Effect of Colestipol on Sterol Metabolism in the Rat MAKOTO TAKAHASHI , 1 AMAR N. SARWAL, 3 ROBERTF. RAICHT2, 3 and B E R T R A M I . COHEN

ABSTRACT

Sterol metabolism studies using isotopic and chromatographic techniques were performed on rats fed" diets supplemented with colestipol (Upjohn). Compared to controls, colestipol altered sterol metabolism dramatically. Bile acid output increased from 7.0 rag/day to 12.2 mg/day (0.42% coles- tipol) and 39.6 mg/day (1.67% colestipol). Daily fecal neutral sterol output and daily endogenous neutral sterol output increased 36% and 55%, respectively, on the 1.67% colestipol diet. Cholesterol absorption was reduced by colestipol feeding. Cholesterol balance increased dramatically with 1.67% colestipol administration (43.5 rag/day vs -1.0 rag/day in controls). Colestipol exerts its effect by binding bile acids and by bile acid depletion interfering with cholesterol absorption.

INTRODUCTION

Colestipol hydrocb_lofide (Colestid, Up- john Co., Kalamazoo, MI), a bile acid seques- tering resin, is a high-molecular weight, insol- uble copolymer of tetraethylenepentamine and epichlorohydrin. It has been reported that long-term administration of colestipol effec- tively lowers serum cholesterol in experimental animals and in man (1-5). This resin binds bile acids in the intestine, prevents their reabsorp- tion and causes a greatly increased fecal excre- tion of acidic steroids which secondarily results in an increased catabolism and synthesis of cholesterol and a reduced plasma cholesterol level ( 1,4,5).

In this communication, we present detailed data which shows the effect of colestipol administration on the sterol metabolism in the rat. Sterol measurements using isotopic and chromatographic techniques were carried out by administering low-dose (0.42%) and high- dose ( 1.67%) colestipol to rats.

MATERIALS AND METHODS

Animals and Diet

Male Sprague-Dawley derived rats weighing 200-225 g were purchased from Charles River Breeding Laboratories, Wilmington, MA. The animals were piaced in metabolic cages and given access to food and water ad libitum. The cages allowed for quantitative feces collection and determination of food intake.

The basal diet consisted of Rockland rat chow (19% protein, 10% fat, 3% fiber, 61% carbohydrate and required vitamins and min-

IThe Public Health Research Institute of The City of New York, Inc., 455 First Avenue, New York, NY 10016; 2Veterans Administration Medical Center, 24th Street and First Avenue, New York, NY 10010; 3New York University Medical Center, 550 First Avenue, New York, NY 10016, and 4author to whom reprint requests should be addressed.

erals) supplemented with 5% corn oil and contained an average of 0.62 mg/g ~sitosterol. This diet was then further supplemented with 0.1% cholesterol by dissolving this cholesterol in ethanol and mixing it with the food. The alcohol was allowed to evaporate. After com- plete dryness, this diet was supplemented with either 0.42% colestipol hydrochloride (for the low-dose colestipol group) or 1.67% colestipol hydrochloride (for the high-dose colestipol group) and was thoroughly mixed.

On the first day of the experimental period, each animal was given an intraperitoneal injection of 10 /2Ci of DL-[2-14C] mevalono- lactone; feeding of the experimental diet was begun several hours later. Control rats were always studied concurrently with the colestipol- fed groups and were maintained under identical conditions. Feces were collected in 3 2-day pools on days 10, 12 and 14 after isotopic labeling and were dried, weighed and ground in a mortar for subsequent neutral sterol and bile acid analyses. Beginning on day 4 of the experi- mental period and every 2 days thereafter, blood was obtained from the tail vein for determination of plasma cholesterol concen- tration and plasma cholesterol specific activity (sp act). At the end of the 14-day experiment, the rats were weighed, anesthetized with Diabutal (Diamond Laboratories, Des Moines, IA), cannulas were inserted into the common bile duct, and bile was collected for 1 hr to determine biliary cholesterol and bile acid concentrations. A section of the fiver was excised for determination of liver cholesterol concentration and sp act as described previ- ously (6,7).

Labeled Compounds

DL-[2-14C]-Mevalonolactone in benzene (Amersham-Searle, Arlington Heights, IL) was found to be greater than 95% pure on silica gel thin layer chromatography (TLC) with acetone/

434

COLESTIPOL IN RATS 435

benzene (1:1, v/v). All solvents were evaporated from the mevalonolactone and the material was redissolved in sterile isotonic saline to give a final concentration of 10 ~tCi/ml.

