol synthesis,whereas distally, in the segments perfused withbile, there was marked suppression of acetate-2-C14incorporation into digitonin precipitable sterols.In segments proximal to the infusion point thesynthesis of digitonin precipitable sterols becameelevated to levels characteristic of the synthetic ac-tivitv of these segments in animals with total bili-ary drainage alone, viz., the gradient of syntheticactivity along the length of the small bowel of ani-mals with bile fistula noted in Figure 3 was againobserved.

Nature has provided an experimental situation

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Small Bowel Segment by Number

FIG. 5. DEPRESSIVE EFFECT OF BILE UPON DIGITON-IN- PRECIPITABLE STEROL SYNTHESIS AT

DIFFERENT LEVELS OF THE SMALL BOWEL. In experiments A and C, bile was infused at twodifferent levels of the small bowel by indwelling intestinal catheters, whereas in experi-ment B bile was allowed to flow from the animal's common duct to its mid-small bowelby an internal plastic fistula. The placement of the intestinal catheters with regard to thesmall bowel segments is indicated by the arrows.

1319


similar to that described above in that the commonbile duct enters the small bowel slightly distal tothe gastroduodenal junction. That short initialsegment of small bowel proximal to the ampullaof Vater should not be perfused with so high aconcentration of bile as those portions of the du-odenum immediately distal to the entrance of thecommon duct. In two normal animals this 1.2-cmsegment of duodenum between the gastroduodenaljunction and the ampulla of Vater was carefullydissected out. The rate of incorporation of acetate-2-C14 into digitonin precipitable sterols was com-pared between slices from this proximal segment(segment A) and slices obtained from three con-tiguous segments of duodenum of similar lengthtaken just distal to the entrance to the commonduct (segments B, C, and D). As shown in Fig-ure 6, in both experiments the proximal segmenthad 15 to 20 times the synthetic activity as didany of the next three segments. This observationadds further evidence for a direct inhibitory effect

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FIG. 6. DIGITONIN PRECIPITABLE STEROL SYNTHESIS IN

SPECIFIC SEGMENTS OF THE DUODENUM. In two separateexperiments the duodenum was divided into four seg-

ments, each approximately 1.2 cm long. The most proxi-mal segment (segment A) was that portion of the du-odenum between the gastroduodenal junction and theampulla of Vater, and the next three sections (segmentsB, C, and D) comprised the remainder of the duodenumbetween the entrance of the common duct and the du-odenal-jejunal junction.

of bile upon the sterol synthetic activity of thesmall bowel mucosa.Time sequence for bile inhibiton of intestinal

digitonin precipitable sterol synthesis. In orderto determine the rapidity with which bile infusioninhibited intestinal sterol synthesis, seven rats wereprepared with bile fistula and indwelling intestinalcatheters placed at the junction of intestinal seg-ments 7 and 8. After 48 hours of biliary drainage,when the small bowel of each animal was presumedto be synthesizing sterols at its uninhibited rate,infusions were begun through the intestinal cathe-ters at a rate of 1.0 ml per hour. Initially, all ani-mals were begun on infusions of isotonic saline,but at different times during the subsequent 72hours, rat whole bile was substituted for the saltsolution. Thus, when the animals were sacrificedall had had their terminal three small bowel seg-ments perfused for a total of 72 hours; in indi-vidual animals the infusate was whole bile for aduration of 0, 3, 6, 12, 24, 48, and 72 hours beforetermination of the experiment. The small bowelof each animal was removed, and the three seg-ments proximal and distal to the infusion pointwere sliced and incubated with acetate-2-C14. Theper cent of inhibition of synthetic activity in theperfused segments was determined by comparingthe mean values of the millimicromoles of acetate-2-C14 incorporated into digitonin precipitable ster-ols by the terminal three segments (segments 8, 9,and 10) with the mean values of the millimicro-moles of acetate-2-C14 incorporated by the threeproximal segments (segments 5, 6, and 7). Thesedata are plotted in Figure 7. No inhibition oc-curred with bile infusions of 6 hours or less; attimes greater than this inhibition became manifestreaching apparent maximal values at about 48hours.

