EXPERIMENTAL AND MOLECULAR PATHOLOGY 25, 142-151 (1976)

Hepatic Cholesterol Metabolism in Vitro in the Obese

Spontaneously Hypertensive, Hyperlipemic

and Atherosclerotic Rat

EUGENE TAN, ANTANAS BUTKUS,~ AND SIMON KOLETSKY

Research Diuision, Cleveland Clinic Foundation, Cleveland, Ohio 44106, and Department of Pathology, Case Western Reserve University School of Medicine,

Cleveland, Ohio 44106

Received October 27, 1975

Hepatic cholesterol synthesis and esterification as well as cholesteryl ester hydrol- ysis and cholesterol-7ol-hydroxylation in vitro were studied in a strain of spon- taneously hypertensive rats (SHR) in which phenotypic obesity is inherited as a recessive trait. The obese rats also develop hyperlipemia and arterial lesions while on Purina Chow diet, but their heterozygous littermates do not. Measurements of cho- lesterol synthesis from [YZ]mevalonate by microsomal fraction of liver homogenates from obese SHR gave evidence of elevated hepatic cholesterogenesis as compared to nonobese SHR. Other experiments in vitro also showed that the obese SHR liver had a decreased cholesterol esterifying capacity and a reduced capacity of cholesterol-i’a- hydroxylation as a function of free cholesterol concentration, as well as a depressed cholesteryl ester hydrolytic capacity as a function of cholesteryl ester concentration when compared with nonobese SHR. These changes in enzymatic activities may, in part, contribute to the elevated total cholesterol concentrations in plasma and liver of these obese SHR.

INTRODUCTION

Cholesterol metabolism and obesity are related to atherosclerosis. Liver is the major site for cholesterol biosynthesis and catabolism. There are two rate-limiting steps in the cholesterol biosynthetic pathway: the first one is 3-hydroxy-3- methylglutaryl CoA reductase, and the second one is believed to be located between mevalonate and cholesterol (Gould and Swyryd, 1966; Ono and Imai, 1971). Bile acids are the major catabolic and excretory products of cholesterol. This pathway contributes more than one-half of the total cholesterol catabolism (Siperstein and Murray, 1955; Berseus et al., 1969; Tyor, 1970). Rate of con- version of cholesterol to bile acids is regulated by cholesterol-7a-hydroxylase (Bjorkhem et al., 1968; Mosbach, 1972; Shefer et al., 1968; Danielsson and Einarsson, 1969; Shefer et al., 1970; Mayer et al., 1972; Bjorkhem et al., 1968). This investigation explores some of the differences in hepatic enzymes responsible for cholesterol synthesis and esterification as well as cholesteryl ester hydrolysis and cholesterol-7a-hydroxylation between genetically obese (0) and nonobese

1 Address correspondence to Antanas Butkus, Research Division, Cleveland Clinic Founda- tion, 9500 Euclid Avenue, Cleveland, Ohio 44106.

142

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

HEPATIC CHOLESTEROL METABOLISM IN RATS 143

(NO) spontaneously hypertensive rats (SHR) (Koletsky, 1972) to possibly pro- vide some explanations for hyperlipemia and tissue lipid accumulation (Butkus et al., 1974).

METHODS

[4:14C] Cholesterol, [4J4C] cholesteryl-oleate, [ 2-14C] mevalonic acid (DBED salt), and [9,10-3H]stearic acid were bought from New England Nuclear. NADP+, G-6-P, G-6-P dehydrogenase, K2HP04, MgClz and albumin were pur- chased from Sigma Chemical Company. Chloroform, methanol, dichloromethane, ether, petroleum ether, sulfuric acid, acetic acid and Cutscum (catlog No. C-547) were products of Fisher Chemical Company.

The purities of the compounds used as substrates were checked immediately before use. If the purity was below 99%, the lipid material would be purified by thin-layer chromatography,

Animals used in this study were littermates of a new strain of spontaneously hypertensive rats (Koletsky, 1972). A mutation in the course of breeding SHR resulted in ,an injurious phenotype which is inherited as a homozygous recessive trait from heterozygous parents, each of which carries the same recessive allele. This phenotype is characterized by obesity and arterial lesions while on Purina Chow diet, while their litter-mates, although hypertensive, do not develop obesity (Koletsky, 1972). Six obese and six nonobese SHRs were used in this study.

