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Intrahepatic cholestasis induced by allo monohydroxy bile acid in rats
GASTROENTEROLOGY
1981;81:242-9
Intrahepatic Cholestasis Induced by Allo Monohydroxy Bile Acid in Rats R. J. VONK, B. TUCHWEBER, D. MASS& A. PEREA, M. AUDET, C. C. ROY, and I. M. YOUSEF Centre de Recherche, HGpital Sainte-Justine, Montreal, Montreal, Quebec, Canada
Little is known about the biologic effect of alio bile acids on the liver. Since the monohydroxy-5/3-cholanic acids are known to induce a well-characterized intrahepatic cholestasis, the effects of 3/Chydroxy5cu-cholanic acid was tested in these studies. The bile ducts of 28 male Wistar rats were cannulated, and bile was collected for 2 h. The rats were then divided into three groups: The first group was injected i.v. with 6 pmol/lOO g body weight of 3/3-hydroxy-5acholanic acid in normal saline containing 7.5% albumin. The second group was injected with a similar concentration of the sulfate ester. The third group (control animals) received only albumin-saline solution. Bile was collected #or 2 h at 15, 30, 60, and 120 min. Bile volume and biliary lipids were measured. The livers were biopsed at 30 min after injection and at the end of the experiment for examination by electron microscopy, and a morphometric analysis was performed. The 3/3-hydroxy-5a-cholanic acid and its sulfate ester significantly reduced bile flow, bile salt, cholesterol, and phospholipid secretion in the 1st hour. However there was a tendency for improvement of these parameters in the 2nd hour after injection. The reduction in bile flow was dose dependent. At 30 min after injection, marked changes were observed at the canalicular pole of the hepatocytes. The canaliculi showed dilatation and partial or total Received August 15, 1979. Accepted August 29, 1980. Address requests for reprints to: Dr. I. M. Yousef, Centre de Recherche, HBpital Sainte-Justine, 3175 Chemin Sainte-Catherine, Montreal, Quebec, Canada H3T lC5. Dr. Vonk’s present address is: Department of Pediatrics, University of Groningen, Holland. This work was supported by grant from the Medical Research Council of Canada, was presented at the 30th Annual Meeting of the American Association for the Study of Liver Disease, November, Chicago, 1979. The authors thank Dr. Martin Carey of Harvard Medical School, for providing a copy of reference 27 before publication. 0 1981 by the American Gastroenterological Association OOlS-5085/81/020242-08.902.50
Departement
de Nutrition,
Universite
de
loss of microvilli. Pericanalicular diverticuli originating from the plasma membrane intruded into the underlying cytoplasm. Amorphous material and clusters of microfilaments were seen around the pericanalicular vacuoles. At 2 h, the hepatocytes showed the same changes described at 30 min but to a lesser degree. These data suggest that (a) allomonohydroxy bile acids have a greater cholestatic potential than 5/&analogues (four times the concentration of 5/?-analogue was required to produce the same degree of cholestasis); (bJ the sulfation does not protect against the cholestatic effect of allomonohydroxy bile acids; and (c) the mechanism of allo-monohydroxy bile acid cholestasis involves an early morphologic change in the bile canalicular membrane. The allo bile acids are derivatives of Sa-cholanic acid, analogues of the common 5/?-bile acids. These bile acids are more prevalent in lower species (1,2). The lower concentrstion of allo bile acids in the higher mammals was explained partially on the basis of lower activity of the microsomal &steroid reductase (1). Whether this difference is evolutionary or protective for mammals is not known. Because of the paucity of these bile acids, it is also not known whether allo bile acids play any role in regulation and control of sterol and/or bile acid synthesis. The question of their toxicity via the diet or other diseases remains to be ascertained. Since the monohydroxy !$-bile acids are known to induce intrahepatic cholestasis in mammals (3-17) by mainly affecting the bile canalicular membrane structure and function (3,8), it became of interest to investigate the biologic effect of similar !%cholanic acids. The data obtained indicated that the allomonohydroxy bile acid is a more potent cholestatic agent than the 5&cholanic acid. The cholestasis induced involves an early change in the bile can-
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Table
1.
Effect of Albumin, 3P-Hydroxy-5a-Cholanic Injections on Bile Flow
After injection
60-120
O-60
243
Acid, and its Sulfated Ester
Preinjection Compound
BILE ACID CHOLESTASIS
o-15
15-30
30-60
60-120 (min)
Albumin
9.2 f1.2
10.0 f0.7
9.1 21.1
9.5 k1.1
9.3 f0.9
8.7 f1.0
3 P-Hydroxy 5 a-Cholanic
9.3 k2.0
9.2 k1.8
5.4” f0.8
1.6" k1.1
1.7",b fl.1
2.9" f1.6
3 B-Sulfated 5 cr-Cholanic
10.6 21.5
10.1 +1.4
5.0” k1.4
3.0” k1.6
2.9”,” f0.6
4.3” k1.4
_
” Significant difference from corresponding albumin period p < 0.05. b Significant difference from corresponding 3/?-sulfated-5a-cholanic acid period p < 0.05. c Significant difference from corresponding 3/I-hydroxy-5a-cholanic acid period p < 0.05. Values are means and standard deviations of six animals and expressed as ~l/lOO g body wt/min.
alicular membrane of the hepatocyte. The data also show that sulfation of the allo-monohydroxy bile acid does not prevent its cholestatic potential.