Possible mechanisms of bile inhibition of sterolsynthesis. When bile was diverted from the gas-trointestinal tract, the experimental animals ab-sorbed fat poorly and developed semisolid, bulkystools. It was possible, therefore, that the stimula-tion of digitonin precipitable sterol synthesis wasonly an indirect consequence of this malabsorption.Alternatively, the absence of bile and presence ofexcess fat in the gut lumen could allow abnormalbacterial overgrowth, and this in turn might en-hance sterol synthesis by the intestinal mucosa.

In order to test both of these possibilities the

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20 -

40 -

60 -

80 -

100 I- I

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Hours of Perfusion of Small Bowel

48

with Bi e

72

FIG. 7. TIME COURSE FOR THE INHIBITION OF INTESTINAL DIGITONIN PRECIPITABLE

STEROL SYNTHESIS BY BILE. Bile was infused into the small bowel of rats via indwellingintestinal catheters (at the junction of segments seven and eight) for varying periods oftime. Then a comparison was made of the DPS synthetic activity in segments proximaland distal to the infusion point. Any decrease in the rate of incorporation of acetate-2-C1'into DPS by the perfused distal segments was expressed as a per cent of the syntheticrate in the uninhibited proximal segments.

experiments illustrated in Figure 8 were carriedout. After a 24-hour fast two animals were oper-

ated upon: a bile fistula was placed in one whilethe second was sham operated. Both were placedin restraining cages and allowed only water for 48hours, at the end of which time they were sacri-ficed and intestinal slices were prepared. The in-fluence of intestinal bile on the incorporation ofacetate-2-C14 into digitonin precipitable sterols isstill apparent in such fasted rats (Figure 8A) even

though the bowels of these animals were essentiallyempty throughout the experimental period. Itseemed unlikely, then, that any product of the mal-digestion of dietary fat acted as a stimulating agentfor sterol synthesis.

In order to test the second possibility, two ani-mals were begun on oral antibiotics (polymixin B,8 mg, and bacitracin, 5,000 U, per 24 hours) for24 hours before being operated upon. Biliary di-version was then performed in one animal whilethe second was sham operated. Both animals were

allowed food and water ad libitum for 48 hours,and in addition, both were given their daily dose ofantibiotics in the drinking water. The animalswere sacrificed at the end of 48 hours, and sterolsynthesis in the intestine was assayed. Quantita-tive bacterial counts were carried out on the in-testinal contents of the sixth intestinal segment.For comparison bacterial counts were also madeof the contents of the sixth intestinal segment of a

sham-operated and fistula-containing rat nottreated with antibiotics. The data in Figure 8B

TABLE V

Bacterial counts in the sixth small bowel segment of controland antibiotic treated rats

Logio bacteria percm of intestine

AntibioticControl treated

Sham-operated animals 7.40 3.78Biliary fistula animals 8.08 3.25

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FIG. 8. EFFECTS OF BILIARY DIVERSION UPON INTESTINAL DIGITONIN PRECIPITABLE STEROL SYN-

THESIS IN FASTED AND ANTIBIOTIC TREATED ANIMALS. These data show the effects of fastingand antibiotic treatment upon the enhancement of small intestinal DPS synthesis produced bybiliary diversion. In experiment A the animals were fasted for 24 hours before operation andthroughout the 48-hour experimental period. Similarly, in experiment B, each animal was be-gun on antibiotics 24 hours before being operated on, and the same dosage of drugs was giventhroughout the 48-hour experimental period.

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show that the small intestine from the animal withbiliary diversion still had enhanced labeling of thedigitonin precipitable sterol even though antibiotictreatment had reduced the bacterial count of itsmid-small bowel by a factor of nearly 105 belowthat found in a similar animal with no antibiotictreatment (Table V) ; furthermore, the bacterialcounts in both the fistula-containing and sham-operated animals were nearly identical. This ex-

periment demonstrates that, at least in these stud-ies, bacterial overgrowth plays no significant rolein the control of sterol synthesis, and it would,therefore, appear that the presence or absence ofbile per se determines the rate of sterol synthesisin the intestine.

