Effect of gemfibrozil on biliary lipid metabolism in normolipemic subjects

Effect of Gemfibrozil on Biliary Lipid Metabolism in Normolipemic Subjects Ottmar Leiss, Klaus von Bergmann, Augostino Gnasso, and Jan Augustin The mechanisms of the lipid-lowering agent gemfibrozil on biliary lipid metabolism were studied in eight normolipemic male volunteers. These measurements were performed before and after 3 months of administration. During administration of gemfibrozil, plasma cholesterol decreased by 19% W < 0.01) and triglycerides by 45% (P < 0.01 I, and HDL cholesterol increased by 10% (P < 0.01). The lithogenic index in gallbladder bile increased from 0.73 to 1.37 (P < 0.05) and in hepatic bile from 0.88 to 1.42 (P < 0.01). The increase in lithogenicity of gallbladder bile and hepatic bile was due to an increased biliary output of cholesterol from 47 to 70 mg/h (P < 0.01) and a decreased output of bile acids from 943 to 884 mg/hr (P -z 0.01). whereas phospholipid output was not altered. The reduction in bile acid output was a result of a significant decrease in chenodeoxycholic acid secretion (r = 0.852: P < 0.01). Cholic acid output was not effected by gemfibrozil. These results suggest that administration of gemifbrozil enhances the possible risk of gallstone formation like clofibrate.

A

VARIETY of drugs have been used to reduce serum cholesterol and triglycerides in an effort to prevent atherosclerosis in patients with hyperlipoproteinemia.‘** One of these agents is clofibrate, which reduces serum triglycerides and, to a minor extent, cholesterol. However, an increased incidence of gallstone formation during clofibrate treatment34 might prevent its widespread use. This possible side effect was predicted by Grundy et al’ and Pertsemlidis et al,’ who demonstrated that clofibrate increases the cholesterol content of human bile. Recently, we have demonstrated that two other fibric acid derivatives, bezafibrate and fenofibrate, influence biliary lipid metabolism in the same manner as clofibrate’ and therefore probably increase the risk for gallstone formation. A relatively new fibric acid derivative, gemfibrozil, a nonhalogenated water-soluble phenoxyvaleric acid derivative,” has now been introduced for treatment of hyperlipidemia. Although this drug has been shown to lower both plasma cholesterol and triglycerides’ I-20 and to raise high-density (HDL) cholesterol levels “-‘3~‘5~‘9 data on its effects on biliary lipid metabolism’ are limited.” In order to elucidate possible actions of gemfibrozil on biliary lipid metabolism, composition of gallbladder bile and hepatic secretion rates of biliary lipids were measured before and after 3 months of administration of gemfibrozil in eight normolipemic volunteers. MATERIALS AND METHODS Volunteers Eight normolipemic male volunteers participated in the study. Age, weight, and ideal body weight of each subject are presented in

From the Department of Medicine, University of Bonn, and Department of Medicine, University of Heidelberg. This work was supported by a grant from Sandor Stiftung fuer therapeutische Forschung. Address reprint requests to Klaus von Bergmann. MD, Department of Medicine, University of Bonn, 5300 Bonn I-Veusberg. Federal Republic of Germany. 0 I985 by Grune & Stratton, Inc. 0026-0495/85/3401~014$01.00/0 74

Table 1. All subjects had normal liver function and no signs of renal disease, thyroid dysfunction, or diabetes mellitus. Their weight remained constant during the study. All subjects had a functioning gallbladder without gallstones as verified by sonography before and after cholecystokinin injection. The study was conducted in accordance with the principles of the Helsinki Declaration, and informed consent was obtained from each subject. Experimental

Design

The study was performed on outpatient basis. Blood for plasma lipids was obtained after an overnight fast two times before, one time after one and two months of the study and twice during the last week. Lipid composition of cbolecystokinin-stimulated fasting duodenal bile (which is hereafter referred to as gallbladder bile) and hepatic secretion of biliary lipids were determined before (period I) and after 3 months of administration of gemfibrozil (I2 x 600 mg/d; period II). During drug administration 800 mg of gemfibrozil were given in the morning of the study before starting measurements of biliary lipid secretion. In another group of eight male volunteers (comparable in body weight), two successive studies of biliary lipid secretion were performed under identical conditions at different time intervals to validate the method in outpatients. The time intervals and the clinical date of these volunteers are given in Table 2. Analytical

