Relationships between squalene and cholesterol in bile: Effect of ursodeoxycholic acid administration in patients with radiolucent gallstones

Relationships between squalene and cholesterol in bile: Effect of ursodeoxycholic acid administration in patients with radiolucent gallstones

Relationships Between Squalene and Cholesterol in Bile: Effect Ursodeoxycholic Acid Administration in Patients With Radiolucent Gallstones Gianfranco ...

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Relationships Between Squalene and Cholesterol in Bile: Effect Ursodeoxycholic Acid Administration in Patients With Radiolucent Gallstones Gianfranco

of

Salvioli, Ruggero Lugli, and John M. Pradelli

Squalene is an obligate intermediate of cholesterol synthesis and plasma squalene to cholesterol ratio correlates significantly with cholesterol synthesis rate in the liver. Sixteen nonobese patients with radiolucent gallstones were randomly allocated into two treatment groups receiving 15 mg/kg/day ursodeoxycholic acid (group A) or 15 mg/kg/day lactose (group B) administered three times daily for 30 days. In group A, biliary squalene to cholesterol ratio was significantly lowered (from 1 .I9 to 0.86, P < 0.02). as was cholesterol saturation (from 1.39 to 0.95, P cc 0.001); levels of plasma very-low-density lipoprotein cholesterol WLDL-C) (from 30 to 26 mg/dL) and plasma VLDL-triglyceride (VLDL-TG) (from 81 to 68 mg/dL) decreased significantly only in the group taking ursodeoxycholic acid. No variations of squalene concentrations and squalene to cholesterol ratio were observed in the plasma of both groups. Biliary cholesterol saturation during ursodeoxycholic acid administration correlated directly with squalene to cholesterol ratio in bile; reduction of these two parameters is accompanied by decreased VLDL-C levels.

QUALENE is an obligate intermediate of cholesterol synthesis. The rate of hepatic cholesterol synthesis, as measured by various methods, correlates with plasma squalene concentration and plasma squalene to cholesterol ratio,’ and these values have been employed to detect changes in cholesterol synthesis under many clinical conditions.’ Biliary cholesterol molar percentage increases in patients with cholesterol gallstones having high prevalence of type IV hyperlipoproteinemia.’ It is noteworthy that serum triglyceride levels correlate negatively with bile acid molar fraction3 and positively with plasma squalene levels? however, the recent National Cooperative Gallstone Study failed to confirm a correlation between serum triglyceride levels and bile acid molar fraction.5 The mechanism controlling biliary cholesterol level still escapes our understanding even though there is evidence indicating that part of the cholesterol neosynthesized by the liver is secreted in bile.‘j It is known that the administration of bile acids, such as chenodeoxycholic (CDCA)’ and ursodeoxycholic (UDCA)’ acids for dissolution of radiolucent gallstones decreases the activity of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoA-R), but it is necessary to consider that the activity of this regulatory enzyme shows a wide range of basal values’ and that its in vitro evaluation is not always indicative of in vivo cholesterol synthesis.‘,” Until now no controlled studies had been performed on biliary concentration of squalene and their variations during bile acid administration. In this context, it seemed of value to us to investigate first the squalene concentration, squalene to cholesterol ratio in bile and in plasma, and cholesterol saturation in bile of patients with radiolucent gallstones treated with ursodeoxycholic acid as litholytic agent compared to a control gallstone group taking placebo; and second, the effect of ursodeoxycholic acid administration on very low

S

Metabolism,

Vol 33. No 7 (July). 1984

density lipoproteins (VLDL), on the basis of observed correlations between hepatic cholesterol synthesis and VLDL concentration.“.” Our study shows that ursodeoxycholic acid administration reduces squalene to cholesterol ratio in bile and also decreases biliary saturation index and VLDL levels. MATERIALS