Materials and Methods 3/3-Hydroxy-5a-cholanic acid sodium salt was obtained from Steroloid (New York, N.Y.). The compound gave a single spot, in various systems of thin-layer chromatography (TLC), with Rf value similar to lithocholic acid. When examined by gas liquid chromatography (GLC), the compound gave a single peak with retention time higher than lithocholic acid when it was derivated as methyl ester acetate and using a 3% OV-225 column (18). Therefore we concluded that the compound does not contain any detectable contaminants. The sulfate ester of the above compound was synthesized in this laboratory using a sodium trioxide complex as previously described (19). The sulfate ester was examined by TLC and GLC and was shown to be a single compound. The bile acid was solubilized in 7.5% albumin in normal saline at a concentration of 3 pmol/ml. Eighteen male Wistar rats obtained from Biobreeding farm (Montreal, Quebec) and weighing 200 + 5 g were used in these studies. The rats were fed laboratory Purina Table 2.
Effect of Albumin, 3/?-Hydroxy-5a-Cholanic Phospholipid Secretion Albumin
Acid and its Sulfated Ester on Biliary Bile Acid, Cholesterol, 3/3-Hydroxy-5cY-cholanic
After injection Biliary lipids Bile salt
Preinjection 146 f21.8
O-60 84 k14.7
chow before the experiments. A biliary fistula was created, and bile was collected for the next 120 min. In six rats 6 pmol/lOO g body wt of 3P-hydroxy-5a-cholanic acid were injected intravenously in the femoral vein, and bile was collected for 2 h at 15, 30, 60, and 120 min after injection. In another six rats treated in a similar manner, 6 pmol/lOO g body wt of the sulfate ester of 3/?-hydroxy-5acholanic acid were injected. The remaining rats were injected with 2 ml of 7.5% albumin/100 g body wt. The dosage used in the present study was based on a preliminary dose-response experiment in which six groups of four rats each were injected with 3, 6, or 9 ymolJlO0 g body wt of the parent compound or its sulfate ester, and bile flow was determined for 1 h after injection. The livers of the animals were biopsied at 30 min after injection. At the end of the experiment the animals were killed, and the livers were autopsied. The liver tissues obtained at 30 min and 2 h after injection were fixed in osmium tetroxide and processed for electron microscopy as previously described (19). Sections 0.5 pm thick were stained with toluidine blue to select midlobular areas for thin sectioning. Morphometric analysis consisted of measurements of volume density (Vv) according to Weibel et al. (20). Approximately 15 hepatocytes were randomly photographed from each block at an original magnifica-
60-120 76 k13.8
3P-Sulfated-5a-cholanic
acid
138 f23.4
O-60
60-120
400 k6.7
24" f4.5
acid
After injection
After injection Preinjection
and
Preinjection 152 f20.9
O-60
60-120
48" k6.6
39O f5.7
Cholesterol
1.38 -co.44
1.29 kO.28
1.43 -to.52
1.49 f0.38
0.73" kO.29
0.48" -co.17
1.28 f0.53
0.64O +0.34
0.59" f0.21
Phospholipids
33.4 f6.9
22.8 k7.0
21.9 f4.8
38.7 k6.4
11.1" 23.8
8.4" f2.9
36.3 27.3
18.41" f4.l
6.4" k2.8
0 Significant difference from corresponding expressed as nmole/lOO g body wt/min.
control value p < 0.05. Values are means f standard
deviation of six samples. Values are
244
GASTROENTEROLOGY Vol. 60, No. 2
VONK ET AL.
Results
tion of x 4700, centering the bile canaliculi in the photographed field. A total of 30 photographs from pericanalicular areas per animal were taken and a total of 150 photographs was obtained from five animals in each group. Fixed areas of 100 pm2 were used for calculation of volume density. Prints were prepared at x 17,ooO magnification and volume densities determined for canalicular lumina, microvilli, pericanalicular vacuoles, and diverticuli as well as for the “Golgi-rich areas” and expressed as ratio of the volume density of a lOO-pm2 area. “Golgi-rich areas” were defined as regions that contained lamellar saccules and associated small and large vesicles. Pericanalicular vacuoles were defined as vacuoles delineated by a single 90-A membrane and surrounded by a material with an electron density similar to that of the pericanalicular cytoplasm. The lumina of these structures contained fine fibrillar material similar to that seen in the bile canaliculi and were devoid of microvilli. Pericanalicular diverticuli were defined as lateral extensions of the canalicular lumen which were usually also devoid of microvilli. Data were analyzed statistically by Student’s t-test. Bile volume, biliary lipids, cholesterol, phospholipids, and bile acids were determined. The biliary phospholipids were measured after extraction of the neutral lipids by chloroform/methanol (2: 1) as described by Bartlett (21); cholesterol was measured by GLC. Biliary bile acids were measured by combined TLC-GLC previously described by this laboratory (18). To determine the recovery of the bile acid injected, allo bile acids were further measured in liver tissue and the blood (19).
Table 3.