Rats were anesthetized with sodium nembutal and exsanguinated. The livers were quickly excised and kept in ice-cold isotonic saline, freed of connective tissue, and weighed. A portion of tissue from each liver (approximately 5-S gm) was washed in cold saline, minced, and homogenized in 2 volumes ,of Krebs- Ringer’s solution (pH 7.4) with a Polytron homogenizer. The homogenate was centrifuged at 10,OOOg for 20 min. After careful removal of the lipid material of the resulting supernatant fraction, the portion containing microsomal and soluble fractions was used as an enzyme source in incubation studies.

Livers from three nonobese spontaneously hypertensive rats (Koletsky, 1972) were used in studying the kinetics (both times ,and concentrations) to obtain guidelines for estimating enzyme levels as determinants of the reactions (choles- terol esterification, cholesteryl ester hydrolysis, and cholesterol-7a-hydroxylation) in the extracts.

Hepatic cholesterol synthesis in &TO from [‘“Cl mevalonate was studied by incubation of 0.04 PCi of o~-[2-~~C]mevalonic acid (DBED salt, 8.3 mCi/mmole) (dibenzylethylene diamine was removed by adding excess quantity of sodium bicarbonate solution to dried DL- [2-14C] mevalonic acid (DBED salt), followed by extraction ‘of free ‘amine with ether and evaporation of a trace amount of ether dissolved in an ,aqueous layer under N2 gas), then the aqueous layer was neutralized with equimolar HCl with 0.2 ml of 10,OOOg supernatant fraction (equivalent to 0.1 gm of fresh liver tissue) at 37°C for 1 hr. At the end of incu- bation, 10 ml of chloroform : methanol (2: 1) was added to the incubation mix- ture. The preparation was vigorously shaken for 1 min and allowed to stand over- night at 2°C b f e ore filtration. The filtrate was washed with 0.02% CaCL solution. The organic phase was saved and taken to complete dryness under Nt. The dried residue was dissolved in 0.1 ml of chloroform and applied to the tic plate coated with silica gel G. A lipid standard containing cholesterol,

144 TAN, BUTKUS AND KOLETSKY

cholesteryl-palmitate, stearic acid, and triolein was also applied to the same plate for identification of corresponding spots. The plate was developed in a solvent system ‘of petroleum ether : diethyl ether : glacial acetic acid ( 80 : 20: 2 ) . The tic plate was ,air-dried and sprayed with dichlorofluorescein solution, by dissolving 400 mgm of dichlorofluorescein in 1 liter of 50% ethanol. The choles- terol spot, detected and marked under uv light, was scraped into scintillation vials, Fifteen milliliters of scintillation fluid (which had a composition of 8.25 gm PPO, 0.25 gm POPOP, 333 ml Triton X-100, and 667 ml toluene) was added to the vial. The sample was then mixed well and counted.

For cholesterol esterification, each incubation mixture had a volume of 0.9 ml, which contained 0.75 &i of [4-W J choIesteroI (48 mCi/mmoIe) (for the sub- strate saturation experiment, the ‘amount of cholesterol ranged from 1.8 to 30 PmoIar concentrations), 4 &i of [9, 10-3H]stearic acid (88.3 mCi/mmoIe), and 0.6 ml of 10,OOOg liver supernatant fraction (equivalent to 0.273 gm fresh liver tissue). Cholesterol was dissolved in hexane. The cholesterol solution was then mixed with Cutscum solution prepared by dissolving Cutscum in acetone. The organic solvents were dried under a stream of nitrogen, ‘and the residue was solubilized by vigorously mixing with 0.1 M, pH 7.4 phosphate buffer (Nicolau et al., 1974). This substrate solution was used in the experiments of cholesterol esterification and cholesterol-7a-hydroxyIation. Final incubation pH was 7.4. Incubation was carried out at 37°C for 2 hr (0.5-4 hr for time-course study) in a shaking water bath. At the end of incubation, 10 ml of chIoroform:methanoI (2:l) was added to the incubation mixture. The sample was then treated the same as the procedures described in the experiment for cholesterol synthesis.