Methods

Total cholesterol and triglycerides in plasma were determined enzymatically according to Roeschlau et al** and Eggstein and Kreutz.” Concentrations of HDL cholesterol were measured in the supernatant after separation of very low-density lipoproteins by ultracentrifugation and subsequent polyanion precipitation of apoprotein-B containing lipoproteins.24 Lipid composition of gallbladder bile and biliary lipid secretion rates were measured by the intestinal perfusion technique of Grundy and Metzger. Z5 The evening before the study the subjects were admitted to the Metabolic Ward where they swallowed a triple lumen tube After an overnight fast the tube was positioned with two proximal outlets adjacent to the ampulla of Vater and a third outlet 10 cm distally just beyond the ligament of Treitz. Cholecystokinin (0.5 IE/kg body weight; Kabi Diagnostica, Studsvik, Sweden) was injected intravenously (IV). Thereafter dark gallbladder bile was obtained (approximately 20 mL), shaken vigorously, and immediately checked for the presence of cholesterol crystals by polarizing microscopy.*6 Three milliliters of bile were mixed immediately with chloroform/methanol (2/l; vol/vol), the rest was returned to the duodenum through the most proximal outlet of the 3-lumen tube. A liquid-formula diet (1,42 cal/kg/h) containing 36% of calories as fat, 16% as protein, and 48% as carbohydrates (Nutrodrip, Wander GmbH, 6522 Osthofen, FRG) together with &sitosterol (Delalande, 5000 Koeln, FRG) as a nonabsorbable marker were infused constantly through the most proximal outlet. After allowing Metabdism,

Vol34, No

1

(January), 1985

GEMFIBROZIL AND BILIARY LIPID METABDUSM

Table 1. Clinical Data of Subjects Psrticipated in the Gemfibrozil Study Age Subject

w

Weight

Height

(kg)

(cml

(%I

103 115

1

33

78

184

2

39

89

186

Ideal Weight*

3

27

70

186

90

4

26

80

191

98

5

24

63

175

93

6

34

74

175

110

7

34

68

176

99

8

33

75

180

104

*Ideal weight = weight (kg)/(height [cm] -

100 -

10%) x 100.

four hours for stabilization

of hepatic bile secretion, hourly samples were obtained for six hours from the second proximal outlet and the distal port by continuous aspiration. Storage of gallbladder bile and samples from the proximal outlet (representing postprandial duodenal bile and is hereafter referred to as hepatic bile or stimulated hepatic bile), as well as separation of bile acids from cholesterol and phospholipids, were performed as described in the National Cooperative Gallstone Study.27 The distal samples were added to 10 mL of ethanol. In every sample, cholesterol and j3-sitosterol were measured by gas liquid chromatography (GLC) as trimethylsilylethers using S-cu-cholestane as an internal standard.“’ The recovery of cholesterol and fl-sitosterol averaged 98% (range 94% to 101%). The coefficient of variations of six replicate determinations was i- 1.3%. In gallbladder bile and every sample from the proximal outlet, bile acids were determined enzymatically’9~30 and phospholipids by the method of Bartlett.” Hourly outputs of cholesterol, bile acids, and phospholipids were then calculated according to equations given by Grundy and Metzger.25 Biliary lipid composition was expressed as molar percent for each lipid component,3* and cholesterol saturation of bile was expressed as lithogenic index (LI) according to Carey and Small” assuming total lipid content for gallbladder bile of 20 g/dL and 7.5 g/dL for hepatic bile. Individual bile acids were determined in every proximal sample by GLC. The conjugated bile acids were deconjugated enzymatically (cholylglycin hydrolase; Sigma Chemicals Co. St Louis, Moa4) and solvolized.‘s After methylation”’ and conversion to trimethylsilylethers the individual bile acids were separated on a fused silica capillary column (SP 2100, 25 m, ID 0.20 to 0.21 mm, Hewlett Packard, Palo Alto, Calif) using an automatic injection system (7671 A Automatic Sampler, Hewlett Packard). The recovery of total individual bile acids by this procedure averaged 94% (range 91% to 97%) on every sample when compared to the amount of total bile acids in bile Table 2. Clinical Data on Control Subjects Studied at Different Time Intervals

(kg)

Id%4*

Period A 1

23

B

Weight (cm)

(%)

70-70

185

92

Time Between Period A and B (mo)

14

2

24

70-70

185

92

9

3

23

84-83

184

110

18

4

24

65-66

178

94

4

5

28

65-65

183

67

4.5

6

22

75-76

192

92

1

7

24

75-75

189

94

5

8

25

77-77

190

95

1.5

*Calculation of ideal weight see footnote Table 1

measured by the 3-ol-hydroxy-steroid-dehydrogenase. The coefficient of variations of six replicate determinations of a pooled sample for DCA, CDCA, and CA averaged 0.1%. 0.4%, and OS%, respectively.

Statistics The results ences between test for paired the method of

are expressed as the mean +/ - SD. Statistic differgroups were calculated using Wilcoxon’s signed ranks observations. Linear regressions were calculated by least squares.”