AND

METHODS

The study consisted of 16 nonobese patients with radiolucent gallstones (I I female and 4 male) who gave informed consent; basal data are given in Tables I and 2. Patients received regular diet (30 Cal/kg) for ten days prior to and during the trial. Foods containing high concentrations of squalene were not given; energy content was supplied as 30% fat, 20’S protein, and 50% carbohydrate. Daily intake of cholesterol was about 500 mg. No drugs affecting liver function or lipid metabolism were given during the trial. Experimentalprocedure. Patients were randomly allocated into two groups: group A received I5 mg/kg/day ursodeoxycholic acid administered three times daily for 30 days; group B received placebo (lactose). Fasting blood samples were collected in tubes containing ethylenediaminetetraacetic acid, disodium salt (Na,EDTA, I mg/ mL) before treatment and on the thirtieth day of therapy. The VLDL fraction was separated by preparative ultracentrifugation at a density of 1.006 g/mL at 4 “C according to the method of Hatch.” using a Beckman L2-65 with Beckman 65 rotor (Beckman Instruments, Palo Alto, CA) for I6 hours at 40,000 rpm. Bile-rich aspirates were obtained at the same times from the duodenum after cerulein infusion (0.01 wg/min) (Farmitalia; Milan, Italy). Anal,vsis of plasma lipids. Plasma levels of total and free cholesterol and triglycerides were assayed with Biochemia (Milan, Italy) enzyme kits. VLDL lipid composition was similarly determined. Subjects.

Anal.vsis ofbiliary lipids. Immediately after aspiration a I mL bile sample was extracted with IO mL 2:1 chloroform-methanol (vol/vol); cholesterol and phospholipidJ4 concentrations were deter-

From Generale Address Ospedale

the

University

e Terapia reprint Estense.

of

Medica. requests

Modena.

Istituto

di

Clinica

Medica

Insegnamento

di Semeiotica

Medica

to Dr G. Salvioli,

Universith

di Modena.

Viole Vittorio

8 1984 by Grune & Stratton,

Veneto, 9, 41100

Modena.

1tnl.v.

Inc.

0026-0495/84/3307-0011$03.00/0

641

642

SALVIOLI, LUGLI. AND PRADELLI

Table 1. Clinical and Biochemical

Data Regarding Patients

With Radiolucent

Gallstones

Taking Ursodeoxycholic Bile

PlWlM

Patient

Sex

1

Age

RBW

52

98

58

2

43

3 4

118 101

60

5

51

6

50

88 105

VLDL-TG (mg/dL)

SQ kI/dL)

SQK Ratio

C

PL BS (mol/L %)

CS

SO Ira/dL)

C (mg/dL)

SQIC Ratio

b

217

76

18

41

53.2

0.245

10.7

22.8

66.5

1.42

180

160

1.12

a

217

50

15

18

52.4

0.223

8.1

24.9

67.0

1.04

204

182

1.12

b

219

256

42

164

42.7

0.194

7.8

16.9

75.3

1.28

110

88

1.25

a

220

175

34

125

40.6

0.184

6.1

17.3

76.5

1.00

162

180

0.90

b

195

97

27

64

19.9

0.102

9.5

19.4

71.1

1.42

196

175

1.12

a

190

88

20

58

17.4

0.09 1

6.2

21.1

72.7

0.89

260

351

0.74

b

234

132

39

88

28.7

0.293

11.1

20.8

68.1

1.55

126

80

1.58

a

235

86

32

69

25.8

0.109

6.0

23.1

70.9

0.81

108

169

0.64

b

258

139

36

86

32.5

0.125

8.4

24.2

67.4

1.10

128

166

0.77

a

268

132

34

78

37.0

0.138

5.9

23.6

70.5

0.79

201

359

0.56

256

107

25

64

39.5

0.154

9.6

20.8

69.6

1.36

176

163

1.08

a

268

109

25

64

42.0

0.153

7.1

21.4

71.5

1.00

210

210

1.00

b

207

128

30

76

32.7

0.158

11.4

24.2

64.4

1.47

224

188

1.19

a

215

126

24

72

30.2

0.140

8.8

21.6

69.8

1.20

176

176

1.00

b

207

116

25

69

21.7

0.107

10.8

18.2

71.0

1.55

218

157

1.39

a

197

110

22

64

18.5

0.097

7.1

24.7

68.2

0.93

184

798

0.93

103

223

131

30

81

33.8

0.172

1.39

170

147

1.19

9

24

74

8

36

11.0

0.067

0.15

43

40

0.23

mean

228

109

26.

