Lithocholate cholestasis—Sulfated glycolithocholate-induced intrahepatic cholestasis in rats

GASTROENTEROLOGY

1981:80:233-41

Lithocholate Cholestasis-Sulfated Glycolithocholate-Induced Intrahepatic Cholestasis in Rats I.M. YOUSEF, B. TUCHWEBER, M. AUDET, and C.C. ROY

R.J. VONK,

Centre de Recherche Pediatrique, H6pital Ste-Justine, Universite de Montreal, Montreal, QuBbec,Canada

The intrahepatic cholestasis induced by lithocholic acid and its conjugates is characterized by specific morphological and biochemical changes in the bile canalicular membrane. Although sulfation of lithochelates has been suggested as an important detoxification mechanism, there is as yet little experimental evidence to support this contention. A bile jistula was created in male Wistar rats and 2 h later, they were given i.v. (24 ~moles/lOO g body wt) one of the following: lithocholic acid sulfate, its taurine or glycine conjugate. Taurolithocholic acid sulfateinjected animals showed no change in bile flow monitored every 15 min over a 2-h period when compared to controls who received a bile acid-free solution of 7.5% albumin in 0.45% NaCl. The response to lithocholic acid sulfate was variable and characterized by a 20% decrease in bile flow limited to the first 30 min. In contrast, glycolithocholic acid sulfate reduced bile jlow by more than 60% in the first 30 min in a dose-dependent manner. Thereafter, bile flow gradually returned to 70% of the control level. Lithocholic acid sulfate was mainly excreted as a taurine conjugate. Taurolithocholic acid sulfate rapidly appeared in bile in contrast to the delayed appearance of glycolithocholic acid sulfate. Lithocholic acid sulfate and taurolithocholic acid sulfate did not lead to any electron microscopy changes in Received August l&1979. Accepted September 2,1989. Address requests for reprints to: Dr. I.M. Yousef, Centre de Recherche Pediatrique, HGpital Ste-Justine, 3175 Chemin SteCatherine, Montreal, Quebec, Canada H3T lC5. This work was supported by a grant from the Medical Research Council of Canada and was presented to the American Gastroenterological Association’s annual meeting, New Orleans, May, 1979. Dr. Vonk’s present address is: Department of Pediatrics, University of Gronigen, Holland. The authors wish to thank Dr. Martin Carey of the Peter Bent Brigham Hospital for providing a reprint of reference 42, and for helpful discussions. 0 1981 by the American Gastroenterological Association 0016-5065/61/020233-09$02.50

D. MASSE,

and Department

de Nutrition,

hepatocytes. However, glycolithocholic acid sulfate induced the formation of single membrane bound cytoplasmic vacuoles containing material of low electron density which were evident as early as 10 min after injection. The percent of hepatocytes with cytoplasmic vacuoles and the percent volume density of vacuoles reached its maximum at 60 min after injection. At 2 h, these values did not differ significantly from controls. The bile canaliculi appeared normal and contained canalicular microvilli. These data suggest that sulfated lithocholic acid conjugated with glycine is cholestatic in rats and that the mechanism may differ from the cholestasis induced by lithocholic acid. Lithocholic acid (LCA) and its conjugates are known to induce intrahepatic cholestasis in a variety of experimental animals (1-15). The significance of this cholestasis in humans has not been evaluated because LCA is usually a minor component of normal human bile (16). Furthermore, it has been claimed, albeit with little experimental evidence, that humans are protected from the hepatotoxicity of LCA by the efficient sulfation of this bile acid (17,16). Sulfation of LCA renders the bile acid more water-soluble and promotes its renal and fecal excretion (18). However, there is a renewed interest in the potential hepatotoxicity of LCA because this bile acid is formed mainly from the bacterial 7a-dehydroxylation of chenodeoxycholic acid (CDCA) (19) which is used for the dissolution of gallstones (20,21). Monohydroxy bile acids (LCA and 3P-hydroxy+cholenoic) have been identified in human amniotic fluid (22) meconium (23,24), fetal bile (25) as well as in the bile of neonates with cholestasis or biliary atresia (26). The 3/l form has also been identified as a major bile acid in the urine of patients with hepatoma (26). The consistency with which LCA induces cholestasis offers an important model for new in-

234

YOUSEF ETAL.

sights into the mechanism of bile formation and the understanding of the pathogenesis of secretory failure by the liver. The mechanisms involved in LCA cholestasis have been discussed in previous publications (1,4,8,27-30). However, there is very little experimental evidence concerning hepatic response to the sulfated lithocholate compounds (31-33). The purpose of this study was to test the effect of sulfated lithocholates on liver structure and function with regard to biliary secretion. The results confirm that sulfation of LCA protects against LCA toxicity, but suggest that this protection depends on the type of LCA conjugates produced: Glycolithocholic acid sulfate (G-LCA-S) is cholestatic and taurolithocholic acid (T-LCA-S) is not.