RESULTS Plasma Lipids

Gemfibrozil lowered plasma cholesterol and triglycerides in the subjects of this study from 196 mg/dL + 39 (SD) to 159 mg/dL f 30 (mean reduction = 19%; P < 0.01) and from 126 mg/dL t 53 to 68 mg/dL + 18 (mean reduction = 46%; P -c0.01) respectively; HDL cholesterol was raised significantly from 51 mg/dL f 10 to 56 mg/dL * 13 (mean increase = 10%; P < 0.01). Gallbladder

Bile Composition

Table 3 summarizes the molar composition of biliary lipids and the LI during control period and gemfibrozil administration. Mole percent of cholesterol increased in every subject (mean increase = 118%; P < 0.05), phospholipids in five of six (mean increase = 29%; P -c0.05) whereas mole percent of bile acids decreased on the average by 14% (P -c0.05). These changes in biliary lipid composition were followed by a marked increase in lithogenicity of gallbladder bile. During control period gallbladder bile was undersaturated with cholesterol. The LI of gallbladder bile increased by 88% (P < 0.05) and bile became supersaturated in every subject; however, no cholesterol crystals could be detected in the fresh gallbladder bile. Biliary Lipid Secretion and Composition Postprandial

of

Duodenal Bile

In stimulated postprandial duodenal bile (in the following context substituted by hepatic bile), mole percent cholesterol increased in every subject during gemfibrozil administration (mean increase 82%; P < 0.01) and mole percent bile acids decreased (mean decrease 9%; P -c0.05). Relative amounts of phospholipids were not changed consistently. Hepatic bile was undersaturated in every subject during the control period, and it became supersaturated in all during treatment (mean increase = 65%; P < 0.01; Table 4). Administration of gemfibrozil increased hepatic secretion of cholesterol by 49% (P -c0.01) and reduced bile acid output (mean decrease 26%; P < O.Ol),

76

LEISS ET AL

Table 3.

Effect of Gemfibrozil

on Lipid Composition

of Gallbladder

Lioid Composition

Bile

of Gallbladder Bile

Bile Acids Subject

Period

I

1

Cholesterol

Lecithin

4.6

76.2 -

19.2 -

0.63 _*

5.3

72.0 -

22.7

0.65 _*

II

I

2

II 3

4

5

6

7

8

Mean

(3-8)

2 SD

Mean l-8

r SD

LI

(molar %)

(20%

solids)

I

6.8

73.3

19.9

0.89

II

13.6

62.8

23.6

1.56

I

5.0

77.4

17.6

0.72

II

8.8

76.3

15.0

1.37

I

5.0

78.7

16.4

0.77

II

8.9

70.9

20.2

1.22

I

5.7

76.8

17.5

0.83

II

15.0

62.8

22.2

1.77

I

3.7

81.8

14.5

0.6 1

II

7.4

75.1

17.5

1.05

I

3.7

80.2

16.0

0.58

II

11.6

55.1

33.2

1.25

I

5.0 + 1.2

78.0

f 3.0

17.0 + 1.8

0.73

II

10.9 * 3.0

67.2

+ 8.3

22.0

1.37 f 0.26

(P < 0.05)

(P < 0.05)

(P < 0.05)

77.1

18.0 + 2.6

I

5.0 + 1.0

* 3.3

+ 6.4

+ 0.12

(P to.051 0.71

+ 0.11

*Samples broken inthe centrifuge. Table 4.

Effect of Gemfibrozil

on Lipid Composition

of Stimulated

Lipid Composition

Hepatic Bile

of Hepatic Bile LI

Bile Acids Subject

Period

1

2

3

4

5

6

7

8

MEAN

+

SD

Cholesterol

hllolar %)

Lecithin

(7.5%

solids)

I

6.2

70.7

23.1

0.90

II

7.2

73.1

19.6

1.14

I

5.5

89.6

24.9

0.77

II

9.3

66.0

24.8

1.26

I

5.2

79.2

15.6

0.97

II

11.6

56.2

32.2

1.51

I

3.9

84.1

12.0

0.88

II

9.6

73.0

17.4

1.62

I

4.0

79.7

16.2

0.75

II

7.8

73.5

18.7

1.30

I

5.4

76.7

17.9

0.94

II

12.8

66.0

21.2

1.85

I

4.6

77.6

17.8

0.79

II

7.7

75.9

16.4

1.38

I

4.7

78.4

18.9

0.85

II

7.0

77.8

15.2

1.32

I

5.0 k 0.8

II

9.1 * 2.1

77.0 + 4.8 70.2 YL7.1

18.0 % 4.1 20.7 -t5.5

0.86 + 0.08 1.42 + 0.23

(Pi 0.01)

(P< 0.05)

(NS)

(f
77

GEMFIBROZIL AND BILIARY LIPID METABOLISM

Table 5. Effect of Gemfibrozil

on Outputs of Biliary Lipids Biliary Lipid Secretion

respectively, whereas the output of CA was not altered (mean reduction = 7%; NS).