Discussion

Since the initial studies of Srere, Chaikoff,Treitman, and Burstein in 1950, a number of in-vestigators have clearly demonstrated that the

small intestine is capable of actively incorporatingC14-labeled acetate into cholesterol (1, 3, 16, 17);however, very little is known about either thespecific anatomical sites of such sterol synthesis or

the factors that may control cholesterogenesiswithin the gut. For these reasons the investiga-tions reported here were undertaken: first, to de-termine the ability of the various specific portionsof the gastrointestinal tract, from the esophagus tothe terminal colon, to carry out sterol synthesis;second, to examine further the anatomical sites ofsterol synthesis by evaluating the rates of digi-tonin precipitable sterol synthesis in the threelayers of the gut wall; and third, to attempt to de-termine what physiological conditions might nor-

mally regulate sterol synthesis in the intestinaltract.

It is apparent from the results of the presentstudy that there are striking differences in sterolsynthesis at various levels of the gastrointestinal

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tract. The esophagus had a low rate of syntheticactivity, whereas the glandular stomach manifestedapproximately 2.5 times the activity of the esopha-gus. The initial portion of the duodenum betweenthe gastroduodenal junction and the ampulla ofVater synthesized labeled sterols at an active rate,but immediately distal to the entrance of the com-mon duct synthesis was almost totally depressed.Synthetic activity remained low in the jejunum,but increased in a regular manner in the ileumreaching maximal rates in the last 30 cm of thesmall intestine. In the large bowel, the cecumusually had the least active rate of sterol synthesis,and, as was found in the small bowel, more distalcolon segments had more active acetate incorpora-tion rates. Such marked differences in syntheticactivity along the length of the gastrointestinaltract not only implied anatomic variation in thedistribution of cells responsible for sterol synthe-sis, but also raised the possibility of the presence oflocal control mechanisms as a possible explanationfor these variations.When the small bowel wall was divided into

three tissue layers consisting of villi, crypts, andsmooth muscle, the major portion of the sterol syn-thetic activity was clearly localized to the intestinalcrypts. Since histologically the crypts accountfor approximately one-fourth of the tissue mass ofthe gut wall, one can calculate that in the ileumfrom 400 to 450 mpmoles of acetate-2-C4 is in-corporated into digitonin precipitable sterols pergram of crypt tissue; this is a rate of sterol syn-thesis which equals or exceeds that found in theliver. Surprisingly, the mature epithelial cells ofthe villi consistently manifested less than 1% ofthe digitonin precipitable sterol synthetic activityfound in the whole-wall preparation even thoughunder the conditions of the experiment this tis-sue layer actively oxidized acetate-2-C1l to carbondioxide.

This pattern of distribution for sterol syntheticactivity transversely through the bowel wall isparticularly interesting when considered in thecontext of our present knowledge of epithelial cellgeneration and turnover. It is now well estab-lished that cells of the mucosal surface of the gas-trointestinal tract are being continuously replaced(18). The renewal rate is so rapid (32 to 38hours for the rat) that at any one time in the smallbowel of the rat approximately 3% of the epi-

thelial cells are undergoing mitosis (19). Thesedividing cells are found almost exclusively in theintestinal crypts and under normal conditions arenot seen higher up on the intestinal villi. It isknown that the epithelial cells newly formed inthe crypts continuously migrate upward to pro-vide the mucosal cells of the upper villi. By his-tochemical techniques Padykula has shown thatthese cells achieve enzymatic maturity only afterthey have moved up out of the crypt zone into thebase of the villus (20). During this migrationstructural changes also occur as the blunt, imma-ture microvilli of the crypt cells gradually becomethe slender, elongated microvilli typical of cellsnear the apex of the villus (21). Hence, cells areformed in the intestinal crypt, move upward andmature into the functionally competent absorptivecells of the villus, and finally, are sloughed into theintestinal lumen from the villus tip at the extrusionzone.Our observation that cholesterol synthesis is