Reference Compounds

Cholesterol (U.S.P., Nutritional Biochem- icals Corp., Cleveland, OH) was recrystallized from ethanol. C'olestipol hydrochloride (Coles- tid R) was obtained as a gift from the Upjohn Co. (Kalamazoo, MI). 5a-Cholestane (Applied Science Laboratories, State College, PA) was used as an internal standard for the gas liquid chromatographic (GLC) separations of the neutral sterols and acidic steroids after prepa- rations of trimethylsilylether derivatives.

3a,7a-Dihydroxy-12-keto-5~cholanoic acid, used as internal standard for the bile acid analyses, was synthesized according to the Fieser and Rajagopalan procedure (8).

Thin Layer Chromatography (TLC)

The thin layer chromatographic separations were carried out exactly as previously described (9-1 1).

Gas Liquid Chromatography (GLC)

The methods and conditions of all GLC analyses have been described in detail (9-11). All analyses were carried out on a Hewlett- Packard 7610A gas chromatograph.

Methods for the Isolation and Quantitation of Neutral and Acidic Steroids from Feces

An aliquot of 0.5-1.0 g powdered fecal material from 2-day fecal pool was extracted with ethanol (80 ml) for 48 hr. Following extraction, fecal residues were allowed to stand for 15 min in 25 ml of ethanol containing saturated aqueous ammonium carbonate (2 ml) in order to elute bile acids bound to colestipol (1). The ethanolic ammonium carbonate solu- tions were combined with the 80-ml ethanol extracts in reservoir bottles and were evapo- rated to 20 ml under nitrogen. The samples then were refluxed for 1 hr in 1 N NaOH in 95% ethanol (20 ml) (mild hydrolysis). In 4 instances, the fecal residues were allowed to stand for 15 min in ethanol (25 ml) containing saturated aqueous ammonium carbonate (2 ml) to determine if any neutral sterols or plant sterols were bound to colestipol. These samples were evaporated, refluxed for 1 hr in 1 N NaOH in 95% ethanol (20 ml) and analyzed for fecal neutral sterols as described earlier.

The subsequent methods used for the isolation and quanti tat ion of fecal neutral and acidic steroids have been described in detail (10,12,13). The material in each fraction was

analyzed by GLC of the trimethylsilylether (TMSi) derivatives. 5a-Cholestane was used as an internal standard. The recovery of dietary t3-sitosterol was used as an index to correct for losses of neutral sterols. Since the recovery was 90% or better, no corrections for losses of neutral steroids resulting from degradation in the intestinal tract were required.

Quantitation of the fecal bile acids was carried out using methods previously described (6). 3oq7a-Dihydroxy-12-keto-5t3-cholanoic acid served as recovery standard in each sample. Corrections for losses were made as required.

Methods for Determination of the Specific Activity and Concentration of Cholesterol in Plasma and Liver

These methods have been described in detail (6,7).

Radioactivity Measurements

All radioisotopic measurements were made using new scintillation glass vials on a Beckman LS-8000 liquid scintillation system (Beckman Instruments, Fullerton, CA).

The radioactivity in each sample was ob- tained after evaporation of the solvent by the addition of 10-12 ml of 2,5-bis[2-(2-tert- butylbenzoxazolyl)]-thiophene (BBOT), 4 g/~ in toluene. Each sample was corrected for background and quenching effects using appro- priate blanks and standards.

Calculations and Statistics

Acidic steroid and neutral steroid output was determined by chromatographic techniques (combined TLC and GLC). Endogenous neutral sterol production was estimated as previously described (6,7,11,14-16).

Cholesterol absorption was determined as the difference between the dietary intake of cholesterol (determined chromatographically) and the unabsorbed dietary neutral steroid in the feces.

The unabsorbed neutral steroid (mg/day), cholesterol turnover (mg/day), and cholesterol balance (rag/day) were calculated as described below (6,7,15,16): Unabsorbed neutral sterols (rag/day) = daily fecal neutral sterol (rag/day) - daily endogenous neutral sterol (mg/day). Cholesterol turnover (mg/day) = daily fecal endogenous neutral steroid output (isotopic) (rag/day) + daily fecal acidic steroid output (chromatographic) (rag/day); cholesterol bal- ance (mg/day) = cholesterol output-choles- terol input = daily fecal neutral sterols (mg/ day) + daily fecal acidic steroids (rag/day)- daily cholesterol intake (mg/day). Student's t-test was used to determine significance.