Bile Acids Subject

1

2

3

4

5

6

Period

Cholesterol

(mg/hr

+ SD’)

Lecithin

Biliary Lipid Secretion and Composition

of Hepatic

I

52 + 9

781 + 173

393 + 38

Bile in Normal Volunteers

II

63 k 14

820 k 149

267 f 215

1031 k 240

Tables 7 and 8 summarize biliary lipid secretion and composition in eight normal volunteers during two successive studies at different time intervals. The time intervals between the studies ranged from 1 to 18 months (average 6 months). Although, there were minor intraindividual changes of secretion of cholesterol (percent range from +3 to -23), bile acids (percent range from + 24 to - 2 1) and phospholipids (percent range from +36 to -33), no consistent changes could be observed during the two different secretion studies. The same was true for mole percent of biliary lipids and the LI of hepatic bile.

580 r 140

I

63 k 16

II

76 + 6

704 ? 56

415 + 27

I

45 k 6

887 + 173

273 A 39

II

89 2 16

561 + 116

507 + 130

I

38 k 3

+ 97

242 + 27

II

87 k 7

855 r 44

322 k 35

I

38 k 12

976 t 289

307 k 81

II

41 k 16

501 + 209

201 + 86

1074

I

41 + 11

757 + 215

275 k 65

II

103 + 33

701 k 233

353 t 111

I

51 t 7

1109 + 340

II

50 + 8

639 + 119

215+30

at Different Time Intervals

DISCUSSION 7

399 + 76

I

43 + 5

930 + 164

313 + 71

II

54 t 6

771 + 72

236 + 20

Mean i SD

I

47 Yk9

943 f 129

348 k 109

Mean ? SD

II

70 + 22

694 t 125

325 + 106

8

(PC 0.01)

P<

0.01)

INSI

*In all subjects, hourly outputs were measured during a period of a six hour formula infusion (steady state) after an initial infusion period of 4 hours (equilibration period). In each case, six hourly determinations were carried out.

whereas phospholipid secretion was not altered (Table 5). A highly positive correlation was found between hepatic secretion of cholesterol and LI of gallbladder bile (r = 0.850; P < 0.001) and LI of hepatic bile (r = 0.772; P -c0.001; Fig 1). Also, a negative correlation was noted between hepatic secretion of bile acids and LI in gallbladder (r = 0.700; P -c0.05) and LI of hepatic bile (r = 0.680; P -c0.01; Fig 2). individual Bile Acid Composition

and Secretion of

Individual Bile Acids in Hepatic Bile

The results of individual bile acid composition and secretion rates of hepatic bile acids are summarized in Table 6. Mole percent and secretion rates of the minor bile acids, lithocholic and ursodeoxycholic acid, were not altered by gemfibrozil. The relative content of chenodeoxycholic (CDCA) and deoxycholic acid (DCA) was reduced during gemfibrozil administration by 6% and 21%, respectively, but this decrease was of borderline significance (Pbetween 0.05 and 0.10). The increase in mole percent of cholic acid (CA), however, was significant (mean increase = 32%, P -c0.01). In contrast, hepatic secretion rates of CDCA and DCA were reduced by 33% (P < 0.01) and 35% (NS),

The fibric acid derivatives have been used world wide for treatment of hyperlipidemia. These drugs are particularly effective in therapy of hypertriglyceridemia because they stimulate lipoprotein lipase.‘3,38,39 However, in hypercholesterolemic patients without elevated triglyceride levels, the fibric acids also reduce levels of low-density lipoproteins (LDL).“,4”14’ The mechanisms of LDL lowering have not been determined with certainty, but Packard et a14*have reported that they enhance the clearance of circulating LDL. Although all the fibric acids are highly effective in lowering triglyceride levels, the relative potency for reducing LDL seems to differ. The newer derivatives, fenofibrate and bezafibrate, appear to be more effective than clofibrate for reducing plasma LDL.4’.43 Recently a new fibric acid, gemfibrozil, has been introduced in the United States. This drug seems to have a similar potency for decreasing concentrations of both triglycerides and LDL as the other newer derivatives.“-*’ A side effect of clofibrate therapy is an increased risk for cholesterol gallstones,3” which is secondary to an increase in saturation of bile with cholestero1.44 The rise in saturation appears to be related to two factors, namely an enhanced secretion of cholesterol in bile’.* and a decreased synthesis of bile acids.7,45Although earlier studies suggested that newer derivatives, bezafibrate and fenofibrate, do not raise bile saturation,46,47 we have shown recently that both these agents induce a rise in saturation.’ In the present study we have extended our studies to gemfibrozil, the newest available agent. The current studies were carried out in normal subjects who had normal plasma lipids. Nonetheless, even in these subjects, gemfibrozil caused a lowering of serum cholesterol and triglycerides and a rise in HDL

78

LEISS ET AL

0 \A 0

I 20

40

60

CHOLESTEROL

0

80

100

120

100

’ 120

Cmg/hrl

lB 0

20

40

60

CHOLESTEROL

00

Cmg/hrl

Fig 1. Relationship between hepatic secretion of cholesterol and lithogenic index (LI) of gallbladder bile (r = 0.850; P < 0.001: n = 12; upper panel) and LI of hepatic bile (r = 0.772; P < 0.001; n = 16: lower panel).