For cholesteryl ester hydrolysis (Kritchevsky et al., 1968), ,a solution of [ 14C ] cholesteryl-oleate was prepared by homogenizing a mixture containing 45 ml of phosphate buffer (pH 7.4), 225 mgm of bovine albumin, 900 pgm of unlabeled cholesteryl-oleate, and 4 &i of [4J4C] cholesteryl-oleate solution and 0.3 ml of 10,OOOg liver supernatant fraction (equivalent to 0.1364 gm fresh liver tissue). For the substrate saturation experiment, the amount of cholesteryl-oleate ranged from 1.8 to 35 pM concentrations. Incubation was carried out at 37°C for 2 hr (OS-4 hr for time-course study). Light was excluded during incubation and processing. At the end of incubation, 15 ml of chloroform: methanol (2 : 1) was added to the incubation mixture. Then the sample was treated in the same manner as described in the hepatic cholesterol synthesis section except the sol- vent system used for developing tic plate was chI,oroform:methanol: water (60:30:5).

For cholesterol-7a-hydroxylation (Shefer et al., 1968; Mitropoules and BaIa- subramanian, 1972; Nicolau et al., 1974), each incubation mixture had a volume of 0.5 ml, which contained 200 pm01 of KSHPO, buffer (pH 7.4), 15 PmoI MgC12, 3.27 pm01 NADP’, 131.16 pm01 glucose-6-phosphate, 5 units of G-6-P dehydrogenase, 1 &i of [4-14C] cholesterol (48 mCi/mmoIe) (for the substrate saturation experiment, the amount of cholesterol ranged from 0.7-110 pit4 con- centrations), and 0.3 ml of lO,OOOg liver supernatant fraction (equivalent to 0.1364 gm fresh liver tissue). Final incubation pH was 7.4. Incubation was carried out at 37°C for 1 hr (0.5-4 hr for time-course study) in a shaking water bath, with exclusion of light. Air was used as gas phase. At the end of incubation, 9 ml of dichIoromethane:ethanoI (5:l) was added to the incubation mixture.

HEPATIC CHOLESTEROL METABOLISM IN RATS 145

TABLE I

Body Weight, Liver Weights, and Protein Contents in 10,OOOg Liver Supernatant Fractions of Genetically Obese (0) and Nonobese (NO) Spontaneously Hypertensive Rats

0-SHR NO-SHR (n = 6) (n = 6) P<

--

Body weight (gm) 735 f 860 341 i 38 0.025 Liver weight (pm) 33 f 2.9 16 f 1.7 0.01 (Liver weight/body weight) X 100 4.51 i 0.49 4.63 i 0.21 NS Protein (mgm) in 10,OOOg liver

supernantant fractions Per gram liver tissue 40 f 2.9 41 f 4.0 NS In whole liver tissue 1312 f 134 665 f 130 0.05

a Mean f SE.

The mixture was vigorously agitated for 1 min, then 1 ml of Hz0 was added to the sample. The sample was once again vigorously mixed for another minute and allowed to stand overnight at 2°C. After filtration and removal of the aqueous layer, the organic layer was taken to complete dryness. The dried residue was dissolved in 0.1 ml of chloroform and applied to tic plate coated with silica gel G. A lipid standard containing 7a- ,and 7p-hydroxycholesterol and ‘I-keto- cholesterol was also applied to the same plate. The plate was developed in ether at 3°C. The quantitation of lipid classes was done nondestructively on thin-layer chromatography, using a modification of the fluorometric technique developed by Roth ‘and Grossberg (Roth and Grossberg, 1971). Quantitative measure- ments were done by using Zeiss spectrodensitometer (Carl Zeiss Inc., West Germany). The 7a-hydroxycholesterol spot was detected and marked under uv light. Then the sample was treated the same ras the procedures described in the experiment for cholesterol synthesis.

For quantitative determination ‘of liver lipid, 3 gm of liver were taken to homogenize with 150 ml chloroform:methanol (2: 1). After filtration and wash- ing with 0.02% CaClz solution, the filtrate was dried under Na. The dried lipid residue was weighed and dissolved in chloroform to make a lo-ml solution. An aliquot of this solution containing 20-30 mgm of lipid was taken to apply on silicic acid column, which was then eluted with 50 ml of chloroform and 50 ml

I#$:~[; :I( P 0 IO 20 30(yM) 0 I 2 3 4(HRS)

,o [4%-CHOLESTEROL TIME

- a

FIG. 1. Cholesterol esterification appeared to proceed at maximal rate, suggestive of enzyme saturation, with above 13 PM concentration of cholesterol. At 15 FM concentration of choles- terol, the time course of the reaction showed rates to remain essentially constant over a 3-hr period.