Materials and Methods Male Wistar rats, ZOO-250 g (High Oak Ranch, Montreal, Quebec) were used in these studies. They were

fed standard laboratory chow and had free access to water. Under pentobarbital anesthesia, the bile duct and the tail vein were cannulated with PE-10 (polyethylene) tubing (Clay Adams). Bile was collected for 120 min after cannulation of the bile duct. Over a 5-min period, the animals were given a dose of 24 pmoles/lOO g body wt of the disodium salt of either lithocholic acid (LCA-S), taurolithocholic acid (T-LCA-S), or glycolithocholic acid (G-LCA-S). This dose was chosen because previous work with nonsulfated lithocholate has been shown to induce a cholestatic response when given by infusion or as a single bolus (1,2). The three sulfated bile acids were dissolved in a 0.45% NaCl solution containing 7.5% albumin. They were prepared in the laboratory by the method of Jenkins and Sandberg, modified by using sulfur trioxide (34). The parent compounds LCA, T-LCA, and G-LCA were obtained from Calibiochem (San Diego, Calif.) and were more than 99% pure as analyzed by thin-layer gas chromatography techniques (3536). Purity of the sulfated products was verified by thinlayer chromatography by using butanokacetic acid:water (10: 1:1)as a solvent system and then were compared with standards of LCA-S, G-LCA-S, and T-LCA-S obtained from Calbiochem. Both products (produced in the laboratory and Calbiochem standards) gave exactly the same RP values of 0.60,0.53, and 0.26 for LCA-S, G-LCA-S, and TLCA-S, respectively. After solvolysis with acetone-methanol (9 : I) at pH 1 and at 37% for 24 h, hydrolysis, methylation, and acetylation were carried out (Se), and all three products gave a single peak for LCA methyl ester acetate. The products gave a negative reaction with hydroxysteroid dehydrogenase (EC 1.1.1.50) before solvolysis and a positive reaction after solvolysis, indicating that the sulfur group was on the carbon in the 3 position. Furthermore, G-LCA-S gave a positive reaction with the G-LCA-S antibody obtained from Abbott Laboratories. Bile was collected for 15, 30, 60, and 120 min intervals

GASTROENTEROLOGY Vol. 60, No. 2

after the injection. At the end of the experiment, the animals were killed and their livers were removed for examination by electron microscopy. Livers were fixed by immersion for 1 h in Millonig’s osmium fixative at 4°C. Specimens were dehydrated in graded ethanol and embedded in Araldite. Thin sections were cut from centrolobular zones and were stained with uranyl acetate and Reynold’s lead citrate. The livers of 3 rats per group were taken at times indicated above, and from each liver at least 2-3 blocks were sectioned and later examined. Morphologic evaluation was done by two separate investigators without knowledge of treatment. In an additional experiment using G-LCA-S, 3-5 rats were killed or underwent biopsies at 10,20,30, and 60 min after injection. Large areas, O&pm thick resin-embedded tissue sections, stained with toluidine blue were used for light microscopy before trimming the blocks for electron microscopy. Morphometric analysis under light microscopy was used to calculate the average mononuclear hepatocyte volume and the volume of nuclei and of hepatocytic cytoplasmic vacuoles. From each animal, 5 blocks containing centrolobular zones were used, and a minimum of 10 sections per rat was photographed. Light-microscope photomicrographs of random thick sections at a final magnification of x loo0were employed for morphometric analysis. The technique for percent cytoplasmic volume density measurement was similar to methods described previously for rat liver cells (37). Point counting was used to calculate the volume occupied by an object per unit volume of cytoplasm. This was done by placing a coherent double-lattice test system on the photomicrograph and obtaining the number of these points within the cytoplasm (NC) as well as the number of points within a given object @Vi). The percentage of cytoplasm occupied by a given object was calculated as percent cytoplasmic volume density = (NVi/Nc) X loo(36). The means and standard errors were calculated and for group comparisons the Student’s t-test was applied. Bile volume was measured and biliary lipids were quantitated. Bile acids were determined by combined thin-layer gas chromatography and total lithocholate was measured in the liver, plasma, and bile (35,36); cholesterol was determined by gas chromatography and phospholipids after extraction of neutral lipids with chloroform-methanol (2: 1 vol/vol) by the method of Bartlett (39). As G-LCA-S was the only bile acid that significantly reduced bile flow, further experiments were carried out to determine whether the response was dose dependent. For this purpose, two groups of 4 rats each were injected with 6 or 12 pmoles of G-LCA-S per 100 g body wt, and bile flow was measured.