2 0

1.6 u

‘;--::I---

1.2

-i .a

0

.I

A

0 II

, 200

400 BILE

Fig 2. bile acids 0.05; n = 0.880; P <

Relationship between hepatic secretion of and LI in gallbladder bile (r = -0.700: f’ < 12; upper panel) and LI of hepatic bile (r = 0.01; n = 16; lower panel).

0

600

ACIDS

wo

1000

l2m

1000

l2M

Cmg/hrl

1B 0

, 200

400

BILE

MO

ACIDS

Ml0

Cmg/hrl

GEMFIBROZILAND BILIARY LIPID METABOLISM

79

Table 6. Effects of Gemfibrozil on Individual Bile Acid Composition and Secretion of Stimulated Hepatic Bile CDCA

CDCA Penod

Sub&t

1 II 2 II

DCA

CA

DCA

lmolar %)

CA

23

48

23

lBl+

377 iB3

177 +42

27

40

23

226 + 52

327 -t54

188 + 26

Ime/h)

39

25

53

18

258 + 65

547 + 122

185 k41

19

53

25

135 k 11

371+

32

178 k 14

243 +43

27

40

29

357 * 68

262 + 58

II

16

34

49

87 + 16

195 + 50

273 + 57

10

54

33

112 f 10

582 + 48

356 -e38

II

18

42

37

156 + 6

362 k 17

321 + 20

417*133

206 k 54

221 * 90

169 -t73

3

4

34

42

21

329 + 97

II

18

44

34

89 f 38

61

25

11

464 f 129

189 + 53

85 t 25

II

47

31

19

328 + 108

221 + 77

133 k 52

5

6

5

65

28

54 + 9

722 f 111

314 + 49

II

5

59

34

31*5

380 zk72

220 2 42

3

56

39

32 f 7

525 + 86

361+-61

II

3

52

43

24 k 2

401 * 41

328 IV38

24~19

48 + 12

25 r 9

209 + 146

465 + 164

243 k 97

19k14

44-c 10

33 + 10

135 * 102

3lOiB4

226 t 73

7

8

Mean _t SD II

(NS)

(NSI

P<

0.01)

(P
(NS)

INS)

Table 7. Comparison of Composition of Stimulated Hepatic Bile in Normal Subjects in Two Successive Studies at Different Time Intervals Biliarv Lipid Comoositicn

of Heratic

Bile

Bile Acids Subject

5

6

7

8

Mean + SD

Penod

Cholesterol

LI

(molar %I

Lecithin

(7.5%

solids)

A

5.1

70.8

24.1

0.72

a

4.4

69.7

25.9

0.61

A

5.6

69.7

24.6

0.78

B

5.5

66.1

28.5

0.72

A

6.9

66.9

26.2

0.93

a

5.6

70.4

24.0

0.70

A

5.2

75.6

19.2

0.85

a

5.9

76.1

18.0

1.00

A

4.9

80.4

14.7

0.96

B

4.3

78.5

17.2

0.76

A

4.4

76.2

19.4

0.71

B

5.0

77.1

17.8

0.85

A

4.4

71.7

24.0

0.63

8

4.4

74.4

21.2

0.72

A

5.0

77.4

17.5

0.86

a

4.9

77.7

17.4

0.85

A

5.2 f 0.8

73.6 i 4.5

21.2 * 4.1

0.81 k 0.11

a

5.0 + 0.6

73.0 k 4.5

21.3 e 4.4

0.78 * 0.12

(NS)

(NS)

(NS)

(NS)

LEISS ET AL

80

Table 8. Comparison

of Outputs of Biliary Lipids in Normal

Subjects in Two Successive

Studies at Different

Time Intervals

Wary LipidSecretion Lecithin

Period

1

A

29 f 11

548 + 242

266 + 101

8

30 + 5

616 + 106

361 + 85

A

55 k 20

900 * 379

492 k 189

8

53 f 12

841 f 296

549 k 114

A

58 f 6

742 + 126

456 f 72

8

51 + 17

922 * 447

458 f 212

A

45*

851 + 247

338 f 9

8

39 r 10

669

246 k 78

A

45k

8

42 t 4

1007 f 167

345 f 44

A

43 k 6

1268

512 k 392

8

47 -t 12

1001 f 321

A

44 t 9

951 f 201

509 f 169

8

34 + 6

758 5 161

339 + 67

A

52?