0.142

0.95’

169

228

29

37

7

68t 29

33.0

k SD

12.0

0.045

0.13

43

79

48

8

108

62

Before

53

f Afier

VLDL-C (mg/dL)

b

7

110

C TG (mg/dL) (mg/dL)

Acid (Group A)

SD

95

~

6

0.86t 0.19

SQ = squalene: CS = cholesterol saturation: C = cholesterol; TG = triglycerides; VLDL = very low density lipoproteins; R8W = relative body weight: PL = phospholipids; 8s = bile salts; b = before; a = after. ‘P < 0.001. tP < 0.02.

Table 2. Clinical and Biochemical

Data Regarding

Patients With Radiolucent

Gallstones

Taking Placebo (Group B) Bile

Pk.llls Patient

Sex

Age

9

F

60

10

F

44

C TG (mg/dL) (mg/dL)

RBW 110 105

VLDL-C ImgldL)

VLDL-TG (mg/dL)

SO (&dL)

SQK Ratio

C

PL

BS

CS

(mol/L %)

SO (pg/dL)

C (me/dL)

swc Ratio

b

200

148

29

74

21.6

0.108

10.6

22.1

67.5

1 .42

224

171

1.31

a

210

152

32

76

36.4

0.173

11.4

21.6

67.0

1.56

176

138

1.27

b

174

110

17

58

34.8

0.200

14.1

30.6

55.3

1 .68

196

175

1.12

a

165

90

19

60

38.7

0.234

8.9

21.8

69.3

1 .23

226

161

1.40

109

21

58

22.6

0.107

7.9

24.1

68.0

1 .04

216

152

1.42

11

F

46

106

b

210

a

217

100

22

52

27.8

0.128

8.1

25.4

66.5

1.04

184

137

1.34

12

F

58

115

b

208

250

50

184

31.9

0.153

7.8

14.8

77.4

1 .41

184

164

1.12

a

215

215

52

170

29.8

0.138

6.1

15.4

78.5

1 .08

196

215

0.9 1

13

F

51

118

b

247

148

30

82

48.9

0.198

11.2

24.8

64.0

1 .43

208

196

1.06

a

246

159

27

88

41.7

0.169

10.6

21.5

67.9

1 .46

216

193

1.12

b

228

168

20

80

26.8

0.117

9.4

20.8

69.8

1.33

152

119

1.28

a

237

170

22

78

34.8

0.146

8.7

20.4

70.9

1.26

184

139

1.32

178

98

14

38

51.4

0.288

9.1

21.2

69.7

1.28

210

228

0.92 0.81

14

F

64

100

15

M

52

87

b a

182

110

18

47

50.7

0.278

9.2

20.9

69.9

1.30

186

230

16

M

46

91

b

224

136

22

76

38.5

0.172

8.8

24.2

67.0

1.15

184

224

0.82

a

222

142

28

78

39.5

0.178

9.1

22.7

68.2

1.22

206

212

0.97

After

mean

53

104

209

146

25

81

34.5

0.161

1.33

197

179

1.33

f SD

7

11

25

42

11

44

11.0

0.061

0.19

33

36

0.20

mean

212

142

27

80

37.4

0.171

1.27

197

178

1.14

k SD

27

41

10

39

7.1

0.050

0.17

17

39

0.21

SO = squalene; CS = cholesterol saturation: C = cholesterol; TG = triglycerides; VLDL = very low density lipoproteins: RBW = relative body weight; PL = phospholipids; 8S = bile salts; b = before; a - after.

lP
SQUALENE

AND CHOLESTEROL IN BILE

mined on the chloroform phase. Total bile acid concentration was estimated by Sterognost-3oc Kit (Bracco; Milan, Italy). Relative lipid composition of bile was calculated as moles of cholesterol, phospholipids. and bile acids per 100 moles of total lipid: bile cholesterol saturation was determined according to the critical tables of Carey.” Percentages of individual bile acids were evaluated by gas-liquid chromatography, as described previously.‘6 Analysis of squalene in plasma and bile. Squalene concentrations were measured in duplicate on plasma and bile samples according to the method of Liu et al:” 3 mL of plasma or 2 mL of bile was extracted with 30 mL of 1: I chloroform-methanol (vol/vol). The mixture was subjected to prolonged agitation, and the chloroform phase was evaporated, then refluxed with 10 mL 70% methanol plus IO’% KOH at 70 “C for two hours. Known amounts of internal standard were added (&cholestane; Eastman Organic Chemicals, Prodotti Gianni, Milan, Italy), and the saponifiable fraction was extracted three times with 50 mL n-hexane. Following evaporation, the sterol-containing fraction was transferred in petroleum ether to a column containing 5 g neutral aluminium oxide, 200 mesh (BDH, Milan, Italy). activated overnight at 105 “C, to which solution had been added 50 rL distilled water. Hydrocarbon fraction was eluted with petroleum ether and evaporated, and a known amount was assayed in a Packard Becker (Milan, Italy) model 420 gaschromatograph using hydrogen flame detector and U-shaped glass column with 1% Dexil 300 on 100 to 200 mesh Supelcoport (Supelchem, Milan, Italy), kept at 235 “C. Reproducibility of the method was within 5%. Statistical sigmnifcance. Values reported are mean + SD. Statistical analysis was performed using Student’s I test or Student’s paired t test where appropriate. RESULTS