146 TAN, BUTKUS AND KOLETSKY

FIG. 2. Cholesteryl-oleate hydrolysis proceeded at maximal rate with a cholesterol concen- tration above 25 PM. The time course of the reaction at 30 FM concentration of cholesteryl- oleate showed rates to remain constant over a 4-hr periad.

of methanol. The obtained neutral lipid fraction was further fractionated into free cholesterol, cholesteryl ester, triglyceride, free fatty acid, etc. The amount of each individual lipid was determined with gas-liquid chromatography (Butkus and Berrettoni, 1967) as well as spectrodensitometer (Roth and Grossberg, 1971).

Kjeldahl method was used for quantitative determination ,of protein in the residue resulting from each filtration.

RESULTS

As shown in Table I, the differences in the body weight and liver weight between obese and nonobese SHR were significant, but the liver weight as percentage of body weight is similar for the two groups. The protein contents in 10,OOOg liver supernatant fractions in both obese and nonobese SHR were the same based on the gram liver tissue.

Cholesterol esterification ( Fig. l), cholesteryl-oleate hydrolysis (Fig. Z), and cholesterol-7a-hydroxylation (Fig. 3) proceeded at rates that varied with sub- strate concentration up to plateau, suggestive of enzyme saturation. The substrate concentrations in pmoles for the enzyme saturation, as well as for the maximal linear concentration of the reaction for each enzyme are given in legends of Figs. 1-3.

/

. 40.

20.

0 I 2 3 4 (HRS.) TIME

FIG. 3. Cholesterol-7a-hydroxylation appeared to react at maximal rate at a cholesterol con- centration above 40 pM. At 50 pM concentration of cholesterol, the time course of the reaction showed rates to remain essentially constant over a 4-hr period.

HEPATIC CHOLESTEROL METABOLISM IN RATS 147

TABLE II

In Vitro, Hepatic Cholesterol Synthesis from [“Cl-Mevalonate

0-SHR NO-SHR P< (n = 6) (n = 6)

[r4C]Cholesterol (dpm) synthesized from [r%]mevalonate

Per liver tissue gram 5,186 f 990” [%]Cholesterol (dpm/gWLT)b/

cholesterol (mgm/gWLT) 3,028 f 167 In total liver weight 164,988 zk 22,348

1,566 f 172 0.025

1,935 f 52 0.005 24,621 f 830 0.005

a Mean f SE. b gWLT = gram wet liver tissue.

Having obtained guidelines for measurement ,of reaction rates under condi- tions of zero order kinetics (Figs, l-3), rates obtained with extracts of livers from obese vs nonobese rat livers could be compared to ‘obtain estimates of relative amounts of enzymes in the two sources.

Cholesterol synthesis in vitro from [14C]mevalonate as shown in Table II were much higher in the obese SHR than in the nonobese SHR.

Table III shows that cholesterol esterifying activity in the obese SHR was higher than in the nonobese SHR, but in terms of cholesterol esterifying capacity as a function of free cholesterol, the nonobese SHR showed higher capacity.

There was no marked difference between the hepatic cholesteryl ester hydro- lytic activity of obese and nonobese SHRs based on unit liver weight, although the total liver weight of the obese SHR had a higher capacity than nonobese SHR (Table IV). However, a reduced hydrolytic capacity as a function of hepatic cholesteryl ester concentration was observed in obese SHR.

The results in Table V show that the hepatic cholesterol-7a-hydroxylation activity based on unit liver weight or the capacity of cholesterol-7a-hydroxylation as a function of cholesterol present with the obese SHR, was markedly decreased as compared to nonobese SHR.