L1PIDS, VOL. 15, NO. 6

436 M. T A K A H A S H I , A.N. S A R W A L , R.F . R A I C H T A N D B.I. C O H E N

RESULTS

Rats weighing ca. 250 g were injected with DL-[2-14C]mevalonolactone to label their cholesterol pool. The rats were then fed the experimental diets for 14 days. The weight gain, daily food intakes and fecal outputs for all the rats were similar (Table I). Plasma cholesterol levels were not decreased by 14-day colestipol administration (average -+ SD) [low- dose colestipol g roup (group 2) - 61.5 -+ 9.0 rag/100 ml; high-dose colestipol group (group 3), 69.0 -+ 12.1 mg/100 ml; control group (group 1), 65.3 + 17.8 mg/100 ml]. Liver cholesterol levels were slightly decreased by colestipol administration (average -+ SD) (group ~, 2.2 - + 0.1 mg/g; group 3, 2.0 - + 0.2 mg/g; group 1, 3.2 -+ 0.8 mg/g). Biliary cholesterol

levels were increased by colestipol adminis- tration (average +- SD), group 2, 0.13 +- 0.03 mg/ml; group 3, 0.17 -+ 0.03 mg/ml; group 1, 0.08 mg/ml. Biliary bile acid concentrations were not significantly different (group 1, 8.93 -+ 2.17 mg/ml, group 2, 9.51 -+ 3.15 mg/ml; group 3, 7.82 +- 0.51 mg/ml).

The sp act of cholesterol in plasma and liver for the animals in each group on day 14 were similar, but not identical, and indicated that isotopic equilibrium of cholesterol had been achieved in these tissues during this short term metabolism study (Table II). The sp act of chol- esterol in these tissues on day 14 were lowest in the high-dose colestipol group (group 3).

Sterol metabolism data for the rats fed the colestipol are summarized in Table III. Daily

T A B L E I

Weights , F o o d In t akes a n d Fecal O u t p u t s o f Ra tsa , b

Init ial Weigh t F o o d Feca l G r o u p Diet w e i g h t at d e a t h i n t a k e c o u t p u t d

no. (no. o f an ima l s ) (g) (g) ( g / d a y ) ( g / d a y )

1 C o n t r o l d ie t (2) 238-+ 11 328-+ 6 23 .2 + 2.7 4 .2 -+ 0 .2 ( 2 3 0 , 2 4 6 ) ( 3 2 4 , 3 3 2 ) ( 1 8 , 2 7 ) (3 .9 ,4 .4 )

2 0 . 4 2 % C o l e s t i p o l h y d r o c h l o r i d e (4) 2 6 2 +- 3 333-+ 21 20.1 +- 3 .0 3.8 +- 0 .5 ( 2 5 8 , 2 6 6 ) ( 3 1 0 , 3 5 8 ) ( 1 4 , 2 6 ) ( 3 . 0 , 4 . 6 )

3 1 . 6 7 % C o l e s t i p o l h y d r o c h l o r i d e (4) 2 4 9 + 4 321 -+ 14 21 .5 + 3.9 4.5 + 0.6 ( 2 4 4 , 2 5 4 ) ( 3 0 8 , 3 3 8 ) ( 1 1 , 2 6 ) ( 3 . 2 , 5 . 4 )

a R a t s w e r e fed the e x p e r i m e n t a l d ie ts fo r 14 days .

b V a l u e s r e p o r t e d r e p r e s e n t average -+ SD; n u m b e r s in p a r e n t h e s e s r e p r e s e n t t he range .

CValues r e p r e s e n t t he average da i ly f o o d i n t a k e f r o m days 4 -14 o f t he e x p e r i m e n t (5 s a m p l e s per an ima l , g r o u p 1, N=10 , g r o u p s 2 a n d 3, N=20) .

dVa lues r e p r e s e n t t he average da i ly feca l o u t p u t s (3 poo ls per an ima l ) on days 9 -14 o f t he e x p e r i m e n t , g r o u p 1, N=6, g r o u p s 2 a n d 3, N=12 .

T A B L E II

R e p r e s e n t a t i v e Speci f ic Act iv i t i es o f C h o l e s t e r o l in Liver and P lasma o n Day 14 a

Liver P lasma cho l e s t e ro l cho l e s t e ro l

G r o u p sp ac t sp ac t no . A n i m a l b ( d p m / m g ) ( d p m / m g )

1 C o n t r o l

2 0 . 4 2 % Coles t ipo l h y d r o c h l o r i d e

1 .67% Co le s t i po l h y d r o c h l o r i d e

A 1471 1 9 0 3 B 1833 2 4 3 2

A 1 5 1 8 1 7 6 9 B 9 0 9 976 C 1299 1611 D 1642 1545

A 621 5 7 0 B 7 4 5 829 C 6 9 9 6 8 8 D 7 8 4 7 7 7

a D e t e r m i n e d b y c o m b i n e d T L C / G L C t e c h n i q u e s a n d l iqu id m e n t .

b R e p r e s e n t s t h e an ima l s in e a c h g r o u p .

sc in t i l l a t ion c o u n t i n g on d a y 14 o f t he exper i -

LIPIDS, V O L . 1 5, NO. 6

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cholesterol intake was similar in both colestipol and control rats. Daily acidic steroid output was significantly increased by colestipol feed- ing, depending on the dose of colestipol admin- istered (group 1, 7.0 -+ 2.8 rag/day vs group 2, 12.2 + 2.2 mg/day [P < 0.005] ; group 3, 39.6 -+ 5.2 mg/day [P < 0.0051).