Subjects

The two groups have similar mean ages and relative body weights (RBW) (Tables 1 and 2). No changes in body weight were observed during the study. Plasma Lipids and Lipoproteins

Plasma lipid concentrations are the same in group A and B at time zero. Ursodeoxycholic acid administration decreased triglyceride levels from 13 1 to 109 mg/dL; the placebo group shows no variations. VLDLTG levels are reduced by 15% (P < 0.02) (Tables 1 and 2). Total cholesterol level remains unchanged in groups A and B, whereas VLDL-C and VLDL-TG levels decrease significantly (P < 0.001) only in the group taking ursodeoxycholic acid (Tables 1 and 2). Plasma squalene levels are 33.8 pg/dL in group A and 35.5 bg/dL in group B and remain unchanged in both groups during treatment. Squalene to cholesterol ratio decreases only in group A from 0.172 to 0.142 (Tables 1 and 2), but the change is not statistically significant. Lipid Composition

643

unchanged in the placebo group. Squalene concentration is higher in bile than in plasma (170 pg/dL in group A and 197 pg/dL in group B) but displays marked variations. Biliary squalene concentration remains unchanged after treatment, whereas biliary squalene to cholesterol ratio decreases from 1.19 to 0.86 during UDCA administration (P < 0.02). Biliary cholesterol concentration increases in bile-rich aspirates only in the group treated with ursodeoxycholic acid; basal values in groups A and B are similar (Tables 1 and 2). Thus, no correlation is found between squalene to cholesterol ratio in bile and plasma before and after ursodeoxycholic acid administration (r = 0.50 and r = 0.34, respectively). On the contrary, squalene to cholesterol ratio and cholesterol saturation of bile are correlated positively (r = 0.88) only in group A (Fig 1). Ursodeoxycholic acid levels are elevated in group A (Table 3), an indication that a 30 day treatment is sufficient to sharply modify bile acid pool composition. DISCUSSION

To date little information is available on sources of biliary cholesterol; a small fraction seems to be newly synthesized by the liver, whereas the major part probably derives from circulating lipoproteins.h Salen et al’” find increases HMGCoA-R activity in cholesterol gallstone patients compared with controls. Key et alI9 show significant correlations between cholesterol secretion in bile and hepatic HMGCoA-R activity, although Ahlberg et al’ do not find any difference in hepatic HMGCoA-R activity in nonobese gallstone patients with respect to controls; these same 16

.

of Bile

Bile obtained by duodenal intubation is in all cases saturated with cholesterol (Tables 1 and 2). Only UDCA administration lowers bile cholesterol saturation from 1.39 to 0.95 (P < 0.001); it remains

Fig 1. Correlation between saturation index and squalene to cholesterol ratio in bile from patients treated with ursodeoxycholic acid (group A).

644

SALVIOLI, LUGLI, AND PAADELLI

Table 3. Individual Biliary Bile Acids in Patients With Radiolucent

Gallstones

Before and After Ursodeoxycholic

Acid (Group A) and

Placebo (Group B) Administration C

Group A Group B

Before

32.6

r 4.8

CDC 36.8

LC

UDC

k 4.0

1.1 + 0.2

0.9 k 0.2

DC

+ 4.7

28.5

After

12.4 k 2.0

17.9 f 2.5

11.6 + 2.1

1.4 * 0.3

Before

34.5

+ 3.9

34.8

k 5.5

28.8

k 2.8

1.3 + 0.2

0.5 + 0.1

After

38.4

k 5.7

32.6

-c 4.6

27.0

+ 3.4

1.2 f 0.2

0.7 + 0.2

56.6

k 6.4

Mean values k SD. C = Cholic acid; CDC - chenodeoxycholic acid; DC = deoxycholic acid; LC = lithocholic acid: UDC = ursodeoxycholic acid.