Table VI shows that hepatic cholestery1 ester and triglyceride were significantIy increased in the obese SHR relative to nonobese SHR. However, there was no marked difference in liver free cholestrol and phospholipid. The increases in

TABLE III

In Vitro, Hepatic Cholesterol Esterification

OSHR NO-SHR P< (n = 6) (n = 6)

[14C]Cholestryl est,er (pmole) formed from [r4C]cholest,erol

Per liver tissue gram 61 f 1.2” Cholesterol esterification (pmole/gWLTb)/

cholesterol (mgm/gWLT) 36.7 f 2.6 In total liver weight 1983 f 220

50 f 3.5 0.05

61.2 f 3.3 0.005 605 f 258 0.025

a Mean f SE. bgWLT = gram wet liver tissue.

14s TAN, BUTKUS AND KOLETSKY

TABLE IV

In T’itro, Hepatic Cholest,eryl-Oleate Hydrolysis

0-SHR NOSHR (n = 6) (n = 6)

P<

C”C]Cholesterol (pmole) released from [14C]cholesteryl-oleate

Per liver t,issue gram 71,900 f 3,920” 58,627 f 3,042 0.3 < P < .1 Hydrolysis (pmole/gWLTb)/choles-

teryl-ester (mgm/gWLT) 7,322 f 212 53,698 f 2,295 0.001 In total liver wieght 2,322,799 f 120,027 132,994 zk 66,310 0.001

a Mean f SE. b gWLT = gram wet liver tissue.

plasma free cholesterol, cholesteryl ester, triglyceride, and phospholipid were very pronounced in the obese SHR.

DISCUSSION

Absorption, synthesis and catabolism of cholesterol are of importance not only to the total body pools but also to the concentration of plasma cholesterol (Wood et al., 1966). Hypercholesterolemia is also highly correlated to the higher inci- dence of atherosclerosis.

Increased cholesterol synthesis from [14C]mevalonate in the liver of obese SHR (Table II) might suggest that there is an alteration in the rate-limiting step beyond [ 14C] mevalonic acid in the cholesterol biosynthetic pathway in the obese SHR.

One of the major pathways of tissue cholesterol elimination in almost all species is catabolism of cholesterol to the bile acids by liver microsomes and mitochondria, followed by excretion via bile and feces (Siperstein and Murray, 1955; Berseus et al., 1969; Tyor, 1970). Cholesterol-7a-hydroxylation is known as the rate-limiting step in conversion of cholesterol to bile acid (Bjorkhem et al., 1968; Mosbach 1972; Shefer et al., 1968; Danielsson and Einarssen, 1969; Shefer et al., 1970; Mayer et al., 1972; Bjorkhem et al., 1972) and hepatic synthesis of bile acids from cholesterol usually contributes more than half of total cholesterol catabolism (Tyor, 1970).

TABLIX V

In F’itro, Hepatic Cholesterol-7a-Hydroxylation

0-SHB NO-SHR P< (n = 6) (n = 6)

7n-[%]Hydroxycholesterol (pmole) formed from [%]cholesterol

Per liver tissue gram 62 f 2.3a 102 f 3.3 0.001 7a-Hydroxylation (pmole/gWLT*)/

cholesterol (mgm/gWLT) 38.5 f 2.0 127.7 f 2.1 0.001 In tot,al liver weight 202j r!c 163 1639 + 207 0.4 (NS)

a Mean & SE. bgWLT = gram wet liver tissue.

TABLE VI

Liver lipids” Plasma lipid@ -

0-SHR NO-SHR OvsNO O-SHR NO-SHR OvsNO (n = 6) (n = 6) (n = 6) (n = 6)

Free cholesterol (FC) 1.70 zt 0.370 0.80 f 0.16 NS 64% 5 29f 2 P < 0.001 Cholesteryl ester (CE) 9.44 l 1.40 1.11 f 0.24 P < 0.001 535 f 53 30+ 5 P < 0.001 Triglyceride (TG) 28.60 f 9.00 2.40 f 0.47 P < 0.02 842 f 110 30 f12 P < 0.001 Phospholipid (PL) 26.20 f 1.70 29.20 zt 1.10 NS 350 f 31 137 f 16 P < 0.005

0 Milliirams of lipids per gram liver weight. b Milligrams of lipids per 100 ml of plasmrt. c Mean f SE.

Inclusion ‘of cytosol (soluble) fraction in the incubation media was due to adopting part of van Cantford’s method (van Cantford et al., 1975) and soluble fraction contains sterol carrier protein which might facilitate mobilization of sterol material (Ritter et al., 1971 and 1973).