8

55 f 5

A

46k

8

43 k 9

2

3

4

5

6

7

8

Mean k SD

Cholesterol

BileAcids (me/h f SD*)

Subject

13

13

(NS)

14

18

+ 234

982 f 358

1047

+ 835

f. 242

1174 f 217

286 + 114

343 + 85

378 + 138 411 + 50

911 + 212

405 f 101

874 f 189

382 + 91

NW

(NS)

*In all subjects, hourly outputs were measured during a period of a six hour formula infusion (steady state) after an initial infusion period of four hours (equilibration period). In each case, six hourly determinations were carried out.

cholesterol. Furthermore, the drug induced a marked increase in saturation of gallbladder bile and stimulated hepatic bile. These results extend the observations made by Hall et al,*’ who found a significant rise in biliary cholesterol saturation in ten healthy volunteers after four weeks of treatment with clofibrate (2 mg/d) and a similar trend with gemfibrozil. Why those authors could not find a significant increase with gemfibrozil remains unclear, but it might be due to the shorter time period of the study. The consistency of the present studies, however, leaves little doubt that gemfibrozil, like the other fibric acids, has an adverse effect on bile composition. By what mechanisms does gemfibrozil increase bile saturation? Two factors appeared to be involved. There was a positive correlation between cholesterol secretion and LI in gallbladder bile and hepatic bile and a negative correlation between bile acid output and LI of both types of bile. The increased secretion of biliary cholesterol on gemfibrozil is in, accord with high-biliary outputs of cholesterol during treatment of

hyperlipidemic patients with clofibrate and bezafibrate.‘,’ The decrease in outputs of bile acids in bile might be a result of a reduction in synthesis of bile acids, which has been demonstrated for c10fibrate,7*45 but to date measurements of hepatic secretion of bile acids have not been performed in patients treated with clofibrate. Estimates of secretion rates of bile acids before and during bezafibrate treatment of three hyperlipidemic patients did not reveal the marked reduction in bile acid secretion noted for gemfibrozil.g Since biliary lipid output at different time intervals in a control group (Tables 7 and 8) remained almost unchanged, the observed change in cholesterol and bile acid output during gemfibrozil was entirely due to administration of this drug. Of particular interest is the finding of the action of gemfibrozil on CDCA. For secretion rates only those for CDCA were significantly lower during drug therapy. Also, a significant correlation between total bile acid secretion and CDCA output during gemfibrozil administration was observed (r = 0.852; P -c0.01). Gemfibrozil seemingly did not alter metabolism of CA. Since biliary cholesterol outputs are affected by the amount of CDCA secreted in bile48,4git is interesting to speculate that part of the increase in biliary cholesterol on gemfibrozil could be due to a reduction of hepatic secretion of CDCA. What is the origin of the excess cholesterol in bile during administration of gemfibrozil? Previously, Grundy et al’ observed an increased fecal excretion of cholesterol in patients treated with clofibrate, and this increment exceeded the decrement in bile acid excretion. This observation combined with isotope kinetic results led these workers to suggest that the excess cholesterol excretion was due to mobilization of cholesterol from tissue pools and not from enhanced synthesis of cholesterol. Recently, fecal steroids also have been measured during gemfibrozil therapy by Kesaniemi and Grundy. 5o Gemfibrozil raised the output of fecal cholesterol, but in contrast to the findings with clofibrate, the increment did not exceed the reduction in bile acid excretions. Therefore, this lack of net change in steroid outputs on gemfibrozil raises the possibility that the drug merely changes the distribution of fecal steroids without changing the total cholesterol synthesis. Plasma squalene, as an indicator of cholesterol synthesis,5’952levels fall during gemfibrozil treatment (unpublished results) suggesting that cholesterol synthesis indeed is decreased. The reduced output of bile acids also supports a reduction in cholesterol synthesis. If a reduced synthesis does occur, the excess cholesterol in feces would represent mobilization of cholesterol from preexisting tissue pools, as originally suggested by Grundy et al.’

GEMFIBROZIL

81

AND BILIARY LIPID METABOLISM

If gemfibrozil increases saturation of bile, will it also increase the risk for gallstones? The reported higher incidence of gallstones during clofibrate therapy3d implies that a higher risk also would occur with gemfibrozil. However, the majority of patients with clofibrate treatment do not develop gallstones. Recently, Sedaghat et als3 reported that while clofibrate raises saturation of bile, it does not consistently induce development of cholesterol crystals, a postulated key step in the formation of gallstones. In the current study we likewise were unable to detect cholesterol crystals in fresh bile during treatment with gemfibrozil.

In summary, this study with gemfibrozil, as those carried out in our laboratory with bezafibrate and fenofibrate,’ demonstrates that these fibric acid derivatives raise saturation of bile. The current investigation shows conclusively that increased saturation is due to both enhanced secretion of biliary cholesterol and reduced secretion of bile acids. ACKNOWLEDGMENT The authors wish to thank P. Pitters, G. Westphal, for excellent technical assistance.

and, S. Weiner

REFERENCES 1. Hunninghake DB, Probstfield JL: Drug treatment of hyperlipoproteinaemia, in Rifkind BM, Levy RI (eds): HyperlipidemiaDiagnosis and Therapy. New York, Grune and Stratton, Inc., 1977, pp 327-362 2. Levy RJ: Drugs used in the treatment of hyperlipoproteinaemias, in Goodman Gilman A, Goodman LS, Gilman A (eds): Goodman and Gilman’s The Pharmacological Basis of Therapeutics (ed 6). New York, Macmillan Publishing Co, 1980, pp 834-847 3. The Coronary Drug Project Research Project. Clofibrate and niacin in coronary 231:360-381. 1975 4. Cooper J, Geizerova Lancet 1:1083, 1975

Group: Coronary Drug heart disease. JAMA

H, Oliver MF: Clofibrate

and gallstones.