Results Bile Flow Table 1 shows the effect of injection of LCAS, T-LCA-S, and G-LCA-S on biliary secretion. After LCA-S injection, bile flow was reduced by about 20% within the first 30 min, but it returned to control lev-

February

Table

1981

I.

SULFATED LITHOCHOLATE

Effect of Albumin, Lithocholic injection on Bile Flow

Albumin (n = 6)

Time Preinjection O-12.0 min After injection O-15min 15-30min 30-60 min 60-120 min

Acid Sulfate,

6.94 f 0.87 7.00* 6.56 f 6.74 f 6.26 f

0 Values are ~l/lOO g body wt/min

0.91 0.77 0.88 0.78

Taurolithocholic

Acid Sulfate

Lithocholic acid sulfate (n = 8)

and GJycoJithochoJic

Taurolithocholic sulfate (n= 8)

acid

235

CHOILESTASIS

Acid Sulfate

Glycolithocholic sulfate (n= 8)

acid

7.06f 0.89

7.05f 0.80

:7.10 f

6.52f 5.89 f 6.92 f 8.26 f

7.30f 0.54 7.81 f 0.98 6.75 zt 0.76 6.05 f 0.63

2.34f 0.43b 2.97 & 0.63b 4.74 f 0.63b 5.02 f 0.50”

(means + SD). b Significantly

els thereafter. Taurolithocholic acid sulfate did not affect bile flow, although a slight increase (not significant) was observed in the first 30 min after injection. On the other hand, G-LCA-S injection significantly (p < 0.05) reduced bile flow during the entire duration of the experiment; however, the reduction in bile flow was more impressive (60%-70% of control values) during the first 30 min after the injection. Figure 1 shows that the reduction in bile flow following injection of G-LCA-S was dose dependent. BiJiary Lipid Secretion Examination of biliary bile acids by thin-layer chromatography showed that LCA-S and T-LCA-S were secreted as taurine conjugates and that only trace amounts of G-LCA-S could be detected in the case of LCA-S injection. After G-LCA-S treatment, G-LCA-S was secreted without any change. Table 2 shows the total and individual bile acids secreted during the three experiments. Total bile acid secretion was significantly increased (p < 0.05) during the experiments with LCA-S and T-LCA-S when compared to the control (albumin) group. The augmentation of bile acid secretion was mainly due to the increased secretion of LCA and accounted for 79% and 66% of the injected LCA and T-LCA, respectively. In the G-LCA-S experiments, total bile acid secretion was not significantly changed during the first 60 min; however, there was a significant increase in the last 60 min. This increase was also due to LCA secretion, as 46% of the amount injected was recovered. There was no significant change in the other biliary bile acids with the exception of muricholic acid, which was increased in the three bile acid-treated groups as compared to the albumin group. Table 3 shows biliary cholesterol and phospholipid secretion. LCA-S and T-LCA-S did not lead to significant changes in the bihary secretion of choles-

0.91 0.70 0.84 0.73

different from corresponding

1.03

control value p c 0.05.

terol or of phospholipids. The ratios of bile acid to cholesterol and to phospholipid were significantly increased as the augmented output of biliary bile acids was not associated with a proportionate increase in cholesterol or phospholipid secretion. In contrast, G-LCA-S significantly increased biliary cholesterol secretion, and this led to an unchanged ratio of bile acid to cholesterol. It had no effect on phospholipid secretion. The reason for increased cholesterol secretion in G-LCA-S remains to be elucidated. Table 4 shows the amount of LCA recovered in liver, plasma, and bile at the end of the experiment. Eighty-nine percent to 95% of the lithocholate injected was recovered with no significant differences between the LCA-S-, T-LCA-S-, and G-LCA-S-injetted animals. This indicates that the extrahepatic

so-

T\\

6

24

G -LC E-3

dose

Figure 1. Percent reduction in bile flow following administration of 6, 12, and 24 moles G-LCA-S/100 g body wt. The solid line corresponds to the 0-30-min period after injection, and the broken line to the 30-60-min period after injection.