The observed significant reduction of cholesterol-7a-hydroxylation potential in unit liver weight (Table V) in the obese SHR, and the insignificant increase in the hepatic free cholesterol concentration (Table VI) in the obese SHR, as well as the indifferent protein content in the 10,600g liver supematant fractions (Table I ) in obese and nonobese SHRs indicate the cholesterol-7a-hydroxylation system may be inadequate for maintaining normal cholesterol catabolism in the obese SHR.

The observed increase in potential hepatic cholesterol synthesis (Table II) as well as reduced catabolism (cholesterol-7a-hydroxylation) (Table V) in the obese SHR would predict a higher concentration of free cholesterol in the livers of these ‘animals as compared to the nonobese SHR. This prediction may be explained in part by the following reasons:

1. Cholesterol synthetic capacity as a function of free cholesterol concentra- tion in the liver of obese SHR was increased (Table II). The ratio of OB/NO was 1.57, or the contribution of free cholesterol to liver free cholesterol concen- tration by hepatic cholesterol synthesis in obese SHR was 57% more than that in nonobese SHR.

2. Liver cholesterol esterifying capacity as a function of liver free cholesterol concentration was decreased in obese SHR (Table III), The ratio of OB/NO was 0.6, or the depletion of liver free cholesterol concentration by hepatic cholesterol esterifkation in obese SHR was 40% less than that in nonobese SHR.

3. The capacity of the hepatic cholesteryl ester hydrolysis as a function of liver cholesteryl ester concentration in obese SHR was reduced (Table IV). The ratio of OB/NO was 0.136, or free cholesterol contributed by the hepatic choles- teryl ester hydrolysis to liver free cholesterol concentration in obese SHR was 86.4% less than that in nonobese SHR.

4. Hepatic capacity of cholesterol-7a-hydroxylation as a function of liver free cholesterol concentration in obese SHR was depressed (Table V). The ratio of OB/NO was 0.3, or the loss of free cholesterol by catabolism from liver free cholesterol concentration in obese SHR was 70% less th.an that in nonobese SHR. Summation of the effects of aforementioned factors in contribution and depletion of free cholesterol on hepatic free cholesterol concentration resulted in a 80.670 increase in free cholesterol concentration in the liver of obese SHR as compared to

150 TAN, BUTKUS AND KOLETSKY

th,at of nonobese SHR. This coincides with the data found (Table VI), although the difference in liver free cholesterol concentrations between obese and non- obese SHR was insignificant because of large variability among liver lipids of the obese SHR group. The significant reduction in the enzymatic activities of cholesteryl ester hydrolysis ‘and cholesterol-7or-hydroxylation is probably one of the mechanisms leading to accumulation of a large quantity of lipid material, especially cholesteryl ester, in the obese SHR.

Whether the excretion of cholesteryl esters with lipoproteins from the liver is impaired by the fact that structurally abnormal cholesteryl esters are produced is at present under investigation.

ACKNOWLEDGMENTS

This work was supported in part by NIH Grant No. HL-6835 and Grant No. 8542 from the American Heart Association of Northeast Ohio, Inc.

REFERENCES

BERSEUS, O., DANJELSSON, H., and EINARSSON, K. (1969). Enzymatic transformation of the the steroid nucleus in bile and acid synthesis. In “Methods in Enzymology, Steroids, and Terpenoids” (R. B. Clayton, ed.), Vol. 15, pp. 551-562. Academic Press, New York.

BJonKmhr, I., DANIELSSON, H., and EINARSSON, K. ( 1968). On the metabolism of cholesterol in rat liver homogenates. Eur. J. Biochem. 4, 453-463.

BJORKHEM, I., DANIELSSON, H., EIXARSSON, K., and JOHANSSON, G. ( 1968). Formation of bile acid in man: Conversion of cholesterol into 5p-cholestane-3a,7ol,12a-triol in liver homo- genates. J. C&n. Inuest. 47, 1573-1582.

BUTKUS, A., and BERRETTONI, J. N. (1967). Quantitative and qualitative lipid correlation in experimental endogenous hyperlipemia. Lipids 2, 212.