Effect of Albumin, wt/min)
3/3-Hydroxy-ScY-Cholanic Albumin
Biliary
In the dose-response experiment, the percent of reduction in bile flow, 1 h after the injection of 3, 6, and 9 pmol of 3/Shydroxy-5cr-cholanic acid, was 34.6 f 3.5, 72.1 + 5.7, and 64.2 f 9.1, respectively. These values were not significantly different from those values obtained when similar doses of the sulfate ester of the bile acid were injected. The percent of reductiqn in bile flow in the sulfate ester experiments were 29.7 f 2.6, 65.6 + 4.9, and 79.0 + 7.3, respectively. Table 1 shows the effect of intravenous injection of 6 ~mol/lOO g body wt of 3@-hydroxy-5a-cholanic
acid and its sulfate ester on biliary secretion. There was a significant and continuous reduction of bile flow for the first 60 min after the injection. Thereafter, the flow started to rise, but remained significantly lower than the control values. No significant differences were observed in bile flow between rats treated with the 3/?-hydroxy-k-cholanic acid and its sulfate ester except for the time period 30-60 min after injection. There was a significant reduction in the biliary secretion of bile acid, cholesterol, and phospholipid in the treated groups compared with the control (Table 2). The secretion of 3/3-hydroxy-5cw-cholanic acid ac-
Acid and its Sulfated Ester on Biliary Bile Acids (nmol/lOO g body 3/&Hydroxy-Sa-cholanic acid
After injection Biliary bile acids
O-60
60-120
Preinjection
3 P-Hydroxy-5a-cholanic acid
ND
ND
ND
ND
Another allo bile acid
ND
Cholic acid
ND
3P-Sulfated-5a-cholanic acid After injection
After injection
Preinjection
ND
Secretion
ND
O-60 4
60-120 6
f0.7
9~2.3
3 fl.O
6 f1.5
Preinjection ND
ND
o-60 6
60-120 9
~~1.6
rt2.4
5 f1.0
12 f1.6
65 ~16.3
52
50
63
26a
f10.4
f11.7
f14.9
f4.6
9O f2.6
91 f20.3
26O f5.7
140 f3.9
26 f4.6
12 f3.1
10 f2.7
23 f4.9
5O fl.1
1” f0.2
26 i5.4
5O f1.3
2O f0.6
12 f3.1
5
2
12
f2.6
f0.9
f2.6
1” f0.3
Trace
13 f2.7
2O f0.7
1 f0.4
Hyo-urso-deoxycholic acid
10 f2.4
2 f0.6
1 f0.4
10 f2.9
2 +0.4
Trace
9 f2.0
2 f0.6
Trace
Muricholic acids
11 f2.3
9 f3.1
11 f2.7
11 f2.4
Trace
Trace
13 zt3.3
Trace
Trace
Deoxycholic
acid
Chenodeoxycholic
acid
a Significant difference from corresponding control values p < 0.05. Values are means and standard errors of three samples. ND = not detectable. Lithocholic acid was not detected in these biles. Another allo bile acid was tentatively identified (by GLC) as a dihydroxyallo-bile acid.
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Table 4. Percentage Distribution of Allo Bile Acids in Bile, Liver, and Blood 3/3-Hydroxy-5acholanic acid Total Bile Liver Blood
94.1 21.4 69.1 3.6
+ f f f
2.8 2.7 4.5 0.9
Sulfate ester 92.8 32.4 59.1 1.3
k f + f
3.1 3.4” 4.7” 0.40
Values are means and standard errors of six samples group. o Significantly different at p < 0.05.
in each
counted for 21% of the amount injected as compared with 32% when the sulfate ester was administered. In both cases, 50% of the amount secreted was a dihydroxy allo bile acid as tentatively identified by GLC. The allo bile acids secreted in the bile were conjugated with taurine, as judged by TLC. There
BILE ACID CHOLESTASIS
245
was also a significant reduction in the secretion of all bile acids as compared with controls (Table 3). Table 4 shows that 93-94% of the bile acid injected was recovered in the bile, liver, and blood. The liver and the blood contents were higher, and the bile content was lower in the Zlghydroxy-5a-cholanic acid as compared with its sulfate ester. Electron Microscopy Qualitative analysis showed that 30 min after the injection of 3/3-hydroxy-5a-cholanic acid or its sulfate ester, the hepatocytes were characterized by the appearance of a moderate number of single membrane-bound cytoplasmic vacuoles of various sizes which appeared predominantly in the sinusoidal pole of the hepatocytes. These vacuoles contained sparse electron-dense material of low opacity
Figure 1. Liver tissue from rat given 38-hydroxy5a-cholanic acid. At 30 min after injection, cytoplasmic vacuoles were seen on the sinusoidal pole of the hepatocytes. A. The vacuoles are membranebound and contained sparse electrondense material of low opacity. B. Sulfate ester of the same compound produced similar morphologic changes (X 14,100).
246
VONK ET AL.
GASTROENTEROLOGY Vol. 80, No. 2
Figure 2. Liver tissue from rat treated as Figure 1. The bile canaliculus is dilated and devoid of microvilli. Note the large diverticulum with an electron-dense membrane extended from the bile canalicular lumen into the cell matrix (x 14,200).