Daily neutral steroid output of high-dose colestipol group (24.5 -+ 4.6 mg/day) increased, compared to that of either control group (15.4 + 3.5 mg/day) or low-dose colestipol group (15.6 • 2.9 mg/day) (P < 0.005). Four fecal extracts were analyzed for the possible binding of fecal neutral sterols to colestipol itself. No neutral or plant sterols could be detected in these extracts. Similarly, daily endogenous neutral steroid output increased only in the high-dose colestipol group (group 3, 11.6 -+ 4.0 mg/day vs control, 5.5 -+ 2.4 mg/day [P < 0.0051; group 2, 6.2 + 1.9 mg/ day). On the other hand, daily dietary choles- terol absorption was reduced by colestipol feeding, depending on the dose of colestipol administered (group 2, 10.0 • 3.0 rag/day and group 3, 7.7 • 5.3 mg/day vs group 1, 13.6 i 2.6 mg/day [P < 0.025]). Moderate increases in cholesterol turnover for low-dose colestipol- fed ratswere observed (from 12.6 -+ 5.1 rag/day to 18.4 • 3.8 mg/day), group 1 vs group 2 (P < 0.01).

Similarly, moderate increases in cholesterol balance for low-dose colestipol-fed rats was observed (from -1.0 -+ 6.7 mg/day to 8.4 -+ 3.7 mg/day), group 1 vs group 2 (P < 0.005). In contrast, large increases in cholesterol turnover and cholesterol balance for high-dose colestipol- fed rats were observed (from 12.6 • 5.1 mg/day to 51.2 -+ 7.4 mg/day, and from 1.0 • 6.7 mg/day to 43.5 -+ 7.7 rag/day, respectively), group 1 vs group 3 ( P < 0.005).

DISCUSSION

This study reports the effects of 0.42% colestipol (equivalent 500 mg/kg/day) and 1.67% colestipol (equivalent 2000 mg/kg/day) administration on: (a) bile acid synthesis; (b) cholesterol absorption and (c) cholesterol balance.

Colestipol binds bile acids and prevents their reabsorption in the intestine (1). This results in a greatly increased fecal excretion of bile acids. As expected, the daily fecal acidic steroid output was greatly increased by colestipol administration (0.42% colestipol administra- tion, 1.7-fold; 1.67% colestipol administration, 5.7-fold, compared to control group). This suggests that bile acid synthesis from choles- terol in the liver was remarkably accentuated

L I P I D S , V O L . 1 5 , N O . 6

438 M. TAKAHASHI, A.N. SARWAL, R.F. RAICHT AND B.I. COHEN

by colestipol administration. It has been re- ported that the maximal synthesis rate for taurocholic acid in a bile fistula rat is 15 mg/ 100 g rat/day (16). Thus, a fecal bile acid excretion rate of 39.6 mg/rat/day (average wt 321 g) is within the limits for synthesis by the animal. It is well known that cholesterol is absorbed from the intestine by micelle forma- tion with bile acids (17). In this experiment, intestinal absorption of exogenous (dietary) cholesterol was suppressed and the daily endogenous neutral steroid output was in- creased by colestipol administration. These findings suggest that intestinal absorption of both exogenous (dietary) and endogenous cholesterol was decreased by colestipol adminis- tration because there were not enough bile acids in the intestine to form micelles indis- pensable for the intestinal cholesterol absorp- tion. No neutral sterols were found bound to colestipol suggesting that indeed the lack of bile acids was responsible for the decreased cholesterol absorption. Cholesterol turnover which reflects the daily fecal excretion of the materials related to endogenous cholesterol was increased dose-dependently, accompanied by increased daily acidic steroid output and increased daily endogenous neutral steroid output. Consequently, colestipol administration caused marked increases (dose-dependent) in cholesterol balance, a measurement which reflects relative cholesterol synthesis.

In summary, colestipol caused a greatly increased fecal excretion of bile acids and consequently, the catabolism of cholesterol was remarkably stimulated. Meanwhile, intes- tinal absorption of both exogenous and endoge- nous cholesterol was decreased by colestipol administration.

ACKNOWLEDGMENTS

This work was supported by grants CA-1865l and CA-27 from the National Large Bowel Cancer Project through the National Institute of Health and a grant from the Veterans Administration.

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[Received September 28, 1979]

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