Bwr~cus, A., KOLETSICY, S., and BUMPUS, F. M. ( 1974). Tissue lipids of obese and nonobese spontaneously hypertensive rats (Abstract). Ital. Sost. Gras. 51, 44.

BUTKUS, A., TAN, E., and KOLETSKY, S. (1974). Lipid metabolism in spontaneously obese, hypertensive, and atherosclerotic rat. Circulation 50, 111-261.

DANIELSSON, H., and EINARSSON, K. (1969). Formation and metabolism of bile acids. In “The Biological Basis of Medicine” (E. E. Bittar and N. Bittar, eds.), Vol. 5, pp. 279-315. Academic Press, New York.

GOULD, R. G., and SU”IRYD, E. A. ( 1966). Sites of control of hepatic cholesterol biosynthesis. J. Lipid Res. 7, 698-707.

KOLETSKY, S. ( 1972). New type of spontaneously hypertensive rats with hyperlipemia and endocrine gland defects. In “Spontaneous Hypertension-Its Pathogenesis and Complica- tions” (K. Okamoto, ed.), pp. 194-197. Igaku Shoin Ltd., Tokyo.

KRITCHXVSKY, D., TEPPEH, S. A., and ROTHBLAT, G. H. (1968). Effects of bile salts on hydrol- ysis of cholesteryl oleate by rabbit aorta. Lipids 3, 454-455.

MAYER, D., Koss, F. W., and GLASENAPP, A. ( 1972). Bestimung der cholesterin-7a-hydrol- ylase aktivitat in der ratenleber. Hoppe-Seyler’s Z. Physiol. Chem. 353, 921-932.

MITROPOULOS, K. A., and BALASUBRAMANIAM, S. ( 1972). Cholesterol-7cu-hydroxylase in rat liver microsomal preparations. Biochem. J. 128, l-9.

MOsBACH, E. H. ( 1972). Hepatic synthesis of bile acid. Arch. Intern. Med. 130, 478-487. NICoLAu, G., SHEFER, S., SALEN, G., and MOSBACH, E. H. (1974). Determination of hepatic

cholesterol-7n-hydroxylase activity in man. J. Lipid Res. 15, 146-151. ONO, T., and IMAI, Y. ( 1971). Effects of steroid hormones on synthesis of cholesterol. J.

Biochem. 70, 45-54. RITTER, M. C., and DE~~PSEY, M. C. ( 1971). Specificity and role in cholesterol biosynthesis

of a squalene and sterol carrier protein. J. Biol. Cem. 246, 1536-1539. RIPER, M. C., and DERIPSEY, M. E. ( 1973). S 1 1 c ua ene and sterol carrier protein: Structural

properties, lipid-binding, and function in cholesterol biosynthesis. Proc. Nat. Acad. Sci. USA 70, 265-269.

HEPATIC CHOLESTEROL METABOLISM IN RATS 151

ROCH, L. A., and GROSSBERG, S. E. ( 1971). Rapid fluorometric micromethod for in situ quan- titation of lipids on thin-layer chromatograms. Ad. Biochem. 41, 105-115.

SHEFER, S., HAUSER, S., BEKERSKY, I., and MOSBACH, E. H. ( 1970). Biochemical site of regulation of bile acid synthesis in the rat. J. Lipid Res. 11, 494411.

SHEFER, S., HAWSER, S., and MOSBACH, E. H. (1968). Seven-alpha-hydroxylation of choles- terol by rat liver microsomes. J, Lipid Res. 9, 328-333.

SIPERSTEIN, M. C., and MURRAY, A. M. (1955). Cholesterol metabolism in man. j. Clin. Invest. 34, 1449-1453.

TYOR, M. P. ( 1970). Bile salt metabolism and ileum. In Viewpoints on Digestive Disease.” Vol. 2, No. 1.

VAN CANTFORD, J., and GIELEN, J. (1975). Cholesterol-7whydroxylase: 2. Biochemical prop- erties and participation of endogenous cholesterol in the assay in vitro. Eur. J. Biochem. 55, 33-40.

WOOD, P. D. S., SHIODA, R., and KINSELL, L. W. ( 1966). Dietary regulation of cholesterol metabolism. Lancet 2, 604-607.