(Figure 1 A and B). The most conspicuous change, however, was noted at the biliary pole of the hepatocytes; here, the canaliculi were dilated, and partial or total loss of microvilli was observed. Moreover, pericanalicular diverticuli originating from the plasma membrane intruded into the underlying cytoplasm (Figure 2). The pericanalicular vacuoles were prominent around canaliculi which exhibited a total loss of microvilli (Figure 3). Amorphous material and clusters of microfilaments were also seen around some of the pericanalicular vacuoles and close to the inner surface of the plasma membrane. At 2 h after injection, the hepatocytes showed similar vacuolation, and the bile canaliculi were still dilated and tortuous; however, some canaliculi began
to acquire a normal appearance (Figure 4). The hepatocytic ultrastructure was unaffected after injection of albumin alone. Quantitative analysis of pericanalicular cytoplasm revealed that 3&hydroxy-5a-cholanic acid and its sulfate ester resulted in a significant increase in volume densities for the canalicular lumina and for the pericanalicular vacuoles accompanied by a decrease for the canalicular microvilli. However, these changes were significantly greater at 30 min than at 2 h after injection and more pronounced with the 3/3hydroxy-5cr-cholanic acid than with its sulfate ester. Volume densities for the “Golgi-rich areas” remained unchanged after the various treatments (Table 5).
Figure 3. Liver tissue from rats treated as in Figures 1 and 2. The biliary pole of the hepatocyte exhibits marked changes. The bile canaliculus is dilated and devoid of microvilli, and numerous pericanalicular vacuoles are observed. Also observed is the thickening of the pericanalicular web (X 9400).
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247
Figure 4. Hepatocytes from rats given 3/l-hydroxySa-cholanic acid and sacrificed 2 h after injection. A. One bile canaliculus appears moderately dilated and devoid of microvilli, while another canaliculus was relatively normal. B. Pericanalicular vacuoles are also noted (X 14,lM1).
Discussion The data obtained in this study show that injection of 3/?-hydroxy-5a-cholanic acid or its sulfate ester induces intrahepatic cholestasis in rats. The cholestasis produced was characterized by a significant reduction in bile flow and biliary lipids. Morphologically, the bile canaliculi were dilated; the microvilli disappeared; and numerous pericanalicular vacuoles and diverticuli were aiso noted. The location and morphology of pericanalicular vacuoles was similar to that observed in cholestatic experiments (19,22,23) and in choleretic studies (24). However, it is evident from the morphometric analysis that the volume densities for the pericanalicular vacuoles and diverticuli are considerably greater in the present experimental conditions. The origin and function of these vacuoles is not known; however,
Layden and Boyer (24) indicated that the pericanalicular vacuoles or diverticuli might be correlated with an increase in bile flow. Recently Boyer et al. (25) suggested that these cytoplasmic vacuoles may originate from the Golgi complex. Jones et al. (26) observed somewhat similar structures and suggested that they may be involved in the transport of certain bile substances through the liver cell and that these vacuoles may originate from the sinusoidal surface of the liver cell and then translocate to the bile canaliculi. The presence of cytoplasmic vacuoles and diverticuli (Figure 2) in these studies suggest that the pericanalicular vacuoles are not necessarily related to an increase in bile flow. Since these vacuoles surrounded the canaliculi which were devoid of microvilli (Figure 3), it appears possible that they may result from the dilatation and disappearance of the microvilli itself. Measurements of
248
VONK ET AL.
Table
5.
Volume
Densities
GASTROENTEROLOGY
of Pericanalicular
Vol. 86, No. 2
Structures in Liver from Rats Given 3/3-Hydroxy Sa-Cholanic Acid and its
Sulfated Ester 38-Hydroxy-5a-cholanic
acid
structures
Bile canaliculus lumina Microvilli Vacuoles and diverticuli Golgi apparatus
Albumin” 0.55 0.50 0.018 2.41
* f f f
0.09 0.04 0.010 0.17
30 min 1.02 0.11 1.22 2.58
f f f f
0.15s 0.02s 0.30s 0.22
120 0.89 0.18 0.89 2.48
acid
Time after injection
Time after injection Pericanalicular
3/%Sulfated&r-cholanic
min
* * 2 *
O.llb 0.04s 0.17 0.19
30 min 1.32 zt 0.21b 0.21 f 0.04b.c.d 0.83 f O.llb*” 2.37 f 0.20
120 min 0.93 0.40 0.20 2.23
f * f f
0.15s 0.07d 0.07b.d 0.15
a Values are from livers taken 30 min after injection. Values at 120 min were similar. Values represent the means f SEM. b Significant difference from control value p < 0.05. c Significant difference from value at 120 min p < 0.05. d Significant difference from corresponding 3 P-hydroxy-5a period p < 0.05.