26.2 f 4.7 12.2 * 2.2 9.1 k 2.4 84.9 + 15.1 10.7 + 2.4 10.5 f 3.6 2.4 f 1.1 0.6 f 0.04 55.7 f 8.6 10.6 f 4.3

80.0 + 6.7 ND

60-120

as nmole/lOO g body weight/min

0 Values are expressed

f 7.0

+0.28 31.7

1.45

f 8.0

f 0.27 37.2

1.46

O-60

f 4.6

f 0.32 28.2

1.48

60-120

(min)

and correspond

Postinjection

Phospholipids

Cholesterol

Preinjection

Albumin

21.9 f 3.6 15.2 f 3.9 11.6 f 1.3 57.4 f 17.2 17.4 + 6.3

ND

123.3 * 21.1

Preinfusion

f 24.7 6.3 f 1.9 6.gb f 2.2 6.9 f 0.9 43.2 f 0.67 19.4b f 5.4

272.6

358.4b f 25.2

O-60

Postinfusion

f 4.7 6.2 f 1.6 3.6 f 2.2 1.5 f 0.8 47.4 f 5.6 16.gb * 5.4

42.0

119.5b It 7.7

60-120

(min)

Lithocholic acid sulfate

23.7 f 5.4 14.6 f 3.5 11.6 f 1.8 95.6 f 19.4 28.2 f 9.4

ND

173.8 f 20.7

f 6.5

f 0.52 36.7

1.68

O-60

Postinjection

f 7.3

f 0.55 37.7

1.82

60-120

(min)

acid sulfate

122.2b + 10.6 40.0 + 4.5 6.6 f 0.26 7.7 f 2.0 2.3 rt0.5 44.2 f 8.7 18.1b f 0.62

O-60

Postinfusion

226.7b f. 12.1 152.1 f 6.5 6.4 f 2.4 3.2 f 2.0 2.0 f 0.9 44.2 f 9.8 17.0b f 6.3

60-120

(min)

f 5.7

* 0.42 34.8

1.65

injection

Pre-

f 5.4

f 0.63 31.7

1.78

O-60

* 6.7

f 0.73 34.8

1.77

Preinjection

* 0.63 37.0 f 7.0 control value p < 0.05.

3.35b f 0.42 36.3 f 5.7

60-120 2.53b

O-60

(min)

acid sulfate Postinjection

Glycolithocholic

different from corresponding

f 6.7

f 0.50 32.6

1.69

60-120

(min)

acid sulfate Postinjection

Taurolithocholic

and

control value p < 0.05. ’ ND = Not

26.7 f 5.3 9.6 * 4.6 10.8 f 2.4 59.2 f 8.2 23.8 f 0.83

132.2b f 16.3 ND

Preinfusion

Glycolithocholic acid sulfate

Acid Sulfate Injection on the BiJiary Secretion of Cholesterol

to X f SEM from 3 to 4 animals in each group. b Significantly

f 6.9

* 0.43 38.3

1.80

Preinjection

Lithocholic

Acid Sulfate and GJycoJithochoJic

f 10.0 7.6 f 1.6 2.6 rt0.6 2.1 f 0.9 46.8 f 0.98 13.8 f 5.4

64.6

137.5b * 11.4

IX-120

(min)

different from corresponding

f 24.5 8.5 f 2.4 7.6 f 3.5 2.0 + 0.6 44.5 f 0.87 14.8b + 6.6

265.4

363.1b f 26.7

O-60

Postinfusion

Taurolithocholic acid sulfate

Acid Sulfate on Total and Individual Bile Acids Secreted”

Preinfusion

Acid Sulfate, and GJycoJithochoJic

to X f SD from 4 animals in each group. b Significantly

Effect of Albumin, Lithocholic Acid Sulfate, Taurolithocholic Phospholipids”

and correspond

12.6 f 0.31 5.3 f 1.2 2.3 f 0.4 50.3 f 0.84 9.2 f 4.0

79.9 * 7.7 ND

O-60

Postinfusion (min)

as nmole/lOO g body wt/min

Biliary components

Table 3.

a Values are expressed detectable.