patients show reduced enzyme activity under chenodeoxycholic acid administration. Similar results have been obtained with ursodeoxycholic acid.‘*20 Of course, it is not feasible to perform serial liver biopsy studies in humans to determine HMGCoA-R activity during drug administration. Hence, the evaluation of cholesterol precursors seems an interesting method for repeated measurements of cholesterol synthesis in clinical investigation. Quantitative assay of cholesterol precursors in bile is of recent investigation: Chenodeoxycholic acid administration reduces plasma and bile concentrations of post-squalene precursors of cholesterol;*’ on the other hand, Begemann et al** find that high doses of chenodeoxycholic acid lower triglyceride synthesis, whereas plasma squalene concentrations increase. The present study demonstrates that values of squalene to cholesterol ratio in bile (but not in plasma) follow the values of cholesterol saturation in bile only in patients receiving ursodeoxycholic acid (Fig 1). Basal squalene levels in bile are not reduced in group A with respect to group B, as seen in Tables 1 and 2; indeed, biliary squalene and cholesterol concentrations (before and after treatment) are probably meaningless in view of possible but unmeasured changes in water content. Nonetheless, the squalene to cholesterol ratio may provide an indication as to whether these two components change in the same direction. The results (Tables 1 and 2) show that squalene to cholesterol ratio in bile is unchanged by placebo and is lowered by ursodeoxycholic acid feeding. These findings support the view that decreased biliary squalene to cholesterol ratio induced by high levels of ursodeoxycholic acid in the bile acid pool may be indicative of reduced cholesterol synthesis; this speculation is supported by the contemporaneous

reduction of VLDL concentrations. However, this hypothesis was not tested by the evaluation of cholesterol synthesis via other methods. In this regard, however, other investigators have not demonstrated differences in hepatic HMGCoA-R activity between gallstone patients and controls,’ and the enzyme activity during ursodeoxycholic acid therapy has been found to be both decreased’ and increased.23 Enhanced cholesterol synthesis is a remarkable metabolic derangement occurring only in obese gallstone patients.24 Our patients are nonobese and normolipemic (except patients 2 and 12) and no differences in biliary squalene to cholesterol ratio and in squalene concentrations are found between normal subjects and nonobese gallstone patients (G Salvioli, unpublished observation). Levels of VLDL-C and VLDL-TG decrease significantly when ursodeoxycholic acid is given. Similar results are obtained in patients with gallstones*‘or type IV hyperlipoproteinemia26 receiving chenodeoxycholic acid. Some studies support the concept of an integrated regulation of cholesterol, bile acids, and lipoprotein metabolism;*’ in fact, VLDL secretion is stimulated when cholesterol synthesis increases” or after bile withdrawal.12 Our results show that ursodeoxycholic acid treatment in gallstone patients reduces plasma VLDL levels and contemporaneously cholesterol saturation and squalene to cholesterol ratio in bile. These findings require further investigations to define whether they indicate variations in cholesterol synthesis. ACKNOWLEDGMENT The authors gratefully tance of Amos Gibertini.

acknowledge

the valuable

technical

assis-

REFERENCES 1. Miettinen TA: Serum squalene and methyl sterols as indicators of cholesterol synthesis in vivo. Life Sci 8:713-721, 1969 2. Einarsson K, Hellstrom K, Kallner M: Gallbladder disease in hyperlipoproteinaemia. Lancet 1:484-487, 1975 3. Van der Linden W, Bergman F: An analysis of data on human hepatic bile. Relationship between main bile components, serum cholesterol and serum triglycerides. Stand J Clin Lab Invest 37:741747, 1977