membrane perimeters for the normal canaliculi and for the canaliculi and pericanalicular vacuoles in control and experimental groups were virtually similar (average: 12 pm). Therefore the pericanalicular vacuoles may represent an inversion of the microvilli from the bile canalicular lumen into the underlying cell cytoplasm. The inversion of the microvilli could result from loss of the canalicular tone due to an alteration of the microfilamentous network. There is evidence in this study to suggest alteration in the filamental network since there occurred a moderate accumulation of amorphous and filamentous material in pericanalicular areas resembling that previously reported in experimental cholestasis (23). The possibility that the bile acid resulted in incorporation of new membrane from intracellular membranes at the maturing face of the Golgi complex is unlikely since Golgi volume densities of the bile acid-treated rats were not significantly different from controls. The data obtained in these studies suggest that the allomonohydroxy bile acid is a more potent cholestatic agent than the 5/3-analogue. The dose of allo bile acid used in these studies is about one-fourth of the 5/3-dose required to produce a similar decrease of bile flow (3,17). Furthermore, the sulfate ester of the monohydroxy-5P-cholanic acid protected the rat liver from the cholestatic effect of the nonsulfated compound (19), presumably due to its conjugation with taurine which is more soluble than the parent compound (27). However, despite that sulfate allomonohydroxy bile acids, conjugated with taurine, the cholestatic effect remained unchanged. This may indicate that the physical properties of sulfate allo bile acids are not different from their parent compound. This is true with regard to their melting point which was 221-224’C for both forms of the bile acid (unpublished data). However, more information will be needed regarding their Kraft point and their solubility at body temperature. Secondly, the data on the sulfated compound suggest that sulfation of the bile acid in its own merit is not necessarily a protective
mechanism against its toxic effect and support previous data obtained by this laboratory (19) and by Carey et al. (27). Thirdly there is morphologic evidence showing that the bile canalicular membrane of the hepatocyte is markedly altered, and that the pericanalicular vacuoles in this model may be an inversion of the microvilli into the hepatocyte which may result from a dilatation of the canalicular membrane. To our knowledge this is the first study on the biologic effect of one of the allo bile acids. Because this compound is not reported to occur in human bile, its significance to human cholestasis is not clear. However, allo bile acids are a major metabolite of cholestanol in mammals such as rats and gerbils (1). Furthermore, the formation of cholestanol from cholesterol in the body is well documented, and allo bile acids were reported in cerebrotendinous xanthomatosis patients (28) and in the urine from rats with ligated bile ducts (1). These findings suggest that through the diet or in some diseases, mammalian liver can synthesize allo bile acids. Because of the high cholestatic potential of these compounds compared with their 5/?-analogues, it might be of importance to investigate their role in human cholestasis. This could be of significance in neonatal cholestasis, because the bile acid synthesis is not as developed as in the adult (29-31).
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20. Weibel ER, Staubli W, Gnagi HR. et al. Correlated morphometric and biochemical studies of the liver cell. I. Morphometric model, stereologic methods and normal morphometric data for rat liver. J Cell Biol 1969:42:68-91. 21. Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959;234:466-8. 22. Jones AL, Schmucker DL, Mooney JS et al. Morphometric analysis of rat hepatocyte after total biliary obstruction. Gastroenterology 1976;71:1050-60. 23. Tuchweber B, Begin G, Badonnel MC, et al. Localization of actin protein in hepatocytes during experimental cholestasis. In: Preisig R, Bircher J, eds. The Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor, 1979:35-42. 24. Layden TJ, Boyer JL. Influence of bile acids on bile canalicular membrane morphology and the lobular gradient in canalicular size. Lab Invest 1978;39:110-19. 25. Boyer JL, Itabashi M, Hruban 2. Formation of pericanalicular vacuoles during sodium dehycholate choleresis: a mechanism for bile transport. In: Preisig R, Bircher J, eds. Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor. 1979:163-78. 26. Jones AL, Renston RH, Schmucker DL, et a.1.A morphological evaluation of the pericanalicular cytoplasm in the rat hepatocyte: demonstrating possible components of the bile secretory apparatus. In: Preisig R, Bircher J, eds. Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor. 1979:63-70. 27. Carey MC, Shu-fung J Wu, Watkins JB. Solution properties of sulfated monohydroxy bile salts. Relative insolubility of glycolithocholate sulfate. Biochim Biophys Acta 1979;575:16-26. 28. Salem G, Mosbach EH. The metabolism of sterols and bile acids in cerebrotendinous xanthomatosis. In: Nair PP, Kritchevsky D, eds. The bile acids: Vol. 3 Pathophysiology. New York: Plenum Press, 1967:115-53. 29. Li JR, Dinh DM, Marai L, et al. Sterol and bile acid metabolism during development. 2. Identification of 3P-hydroxy-5cholenoic acid in newborn and fecal guinea pig. Steroid 1977;30:815-26. 30. Eyssen H, Paumgartner G, Compenolle, et al. Trihydroxycoprastanic trahepatic bile
induodenal
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fluid of two children
anomalies.
Biochem
with
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in-
Acta
1972;273:212-21. 31. Hanson RF, Isenburg JN, Williams GC, et al. The metabolism of 3o, 7a, 12a-trihydroxy-5/I-cholestane-26-oic acid in two siblings with cholestasis due to intrahepatic An apparent inborn error of cholic acid vest 1975;56:577-87.
bile duct synthesis.