most active in the area of epithelial cell generation(the crypts) and is virtually absent in the region ofmature epithelial cells (the villi) would suggestthat sterol synthesis in the gastrointestinal tractmay serve the function of providing structuralcholesterol for the newly forming cells.Although several previous studies have demon-

strated that dietary cholesterol-C14 collected inthe thoracic duct lymph is diluted by endogenouscholesterol (22, 23), there has been until recentlyno unequivocal evidence that cholesterol synthe-sized locally in the intestinal wall contributes toblood cholesterol. The recent studies of Lindsayand Wilson have now amply demonstrated thatcholesterol synthesized in the gut wall does enterthe lymph and eventually becomes part of the cir-culating cholesterol pool (5). Precise details ofhow this process occurs are not known. If, as ispostulated above, newly synthesized intestinal cho-lesterol is primarily used for structural purposes,then the cells containing the sterol must migrate upthe villi, be sloughed into the intestinal lumen, andbe digested before the cholesterol moiety can beabsorbed and incorporated into chylomicrons. Al-ternatively, newly formed cells may contain a cho-lesterol pool that can be transferred directly intothe chylomicrons of the central lacteal as the cellmoves upward out of the crypt. Which of thesepossibilities is correct remains to be elucidated.

1323


Three possible conditions that might influenceintestinal cholesterogenesis were examined in thepresent study; these were the feeding of a highcholesterol diet for 6 weeks, fasting for 48 hours,and diversion of bile out of the gastrointestinaltract. Both cholesterol feeding and fasting areknown to cause prompt suppression of cholesterolsynthesis in the liver. Much data have accrued inthis laboratory to demonstrate that cholesterolfeeding inhibits hepatic cholesterogenesis by afeedback mechanism operating at the point of con-version of /3-hydroxy-fl-methylglutarate to meva-lonic acid (3, 24). Inhibition of this same step incholesterol synthesis has been identified as theprobable cause of the depressed hepatic cholesterolsynthesis in the fasting animal (25).

In contrast to these findings in the liver, feed-back control of cholesterogenesis is absent in theintestine. In 1953, Gould and associates demon-strated in dogs that the feeding of a 1% cholesteroldiet for seven to eleven days suppressed hepaticbut not intestinal cholesterogenesis (16). Simi-larly, Cox, Nelson, Wood, and Taylor could dem-onstrate no inhibition of small bowel cholesterolsynthesis in monkeys fed cholesterol in cream for4 to 6 weeks (17), and a report from this labora-tory showed that in the rat intestine cholestero-genesis was not suppressed by feeding a 5% cho-lesterol diet for as long as 2 weeks (3). Data re-ported in the present study substantiate these find-ings since the rate of incorporation of acetate-2-C14into digitonin precipitable sterols by the smallbowel is not depressed by feeding a 5% cholesteroldiet for as long as 6 weeks. In addition, however,the findings presented here also demonstrate that,unlike liver, the rate of intestinal sterol synthesisis also unaffected by fasting for as long as 48 hours.

Since significant quantities of several sterolsother than cholesterol are present in the intestine,it was conceivable that the previous conditionsmight depress cholesterol synthesis per se but thatsuch an effect would be obscured by compensatorysynthesis of other 3-,8-OH sterols. Labeled digi-tonin precipitable sterols, synthesized by the in-testine of fasted and cholesterol-fed rats, weretherefore fractionated into constituent sterols bythin layer chromatography, and the C14 content ofthe purified cholesterol was shown to be normal.In this way it was unequivocally demonstrated thatthe intestinal synthesis of cholesterol itself con-

tinues at an unaltered rate in the face of either cho-lesterol feeding or fasting.

In striking contrast to the apparent insensitivityof the intestinal sterol synthetic pathway to dietarymanipulations, the presence or absence of bilewithin the gut lumen was shown to have a markedinfluence on the rate of cholesterogenesis by thesmall bowel. When bile was diverted from thegastrointestinal tract, the synthesis of digitoninprecipitable sterols from acetate-2-C14 increasedfrom 2- to 13-fold at different levels of the smallbowel whereas synthetic activity in the stomachand colon remained essentially unchanged. Thesefindings indicate that bile must contain a potent in-hibitor of intestinal sterol synthesis.