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SQUALENE

AND CHOLESTEROL IN BILE

tion of the hepatic bile acid and biliary cholesterol precursor sites. J Clin Invest 6 1:408423, 1978 7. Ahlberg J, Angelin B, and Einarsson K: Hepatic 3-hydroxy3-methyl-glutaryl coenzyme A reductase act*ivity and biliary lipid composition in man: Relation to cholesterol gallstone disease and effects of cholic acid and chenodeoxycholic acid treatment. J Lipid Res 22:410-422.1981 8. Maton PN, Murphy GM, Dowling RH: Ursodeoxycholic acid treatment of gallstones. Dose response study and possible mechanism of action. Lancet 2:1297-l 301, 1977 9. Mitropoulos KA, Venkatesan S: The influence of cholesterol on the activity, on the isothermic kinetics and on the temperature induced kinetics of hydroxy-methyl-glutaryl-coenzyme A reductase. Biochim Biophys Acta 489: 126-142, 1977 10. Nervi FO, Carrella M, Dietschy JM: Dissociation of hydroxy-methyl-glutaryl-coenzyme A reductase activity from the overall rate of cholesterol synthesis in the liver following the intravenous administration of lipid. J Biol Chem 251:3831-3833, 1976 1 I. Witztum JL. Schonfeld G, Weidman SW: The effect of colestipol on the metabolism of very-low-density lipoproteins in man. J Lab Clin Med 88:1008-1018, 1976 12. Nestel PJ, Grundy SM: Changes in plasma triglyceride metabolism during withdrawal of bile. Metabolism 25:1259-l 268, 1976 13. Hatch FT: Practical methods for plasma lipoprotein analysis, in Paoletti R, Kritchevski D (eds): Advances In Lipid Research. New York and London, Academic Press, 1968, pp l-68 14. Bartlett GR: Phosphorus assay in column chromatography. J Biol Chem 234:466-468. 1959 15. Carey MC: Critical tables for calculating the cholesterol saturation of native bile. J Lipid Res 19:945-955, 1978 16. Salvioli G. Salati R: Faecal bile acid loss and bile acid pool size during short term treatment with ursodeoxycholic and chenodeoxycholic acids in patients with radiolucent gallstones. Gut 20:698-704, 1979 17. Liu GCK, Ahrens EH Jr, Schreibman PH. et al: Measure-

645

ment of squalene in human tissues and plasma: validation and application. J Lipid Res 17:38&45, I976 18. Salen G. Nicolau G, Shefer S, et al: Hepatic cholesterol metabolism in patients with gallstones. Gastroenterology 69:676684,1975 19. Key PH. Bonorris GG, Coye MJ, et al: Hepatic cholesterol synthesis: A determinant of cholesterol secretion in gallstone patients. Gastroenterology 72:1182, 1977 (abstract) 20. Salen G, Colalillo A, Verga D, et al: Etfect of high and low doses of ursodeoxycholic acid and gallstone dissolution in humans. Gastroenterology 78:1412-1418, 1980 21. Miettinen TA: Effects of bile acid feeding and depletion on plasma and biliary squalene and methyl sterols, in Paumgartner G, Stiehl A, Gerok W (eds): Bile Acids and Lipids. Lancaster, England, MTP Press, 1981, pp 255-262 22. Begemann F, Papenhagen M, Nielsen S: Effect of chenodeoxycholic acid on interrelations of serum squalene with biliary cholesterol saturation and with endogenous triglyceride synthesis, in Paumgartner G, Stiehl A, Gerok W (eds): Bile Acids and Lipids. Lancaster, England, MTP Press, 1981, pp 2633267 23. Carulli N, Ponz de Leon M, Zironi F, et al: Hepatic cholesterol and bile acid metabolism in subjects with gallstones: comparative effects of short term feeding of chenodeoxycholic and ursodeoxycholic acid. J Lipid Res 21:35-43, 1980 24. Small DM: The etiology and pathogenesis of gallstones. Adv Surg 10:63-85, 1976 25. Bell GD, Lewis B. Petrie A, et al: Serum lipids in cholelithiasis: Effect of chenodeoxycholic acid therapy. Br Med J 3:520-522, 1973 26. Miller NE, Nestel PJ: Triglyceride-lowering effect of chenodeoxycholic acid in patients with endogenous hypertriglyceridemia. Lancet 2:929-93 1, 1974 27. Grundy SM, Salen G: Interruption of the enterohepatic circulation of bile acids in man: Comparative etfects of cholestyramine and ileal exclusion on cholesterol metabolism. J Lab Clin Med 78:94-121, 1971