anomalies. J Clin In-
1981;81:242-9
Intrahepatic Cholestasis Induced by Allo Monohydroxy Bile Acid in Rats R. J. VONK, B. TUCHWEBER, D. MASS& A. PEREA, M. AUDET, C. C. ROY, and I. M. YOUSEF Centre de Recherche, HGpital Sainte-Justine, Montreal, Montreal, Quebec, Canada
Little is known about the biologic effect of alio bile acids on the liver. Since the monohydroxy-5/3-cholanic acids are known to induce a well-characterized intrahepatic cholestasis, the effects of 3/Chydroxy5cu-cholanic acid was tested in these studies. The bile ducts of 28 male Wistar rats were cannulated, and bile was collected for 2 h. The rats were then divided into three groups: The first group was injected i.v. with 6 pmol/lOO g body weight of 3/3-hydroxy-5acholanic acid in normal saline containing 7.5% albumin. The second group was injected with a similar concentration of the sulfate ester. The third group (control animals) received only albumin-saline solution. Bile was collected #or 2 h at 15, 30, 60, and 120 min. Bile volume and biliary lipids were measured. The livers were biopsed at 30 min after injection and at the end of the experiment for examination by electron microscopy, and a morphometric analysis was performed. The 3/3-hydroxy-5a-cholanic acid and its sulfate ester significantly reduced bile flow, bile salt, cholesterol, and phospholipid secretion in the 1st hour. However there was a tendency for improvement of these parameters in the 2nd hour after injection. The reduction in bile flow was dose dependent. At 30 min after injection, marked changes were observed at the canalicular pole of the hepatocytes. The canaliculi showed dilatation and partial or total Received August 15, 1979. Accepted August 29, 1980. Address requests for reprints to: Dr. I. M. Yousef, Centre de Recherche, HBpital Sainte-Justine, 3175 Chemin Sainte-Catherine, Montreal, Quebec, Canada H3T lC5. Dr. Vonk’s present address is: Department of Pediatrics, University of Groningen, Holland. This work was supported by grant from the Medical Research Council of Canada, was presented at the 30th Annual Meeting of the American Association for the Study of Liver Disease, November, Chicago, 1979. The authors thank Dr. Martin Carey of Harvard Medical School, for providing a copy of reference 27 before publication. 0 1981 by the American Gastroenterological Association OOlS-5085/81/020242-08.902.50
Departement
de Nutrition,
Universite
de
loss of microvilli. Pericanalicular diverticuli originating from the plasma membrane intruded into the underlying cytoplasm. Amorphous material and clusters of microfilaments were seen around the pericanalicular vacuoles. At 2 h, the hepatocytes showed the same changes described at 30 min but to a lesser degree. These data suggest that (a) allomonohydroxy bile acids have a greater cholestatic potential than 5/&analogues (four times the concentration of 5/?-analogue was required to produce the same degree of cholestasis); (bJ the sulfation does not protect against the cholestatic effect of allomonohydroxy bile acids; and (c) the mechanism of allo-monohydroxy bile acid cholestasis involves an early morphologic change in the bile canalicular membrane. The allo bile acids are derivatives of Sa-cholanic acid, analogues of the common 5/?-bile acids. These bile acids are more prevalent in lower species (1,2). The lower concentrstion of allo bile acids in the higher mammals was explained partially on the basis of lower activity of the microsomal &steroid reductase (1). Whether this difference is evolutionary or protective for mammals is not known. Because of the paucity of these bile acids, it is also not known whether allo bile acids play any role in regulation and control of sterol and/or bile acid synthesis. The question of their toxicity via the diet or other diseases remains to be ascertained. Since the monohydroxy !$-bile acids are known to induce intrahepatic cholestasis in mammals (3-17) by mainly affecting the bile canalicular membrane structure and function (3,8), it became of interest to investigate the biologic effect of similar !%cholanic acids. The data obtained indicated that the allomonohydroxy bile acid is a more potent cholestatic agent than the 5&cholanic acid. The cholestasis induced involves an early change in the bile can-
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1981
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Table
1.
Effect of Albumin, 3P-Hydroxy-5a-Cholanic Injections on Bile Flow
After injection
60-120
O-60
243
Acid, and its Sulfated Ester
Preinjection Compound
BILE ACID CHOLESTASIS
o-15
15-30
30-60
60-120 (min)
Albumin
9.2 f1.2
10.0 f0.7
9.1 21.1
9.5 k1.1
9.3 f0.9
8.7 f1.0
3 P-Hydroxy 5 a-Cholanic
9.3 k2.0
9.2 k1.8
5.4” f0.8
1.6" k1.1
1.7",b fl.1
2.9" f1.6
3 B-Sulfated 5 cr-Cholanic
10.6 21.5
10.1 +1.4
5.0” k1.4
3.0” k1.6
2.9”,” f0.6
4.3” k1.4
_
” Significant difference from corresponding albumin period p < 0.05. b Significant difference from corresponding 3/?-sulfated-5a-cholanic acid period p < 0.05. c Significant difference from corresponding 3/I-hydroxy-5a-cholanic acid period p < 0.05. Values are means and standard deviations of six animals and expressed as ~l/lOO g body wt/min.
alicular membrane of the hepatocyte. The data also show that sulfation of the allo-monohydroxy bile acid does not prevent its cholestatic potential.
Materials and Methods 3/3-Hydroxy-5a-cholanic acid sodium salt was obtained from Steroloid (New York, N.Y.). The compound gave a single spot, in various systems of thin-layer chromatography (TLC), with Rf value similar to lithocholic acid. When examined by gas liquid chromatography (GLC), the compound gave a single peak with retention time higher than lithocholic acid when it was derivated as methyl ester acetate and using a 3% OV-225 column (18). Therefore we concluded that the compound does not contain any detectable contaminants. The sulfate ester of the above compound was synthesized in this laboratory using a sodium trioxide complex as previously described (19). The sulfate ester was examined by TLC and GLC and was shown to be a single compound. The bile acid was solubilized in 7.5% albumin in normal saline at a concentration of 3 pmol/ml. Eighteen male Wistar rats obtained from Biobreeding farm (Montreal, Quebec) and weighing 200 + 5 g were used in these studies. The rats were fed laboratory Purina Table 2.