Muricholic acids

Cholic acid

Hyoursodeoxycholic acid

Chenodeoxycholic acid

Deoxycholic acid

Lithocholic acid

145.1 f 20.2 ND"

Preinfusion

Albumin

Effect of Albumin, Lithocholic Acid Sulfate, Taurolithocholic

Total bile acids

Table 2.

February

1981

Table 4.

SULFATED

LITHOCHOLATE

CHOLESTASIS

237

Recovery of Lithocholic Acid in Liver, Plasma and Bile in LCA-S-, T-LCA-S-, and G-LCA-S-Jnjected

Animals” Bile acid LCA-S T-LCA-S G-LCA-S

Total recovery 21.70 f 0.74 22.69 f 0.57 21.30 f 1.07

Liver

Plasmab

1.75 f 0.26d.e 0.67 f O.lOC*e 8.55 f 0~30~~~

1.09 f 0.13 0.79 f 0.09e 1.30 f1.14d

Bile 16.65 * 0.71d.e Zl.Ml f OJWe 11.46 k 0.54C.d

o Values are means and standard errors of 6 samples and expressed as pmole/lOO g body wt. b Plasma values are calculated on the basis of 15 ml blood/ZOO g body wt. c Significantly different from LCA-S. d Significantly different from T-LCA-S. e Significantly different from G-LCA-S. p < 0.05.

excretion of sulfated lithocholate in these acute studies is minimal. The concentration of sulfated lithocholate was significantly higher in liver and plasma of G-LCA-S-injected animals, but significantly lower in their bile. Morphology Electron microscopic examination of the liver tissue from control, LCA-S-, and T-LCA-S-injected rats revealed a hepatocytic ultrastructure where all the cytoplasmic organelles appeared normal. However, in G-LCA-S experiments, at 10,20, and 30, and 80 minutes after injection, the most striking change in the hepatocyte was the presence of round cytoplasmic vacuoles, which were bound by a single membrane and contained granular material of low electron density (Figure 2). At 120 min, few vacuoles were evident, and small dense bodies of unidentified nature were mainly seen in the pericanalicular region (Figure 3). Frequency analysis of cytoplasmic vacuoles revealed a significant increase at 10,30, and 80 min after G-LCA-S. The volume density of vacuoles increased gradually, reaching its maximum 80 min after injection. At 2 h, the values did not differ significantly from controls (Figures 4 and 5). Bile canaliculi appeared normal and were lined by regular microvilli. The appearance of normal microvilli in bile canaliculi of G-LCA-S-treatment rats is unusual as in most experimental models of intrahepatic cholestasis, they exhibit characteristic changes (40). All other cytoplasmic organelles were unaltered.

Discussion This study was performed mainly to test the effect of sulfated LCA on the function of the liver. The data confirms that LCA-S and T-LCA-S are not cholestatic (31,33), but shows that G-LCA-S reduces bile flow in a dose-dependent manner and induces morphological changes in the liver cell. The rat conjugates its bile acids mainly with taurine (19) and as LCA-S is also rapidly conjugated

with taurine, its cholestatic potential may have been hindered in these studies. It may be that if the liver is challenged with LCA-S, the response of the liver depends on whether LCA-S is conjugated with taurine or glycine. In fact, a preliminary observation from this laboratory shows that LCA-S is cholestatic in the guinea pig, an animal species in which 90% of bile acids are conjugated with glycine. If these data can be extrapolated to humans in whom there is preferential conjugation of bile acids with glycine, it is possible to theorize that sulfation of LCA does not protect against the hepatotoxicity of tbis compound because G-LCA-S is cholestatic. There is some indirect evidence to support this suggestion. In almost all patients undergoing CDCA theI;apy, there is an increase in serum transaminases (20,21). Moreover, morphologic changes in the liver that consist of sinusoidal dilatation have been recently described in patients undergoing CDCA therapy (41). These abnormalities could be due to the GLCA-S, as biliary LCA was reported to be increased in such patients. Because G-LCA-S is poorly soluble (42), once secreted in bile it is unlikely to be absorbed by the intestinal tract. Its accumulation in the intrahepatic circulation is thus prevented. Therefore, the toxic effect of G-LCA-S in humans would be reduced by its secretion in the feces. The observation that G-LCA-S and not T-LCA-S is cholestatic may be of importance in newborn children who are not breast fed and who are therefore on a taurinedeficient diet (43,44). Perhaps these infants could be more susceptible to monohydroxy bile acid cholestasis despite its sulfation. The mechanism(s) by which G-LCA-S induces intrahepatic cholestasis is not apparent from these studies. Earlier physico-chemical experiments attributed the hepatotoxicity of nonsulfated LCA to its insolubility at body temperature. It was claimed that the formation of precipitates either within the hepatic cell or in the bile canalicular membrane initiated hepatic injury (45). However, recent studies have shown that LCA induces cholestasis by directly affecting the structure and function of the bile canalicular membrane (1,4,8,27). Nevertheless, there

238

YOUSEF ET AL.