Several lines of evidence would suggest that thisbiliary inhibitor acts locally upon the mucosal cellsrather than systemically by absorption from the gutinto the circulation. When bile is infused intovarious sites along the small bowel of an animalwith a biliary fistula, sterol synthesis is enhancedproximal to the infusion point but depressed dis-tally. This is true of infusion at any level of thebowel, even as high as the beginning of the jeju-num. If the inhibitor effected mucosal cell syn-thesis by virtue of its absorption into the blood,sterol synthesis in the intestinal segments proxi-mal to the point of bile infusion should be as de-pressed as segments distal to the infusion point.In fact, in such experiments acetate incorporationinto digitonin precipitable sterols in the unper-fused proximal segments of the intestine alwaysincreased to levels found in similar small bowelsegments of animals with only biliary diversion.Additional support for this argument came fromthe experiment in which simple bile duct ligationwas performed. Despite the resultant biliary re-gurgitation, the small bowel of these rats incor-porated acetate-2-C14 into digitonin precipitablesterols at a markedly accelerated rate. This studywould strongly suggest that the biliary regulationof intestinal sterol synthesis must be exertedthrough a local rather than a systemic action ofbile.When bile is diverted from the gastrointestinal

tract, a defect in fat digestion and absorption isproduced since bile salts are necessary for emulsi-fication and micellar solubilization of the fattyconstituents of chyme. Such a disruption of theabsorptive physiology might indirectly alter sterol

1324


synthesis by the small bowel either as a result ofthe maldigestion and malabsorption of fat per seor because of the bacterial overgrowth in the smallbowel that may occur in malabsorption syndromes.In view of the experimental results shown in Fig-ure 8, neither of these factors appears important inregulating small bowel sterol synthesis. Biliarydiversion enhanced digitonin precipitable sterolsynthesis by the bowel wall even though the ani-mals were fasted throughout the experimental pe-riod, or even when bacterial counts were reduced105-fold by antibiotics administration. These re-sults suggest a direct depressive effect of bile act-ing locally upon the mucosal cells to inhibit in-testinal sterol synthesis.

In view of the variations in sterol synthetic ac-tivity noted in various segments of the small bowelof the intact rat, it is likely that the presence ofbile in the intestine plays a major physiologic rolein regulating the rate of intestinal sterol synthesis.The experimental demonstration that 1) proximalto the entrance of the common duct the duodenumhas very active synthesis of labeled sterol (Figure6); 2) distal to the ampulla of Vater jejunal syn-thetic activity is very low, but can be enhancedover 10-fold by biliary diversion (Figure 3); and3) the relatively active areas of sterol synthesis inthe terminal ileum can be markedly inhibited bythe introduction of bile directly at this level (Fig-ure 4) clearly show the overwhelming importanceof bile in determining variations in the synthesis ofcholesterol along the length of the small bowel.Apparently there is little regurgitation of bile intothe most proximal portion of the duodenum in thenonfasting rat so that synthesis is relatively activein this area. Below the level of the ampulla ofVater in the distal duodenum and proximal jeju-num one finds the highest concentration of bile andthe lowest rate of incorporation of acetate-2-C14into labeled sterols by the bowel wall. More dis-tally there is a progressive rise in synthetic ac-tivity suggesting that the concentration of the in-hibitory constituent of bile is being gradually re-duced, either by absorption out of the gut lumenor by inactivation by bacteria of the lower bowel.

In the animal that has had the normal depres-sive effect of bile eliminated by biliary diversion,however, there persist both the inherent variationsin synthetic activity down the length of the smallbowel as well as the differences between the small