Effect of Albumin, 3/?-Hydroxy-5a-Cholanic Phospholipid Secretion Albumin
Acid and its Sulfated Ester on Biliary Bile Acid, Cholesterol, 3/3-Hydroxy-5cY-cholanic
After injection Biliary lipids Bile salt
Preinjection 146 f21.8
O-60 84 k14.7
chow before the experiments. A biliary fistula was created, and bile was collected for the next 120 min. In six rats 6 pmol/lOO g body wt of 3P-hydroxy-5a-cholanic acid were injected intravenously in the femoral vein, and bile was collected for 2 h at 15, 30, 60, and 120 min after injection. In another six rats treated in a similar manner, 6 pmol/lOO g body wt of the sulfate ester of 3/?-hydroxy-5acholanic acid were injected. The remaining rats were injected with 2 ml of 7.5% albumin/100 g body wt. The dosage used in the present study was based on a preliminary dose-response experiment in which six groups of four rats each were injected with 3, 6, or 9 ymolJlO0 g body wt of the parent compound or its sulfate ester, and bile flow was determined for 1 h after injection. The livers of the animals were biopsied at 30 min after injection. At the end of the experiment the animals were killed, and the livers were autopsied. The liver tissues obtained at 30 min and 2 h after injection were fixed in osmium tetroxide and processed for electron microscopy as previously described (19). Sections 0.5 pm thick were stained with toluidine blue to select midlobular areas for thin sectioning. Morphometric analysis consisted of measurements of volume density (Vv) according to Weibel et al. (20). Approximately 15 hepatocytes were randomly photographed from each block at an original magnifica-
60-120 76 k13.8
3P-Sulfated-5a-cholanic
acid
138 f23.4
O-60
60-120
400 k6.7
24" f4.5
acid
After injection
After injection Preinjection
and
Preinjection 152 f20.9
O-60
60-120
48" k6.6
39O f5.7
Cholesterol
1.38 -co.44
1.29 kO.28
1.43 -to.52
1.49 f0.38
0.73" kO.29
0.48" -co.17
1.28 f0.53
0.64O +0.34
0.59" f0.21
Phospholipids
33.4 f6.9
22.8 k7.0
21.9 f4.8
38.7 k6.4
11.1" 23.8
8.4" f2.9
36.3 27.3
18.41" f4.l
6.4" k2.8
0 Significant difference from corresponding expressed as nmole/lOO g body wt/min.
control value p < 0.05. Values are means f standard
deviation of six samples. Values are
244
GASTROENTEROLOGY Vol. 60, No. 2
VONK ET AL.
Results
tion of x 4700, centering the bile canaliculi in the photographed field. A total of 30 photographs from pericanalicular areas per animal were taken and a total of 150 photographs was obtained from five animals in each group. Fixed areas of 100 pm2 were used for calculation of volume density. Prints were prepared at x 17,ooO magnification and volume densities determined for canalicular lumina, microvilli, pericanalicular vacuoles, and diverticuli as well as for the “Golgi-rich areas” and expressed as ratio of the volume density of a lOO-pm2 area. “Golgi-rich areas” were defined as regions that contained lamellar saccules and associated small and large vesicles. Pericanalicular vacuoles were defined as vacuoles delineated by a single 90-A membrane and surrounded by a material with an electron density similar to that of the pericanalicular cytoplasm. The lumina of these structures contained fine fibrillar material similar to that seen in the bile canaliculi and were devoid of microvilli. Pericanalicular diverticuli were defined as lateral extensions of the canalicular lumen which were usually also devoid of microvilli. Data were analyzed statistically by Student’s t-test. Bile volume, biliary lipids, cholesterol, phospholipids, and bile acids were determined. The biliary phospholipids were measured after extraction of the neutral lipids by chloroform/methanol (2: 1) as described by Bartlett (21); cholesterol was measured by GLC. Biliary bile acids were measured by combined TLC-GLC previously described by this laboratory (18). To determine the recovery of the bile acid injected, allo bile acids were further measured in liver tissue and the blood (19).
Table 3.