GASTROENTEROLOGY

Vol. 80, No. 2

Figure 2. Liver tissue from rats given G-LCA-S at 30 and 88 min after injection. Only cytoplasmic vacuoles could be seen x 18,800 (A) and x 8,808 (B). In Figure ZB, the arrows point to vacuoles surrounded by zin amorphous and electron-dense material.

Figure 3. Liver tissue from rat given G-LCA-S at 120 minutes after injection. Small bodies of various densities are seen in the canalicular region. The bile canalicular membrane appears to be normal x 9,400.

February

SULFATED

1983

is still some support for the hypothesis that precipitation could be a contributory factor. The evidence is largely based on studies in which di- and trihydroxy bile acids were successfully used to prevent It also comes from or reverse LCA cholestasis (4,5). the observation that although sulfation confers an increased solubility to LCA and T-LCA, it does not change that of G-LCA, which could therefore retain its cholestatic properties (42). Although G-LCA-S was found to be cholestatic in the present study, precipitates were not seen either in the liver cell or in the bile canalicular membranes. The bile canalicular membrane structure including the microvilli remained normal, but cytoplasmic vacuoles appeared as early as 10 min following the injection of G-LCA-S. The preservation of normal microvillous structure has been recently reported in North American Indian children with intrahepatic cholestasis (40). These electron microscopic findings suggest that the mechanism of cholestasis induced by G-LCA-S is different from that induced by the nonsulfated LCA. Although bile acid hepatocyte transport is reported to be a carrier-mediated process (46) the presence of vacuoles after injection of G-LCA-S would support the view that endocytosis with intracellular vacuole formation may be a mechanism for the uptake of this and perhaps of other similar compounds, as was shown for the uptake of certain large-molecular-weight solutes (47-49). Jones et al. (50,51), and Boyer et al. (52)provided evidence that certain substances reach the bile through a vesicular transport system that originates at the sinu-

Y

&!

LITHOCHOLATE

Control

CHOLESTASIS

30min 10 min

239

120min 60 min

Figure 5. Volume density of hepatocyte vacuoles in control rats and at various times after G-LCA-S injection.

soidal surface of the liver cell and translocates to the bile canaliculus. Cytoplasmic vacuoles were less frequent by the end of the experiment and, simultaneously, numerous dense bodies were seen in the pericanalicular area. It is tempting to assume that these dense bodies constitute secondary lysosomes resulting from the fusion of vacuoles with lysosomes. The nature and the fate of these dense bodies as well as the role of lysosomes in cholestasis induced by GLCA-S remains to be determined. In conclusion, this study provides evidence that G-LCA-S is cholestatic and that it causes cholestasis by a mechanism different from the one described for the nonsulfated LCA. Further exploration of this cholestatic mechanism may be important for the understanding of bile formation and secretion.

References

Control

30min 10 min

120min 60 min

Figure 4. Percent of hepatocytes containing cytoplasmic vacuoles in control rats, and at various times after G-LCA-S injection.

1. Kakis G, Yousef IM. Pathogenesis of lithocholate- and taurolithocholate-induced intrahepatic cholestasis in rats. Gastroenterology 1978;75:595-697. 2. Bonvicini F, Gautier A, Gandiol D, et al. Cholesterol in acute cholestasis induced by taurolithocholic acid. Lab Invest 1978;38:487-95. 3. Miyai K, Richardson A, Mayr WW, et al. Subcellular pathology of rat liver in cholestasis and choleresis induced by bile salts. 1. Effects of lithocholic, 3 p-hydroxy-5-cholenoic and dehydrocholic acids. Lab Invest 1977;36:249-58. 4. Kakis G, Yousef IM. Mechanism of cholic acid protection in lithocholate-induced intrahepatic cholestasis in rats. Gastroenterology 1980,78:1492-11. 5. Layden TJ, Boyer JL. Taurolithocholate-inducd cholestasis.

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7.

8.

9.

10.

11.

12.

13.

14. 15. 18.

17.

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19.

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21.

22. 23.

24.

25.

26.

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