bowel and the other portions of the gastrointestinaltract. Several possibilities may explain thesevariations: 1) If, as had been postulated, the syn-thesis of sterol is primarily related to new cellformation, then the rate of acetate-2-C14 incorpo-ration into digitonin precipitable sterols for a givenarea of bowel may be a reflection of the cell re-newal rate for that area. 2) Since the intestinalcrypt appeared to be the anatomic site for sterolsynthesis, at least in the small intestine, variationsin the absolute crypt cell mass could account fordifferences in synthetic activity at different levelsof the small bowel. 3) Sterol synthesis in thisstudy was expressed as a function of whole-wallweight; therefore, differences in the ratio of ac-tively synthesizing cells to the synthetically inertportion of the bowel wall could effect the apparentsynthetic activity calculated for different tissuesof the gastrointestinal tract. Which of these fac-tors is most important cannot be determined on thebasis of present knowledge; however, it is note-worthy that there is a good correlation among therelative sterol synthetic rates in the esophagus,stomach, colon, and rectum observed in the presentstudy and the cell turnover rates determined forthese same areas in the rat gastrointestinal tractby Bertalanffy (26). Furthermore, Leblond andStevens showed that in the duodenum 41% of theintestinal epithelial cells are located in the cryptsas opposed to 65%o in the terminal ileum and thatthe cell turnover times in these two areas wereapproximately 38 and 32 hours, respectively (19).Thus, there is a general correlation between a pro-portionately smaller number of epithelial cells inthe crypts, slower cell turnover, and lower sterolsynthetic activity in the proximal small bowel anda greater proportion of epithelial cells in the crypts,a more rapid cell turnover, and more active sterolsynthesis in the terminal small bowel.The clinical importance of the observation that

bile normally contains a potent inhibitor of intesti-nal cholesterol synthesis remains to be elucidated.Since a portion of the circulating cholesterol poolin the intact individual is presumably of intestinalorigin, it is possible that the enhanced syntheticactivity produced by obstruction of the commonbile duct may result in an increased delivery ofcholesterol into the circulation. Such a processcould play a role in the development of the hyper-cholesterolemia commonly associated with pro-

1325


longed biliary obstruction. The clinical signifi-cance of this control mechanism is now underinvestigation.

Summary

These studies provide information concerningthree aspects of sterol synthesis by the gastroin-testinal tract of the rat: 1) the relative syntheticrates along the length of esophagus, stomach, andsmall and large bowel, 2) localization of the siteof cholesterogenesis within the wall of the smallintestine, and 3) possible mechanisms responsiblefor physiologic control of intestinal sterol synthesis.

1) The synthesis of digitonin precipitable sterolsoccurred at every level of the gastrointestinal tract;however, the synthetic rate in the esophagus, prox-imal small bowel, and proximal colon was rela-tively low when compared to the active rates ofsynthesis found in the stomach, ileum, and distalcolon.

2) When the wall of the small intestine was sub-divided into three tissue layers, sterol synthetic ac-tivity was almost exclusively found in the tissuepreparation containing the intestinal crypts; bycontrast, the intestinal villi and smooth musclewere essentially devoid of synthetic activity.

3) Neither cholesterol feeding nor fasting sig-nificantly altered the rate of intestinal sterol syn-thesis. Bile, however, was shown to contain a po-tent inhibitor that acts directly upon the intestinalmucosa to suppress cholesterogenesis. The im-portance of this inhibitor as a determinant of thenormal variation in the rate of sterol synthesisalong the length of the small bowel is discussed.

References

1. Srere, P. A., I. L. Chaikoff, S. S. Treitman, andL. S. Burstein. The extrahepatic synthesis ofcholesterol. J. biol. Chem. 1950, 182, 629.

2. Hotta, S., and I. L. Chaikoff. The role of the liverin the turnover of plasma cholesterol. Arch. Bio-chem. 1955, 56, 28.

3. Siperstein, M. D., and M. J. Guest. Studies on thesite of the feedback;control of cholesterol synthesis.J. clin. Invest. 1960, 39, 642.

4. Sakakida, H., C. C. Shediac, and M. D. Siperstein.Effect of endogenous and exogenous cholesterol onthe feedback control of cholesterol synthesis. J.clin. Invest. 1963, 42, 1521.

5. Lindsey, C. A., and J. D. Wilson. Evidence for acontribution by the intestinal wall to the serumcholesterol of the rat. J. Lipid Res. 1965, 6, 173.