5. Bateson MC, Maclean D, Ross PE, et al: Clofibrate and gallstone induction. Dig Dis 23:623-628, 1978

therapy

6. Palmer RH: Prevalence of gallstones in hyperlipidemia and incidence during treatment with clofibrate and/or cholestyramine. Trans Assoc Am Physicians 91:424432, 1978 7. Grundy SM. Ahrens EH Jr, Salen G, et al: Mechanisms of action of clofibrate on cholesterol metabolism in patients with hyperlipidemia. J Lipid Res 13:53 I-55 1, 1972 8. Pertsemlidis D, Panveliwalla D, Ahrens EH Jr: Effects of clofibrate and of an estrogen-progestin combination on fasting biliary lipids and cholic acid kinetics in man. Gastroenterology 66565-573,

1974

9. von Bergmann

K. Leiss 0: Effect of short-term treatment with bezafibrate and fenofibrate on biliary lipid metabolism in patients with hyperlipidemia. Eur J Clin Invest l4:150-154, 1984 10. Creger PL. Moersch GW, Neuklis WA: Structure relationship of gemfibrozil (CI-719) and related compounds. Sot Med 69(suppl 2):3-5, 1976

activity Proc R

I I. Olsson AG, Roessner S, G Wallding G, et al: Effect of gemfibrozil on lipoprotein concentrations in different types of hyperlipoproteinaemia. Proc R Sot Med 69(suppl 2):28-31, 1976 12. Vessby B, Lithell H. Boberg J, et al: Gemfibrozil as a lipid lowering compound in hypcrlipoproteinaemia. A placebo-controlled cross-over trial. Proc R Sot Med 69(suppl 2):30-37, 1976 13. Nikkila EA. Ylikahri R, Huttenen JK: Gemfibrozil: Effect on serum lipids, lipoproteins, postheparin plasma lipase activities and glucose tolerance in primary hypertriglyceridaemia. Proc R Sot Med 69(suppl2):58-63, 1976 14. Lageder H, lrsigler K: Evaluation of increasing doses of gemtibrozil in hyperlipoproteinaemia. Proc R Sot Med 69(suppl 2):71-75, 1976 15. Janus ED, Costa D, Ononogbu IC, et al: The evaluation of lipoprotein changes during gemfibrozil treatment. Proc R Sot Med 69(suppl 2):76-77, 1976

16. Schwandt P, Weisweiler P, Neureuther G: Serum lipoprotein lipids after gemfibrozil treatment. Artery 5: 117-l 24, 1979 17. Nash DT: Gemfibrozil-a new lipid lowering agent. J Med 11:107-l 16, 1980 18. Kissebah AH, Adams PA, Wynn V: Lipokinetic studies with gemfibrozil (CI-719). Proc R Sot Med 69(suppl2):94-97, 1976 19. Kaukola S, Manninen V, Malkonen M, et al: Gemfibrozil in the treatment of dyslipidaemias in middle-aged male survivors of myocardial infarction. Acta Med Stand 209:69-73, 1981 20. O’Connor RE: Gemfibrozil: Results of clinical studies in the United States, in Ricci G, Paoletti R, Pocchiari F, et al (eds): Therapeutic Selectivity and Risk/Benefit Assessment of Hypolipidemic Drugs. New York, Raven Press, 1982, pp 59-61 21. Hall MJ, Nelson LM, Russel RI, et al: Gemfibrozil-the effect on biliary cholesterol saturation of a new lipid-lowering agent and its comparison with clofibrate. Atherosclerosis 39:5 11.-5 16.

19131 22. Roeschlau P, Bernt E, Gruber W: Enzymatische Bestimmung des Gesamt-Cholesterins im Serum. Z Klin Chem Klin Biochem 12:226-231.1974 23. Eggstein M, Kreutz FH: Eine neue Bestimmung der Neutralfette in Blutserum und Gewebe. Klin Wochenschr 44:262-266, I966 24. Lipid Research Clinic Program Manual of Laboratory Operations: Department of Health, Education of Welfare Publication. NIH 75-628,1974 25. Grundy SM, Metzger AL: A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62:120&1217, 1972 26. Sedaghat A, Grundy SM: Cholesterol crystals and the formation of cholesterol gallstones. N Engl J Med 302: 1274-l 277, 1980 27. Hofmann AF, Grundy SM. Lachin JM, et al: Pretreatment biliary lipid composition in white patients with radiolucent gallstones in the National Cooperative Gallstone Study. Gastroenterology 83:738-752.1982 28. Miettinen TA, Ahrens EH, Grundy SM: Quantitative isolation and gas-liquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 6:411424, 1965 29. Talalay P: Enzymatic analysis of steroid hormones. Methods Biochem Anal 8:114-143, 1960 30. Paumgartner G, Horak W, Probst P, et al: Effect of phenobarbital on bile flow and bile salt excretion in the rat. Naunyn Schmiedebergs Arch Pharmacol270:98-102, 197 1 3 1. Bartlett GR: Phosphorous assay in column chromatography. J Biol Chem 234:466-468, 1959 32. Admirand WH, Small DM: The physiochemical basis of cholesterol gallstone formation in man. J Clin Invest 47:1043-1052, 1968