6. Umbreit, W. W., R. H. Burris, and J. F. Stauffer.Methods for Preparation and Study of Tissues inManometric Techniques, 3rd ed. Minneapolis, Bur-gess, 1957, p. 149.

7. Bray, G. A. A simple efficient liquid scintillator forcounting aqueous solutions in a liquid scintillationcounter. Analyt. Biochem. 1960, 1, 279.

8. Avigan, J., D. S. Goodman, and D. Steinberg. Thin-layer chromatography of sterols and steroids. J.Lipid Res. 1963, 4, 100.

9. Reinke, R. T., and J. D. Wilson. Manuscript in prep-aration.

10. VandenHeuvel, W. J. A., E. D. A. Haahti, and E. C.Horning. A new liquid phase for gas chromato-graphic separations of steroids. J. Amer. chem.Soc. 1961, 83, 1513.

11. Tomkins, G. M., and I. L. Chaikoff. Cholesterolsynthesis by liver. I. Influence of fasting and ofdiet. J. biol. Chem. 1952, 196, 569.

12. Gould, R. G. Lipid metabolism and atherosclerosis.Amer. J. Med. 1951, 11, 209.

13. Tomkins, G. M., H. Sheppard, and I. L. Chaikoff.Cholesterol synthesis by liver. III. Its regulationby ingested cholesterol. J. biol. Chem. 1953, 201,137.

14. Langdon, R. G., and K. Bloch. The effect of somedietary additions on the synthesis of cholesterolfrom acetate in vitro. J. biol./Chem. 1953, 202,77.

15. Frantz, I. D., Jr., H. S. Schneider, and B. T. Hinkel-man. Suppression of hepatic cholesterol synthesisin the rat by cholesterol feeding. J. biol. Chem.1954, 206, 465.

16. Gould, R. G., C. B. Taylor, J. S. Hagerman, I.Warner, and D. J. Campbell. Cholesterol metabo-lism. I. Effect of dietary cholesterol on the syn-thesis of cholesterol in dog tissue in vitro. J. biol.Chem. 1953, 201, 519.

17. Cox, G. E., L. G. Nelson, W. B. Wood, and C. B.Taylor. Effect of dietary cholesterol on cholesterolsynthesis in monkeys' tissue in vitro. Fed. Proc.1954, 13, 31.

18. Bertalanffy, F. D. Cell renewal in the gastroin-testinal tract of man. Gastroenterology 1962, 43,472.

19. Leblond, C. P., and C. E. Stevens. The constant re-newal of the intestinal epithelium in the albino rat.Anat. Rec. 1948, 100, 357.

20. Padykula, H. A. Recent functional interpretations ofintestinal morphology. Fed. Proc. 1962, 21, 873.

21. Trier, J. S. Studies on small intestinal crypt epi-thelium. I. The fine structure of the crypt epi-thelium of the proximal small intestine of fastinghumans. J. Cell Biol. 1963, 18, 599.

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22. Goodman, D. S. The metabolism of chylomicroncholesterol ester in the rat J. clin. Invest. 1962,41, 1886.

23. Swell, L., E. C. Trout, Jr., J. R. Hopper, H. Field,Jr., and C. R. Treadwell. Mechanism of choles-terol absorption. I. Endogenous dilution and esteri-fication of fed cholesterol-4-C'. J. biol. Chem.1958, 232, 1.

24. Siperstein, M. D., and V. M. Fagan. Studies on thefeedback regulation of cholesterol synthesis in Ad-

vances in Enzyme Regulation, G. Weber, Ed. NewYork, Pergamon Press, vol. 2, 1964, p. 249.

25. Bucher, N. L. R., P. Overath, and F. Lynen. f-Hy-droxy-fi-methylglutaryl coenzyme A reductase,cleavage and condensing enzymes in relation tocholesterol formation in rat liver. Biochim. bio-phys. Acta (Amst.) 1960, 40, 491.

26. Bertalanffy, F. D. Mitotic rates and renewal timesof the digestive tract epithelia in the rat. Actaanat. (Basel) 1960, 40, 130.

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cholesterol synthesis by the gastrointestinal tract: local- ization

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