82

33. Carey M, Small DM: The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man. J Clin Invest 61:998-1026, 1978 34. Nair PP, Gordon M, Reback J: The enzymic cleavage of the carbon-nitrogen bound in 3-alpha, ‘I-alpha, 12-alpha-trihydroxy5-beta-cholan-25oylglycine. J Biol Chem 242:7-l 1, 1967 35. Van Berge Henegouwen GP, Allan RN, Hofmann AF, et al: A facile hydrolysis-solvolysis procedure for conjugated bile acid sulfates. J Lipid Res 18:118-122, 1977 36. Ali SS, Javitt NB: Quantitative estimation of bile salts in serum. Can J Biochem 48:1054-1057,197O 37. Snedecor GW, Cochran WG: Statistical methods (ed 6). Ames, Iowa, Iowa State University Press, 1967 38. Tayler KG, Holdsworth G, Galton DJ: Clofibrate increases lipoprotein-lipase activity in adipose tissue of hypertriglyceridaemic patients. Lancet II:1 106-l 107, 1977 39. Vessby B, Lithell H, Ledermann H: Elevated lipoprotein lipase activity in skeletal muscle tissue during treatment of hypertriglyceridaemic patients with bezafibrate. Atherosclerosis 44: 113118,1982 40. Olsson AC, Lang PD: One-year study of the effect of bezatibrate on serum lipoprotein concentrations in hyperlipoproteinaemia. Atherosclerosis 31:429433, 1978 4 1. Roessner S, Oroe L: Fenofibrate therapy of hyperlipoproteinaemia-A dose-response study and a comparison with clofibrate. Atherosclerosis 38:273-282, 1981 42. Packard CJ, Stewart JM, Lorimer AR, et al: Drug-induced modulation of the low density lipoprotein receptor pathway in the treatment of hypercholesterolaemia, in Crepaldi G, Greten H, Schettler G, et al: (eds): Lipoprotein Metabolism and Therapy of Lipid Disorders. Amsterdam-Oxford-Princeton, Excerpta Medica, 1982,~~ 107-112 43. Lageder H: Vergleichende Doppelblindstudie von Bezafibrat

LEIS

ET AL

und Clofibrat bei Patienten mit primaerer Hyperlipoproteinaemie. Wien Klin Wochenschr 92:95-101,198l 44. Angelin B, Einarsson K, Leijd B: Biliary lipid composition during treatment with different hypolipidaemic drugs. Europ J Clin Invest 9:185-190, 1979 45. Einarsson K, Hellstroem K, Kallner M: The effect of clofibrate on the elimination of cholesterol as bile acids in patients with hyperlipoproteinaemia type II and IV. Em J Clin Invest 3:345-351, 1973 46. Schlierf G, Chwat M, Feuerborn E, et al: Biliary and plasma lipids and lipid-lowering chemotherapy-studies with clofibrate, fenofibrate and etofibrate in healthy volunteers. Atherosclerosis 36:323-329, 1980 47. Schlierf G, Fischer H, Roche A, et al: Gallenlipide unter Bezafibrat. Muench Med Wochenschr 122:165-168, 1980 48. Adler RD, Bennion LH, Duane WS, et al: Effects of low dose chenodeoxycholic acid feeding on biliary lipid metabolismk. Gastroenterology 68:326-334, 1975 49. LaRusso NF, Hoffman NE, Hofmann AF, et al: Effect of primary bile acid ingestion on bile acid metabolism and biliary lipid secretion in gallstone patients. Gastroenterology 69: 130 l-l 3 14, 1975 50. Kesaniemi YA, Grundy SM: Influence of gemfibrozil on metabolism of cholesterol and plasma triglycerides in man. JAMA 251:2241-2246,1984 51. Miettinen TA: Serum squalene and methyl sterols as indicators of cholesterol synthesis in vivo. Life Sci 8:7 13-721, 1969 52. Nestel PJ, Kudchodkar BJ: Plasma squalene as an index of cholesterol synthesis. Clin Sci Mol Med 49:621624, 1975 53. Sedaghat A, Kesaniemi YA, Grundy SM: Cholesterol crystals-a crucial link in formation of cholesterol gallstones, in Paumgartner G, Stiehl A, Gerok W (eds): Bile Acids and Cholesterol in Health and Diseases. Lancaster, MTP Press, 1983, pp 43-53