Effect of Albumin, wt/min)
3/3-Hydroxy-ScY-Cholanic Albumin
Biliary
In the dose-response experiment, the percent of reduction in bile flow, 1 h after the injection of 3, 6, and 9 pmol of 3/Shydroxy-5cr-cholanic acid, was 34.6 f 3.5, 72.1 + 5.7, and 64.2 f 9.1, respectively. These values were not significantly different from those values obtained when similar doses of the sulfate ester of the bile acid were injected. The percent of reductiqn in bile flow in the sulfate ester experiments were 29.7 f 2.6, 65.6 + 4.9, and 79.0 + 7.3, respectively. Table 1 shows the effect of intravenous injection of 6 ~mol/lOO g body wt of 3@-hydroxy-5a-cholanic
acid and its sulfate ester on biliary secretion. There was a significant and continuous reduction of bile flow for the first 60 min after the injection. Thereafter, the flow started to rise, but remained significantly lower than the control values. No significant differences were observed in bile flow between rats treated with the 3/?-hydroxy-k-cholanic acid and its sulfate ester except for the time period 30-60 min after injection. There was a significant reduction in the biliary secretion of bile acid, cholesterol, and phospholipid in the treated groups compared with the control (Table 2). The secretion of 3/3-hydroxy-5cw-cholanic acid ac-
Acid and its Sulfated Ester on Biliary Bile Acids (nmol/lOO g body 3/&Hydroxy-Sa-cholanic acid
After injection Biliary bile acids
O-60
60-120
Preinjection
3 P-Hydroxy-5a-cholanic acid
ND
ND
ND
ND
Another allo bile acid
ND
Cholic acid
ND
3P-Sulfated-5a-cholanic acid After injection
After injection
Preinjection
ND
Secretion
ND
O-60 4
60-120 6
f0.7
9~2.3
3 fl.O
6 f1.5
Preinjection ND
ND
o-60 6
60-120 9
~~1.6
rt2.4
5 f1.0
12 f1.6
65 ~16.3
52
50
63
26a
f10.4
f11.7
f14.9
f4.6
9O f2.6
91 f20.3
26O f5.7
140 f3.9
26 f4.6
12 f3.1
10 f2.7
23 f4.9
5O fl.1
1” f0.2
26 i5.4
5O f1.3
2O f0.6
12 f3.1
5
2
12
f2.6
f0.9
f2.6
1” f0.3
Trace
13 f2.7
2O f0.7
1 f0.4
Hyo-urso-deoxycholic acid
10 f2.4
2 f0.6
1 f0.4
10 f2.9
2 +0.4
Trace
9 f2.0
2 f0.6
Trace
Muricholic acids
11 f2.3
9 f3.1
11 f2.7
11 f2.4
Trace
Trace
13 zt3.3
Trace
Trace
Deoxycholic
acid
Chenodeoxycholic
acid
a Significant difference from corresponding control values p < 0.05. Values are means and standard errors of three samples. ND = not detectable. Lithocholic acid was not detected in these biles. Another allo bile acid was tentatively identified (by GLC) as a dihydroxyallo-bile acid.
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Table 4. Percentage Distribution of Allo Bile Acids in Bile, Liver, and Blood 3/3-Hydroxy-5acholanic acid Total Bile Liver Blood
94.1 21.4 69.1 3.6
+ f f f
2.8 2.7 4.5 0.9
Sulfate ester 92.8 32.4 59.1 1.3
k f + f
3.1 3.4” 4.7” 0.40
Values are means and standard errors of six samples group. o Significantly different at p < 0.05.
in each
counted for 21% of the amount injected as compared with 32% when the sulfate ester was administered. In both cases, 50% of the amount secreted was a dihydroxy allo bile acid as tentatively identified by GLC. The allo bile acids secreted in the bile were conjugated with taurine, as judged by TLC. There
BILE ACID CHOLESTASIS
245
was also a significant reduction in the secretion of all bile acids as compared with controls (Table 3). Table 4 shows that 93-94% of the bile acid injected was recovered in the bile, liver, and blood. The liver and the blood contents were higher, and the bile content was lower in the Zlghydroxy-5a-cholanic acid as compared with its sulfate ester. Electron Microscopy Qualitative analysis showed that 30 min after the injection of 3/3-hydroxy-5a-cholanic acid or its sulfate ester, the hepatocytes were characterized by the appearance of a moderate number of single membrane-bound cytoplasmic vacuoles of various sizes which appeared predominantly in the sinusoidal pole of the hepatocytes. These vacuoles contained sparse electron-dense material of low opacity
Figure 1. Liver tissue from rat given 38-hydroxy5a-cholanic acid. At 30 min after injection, cytoplasmic vacuoles were seen on the sinusoidal pole of the hepatocytes. A. The vacuoles are membranebound and contained sparse electrondense material of low opacity. B. Sulfate ester of the same compound produced similar morphologic changes (X 14,100).
246
VONK ET AL.
GASTROENTEROLOGY Vol. 80, No. 2
Figure 2. Liver tissue from rat treated as Figure 1. The bile canaliculus is dilated and devoid of microvilli. Note the large diverticulum with an electron-dense membrane extended from the bile canalicular lumen into the cell matrix (x 14,200).
(Figure 1 A and B). The most conspicuous change, however, was noted at the biliary pole of the hepatocytes; here, the canaliculi were dilated, and partial or total loss of microvilli was observed. Moreover, pericanalicular diverticuli originating from the plasma membrane intruded into the underlying cytoplasm (Figure 2). The pericanalicular vacuoles were prominent around canaliculi which exhibited a total loss of microvilli (Figure 3). Amorphous material and clusters of microfilaments were also seen around some of the pericanalicular vacuoles and close to the inner surface of the plasma membrane. At 2 h after injection, the hepatocytes showed similar vacuolation, and the bile canaliculi were still dilated and tortuous; however, some canaliculi began
to acquire a normal appearance (Figure 4). The hepatocytic ultrastructure was unaffected after injection of albumin alone. Quantitative analysis of pericanalicular cytoplasm revealed that 3&hydroxy-5a-cholanic acid and its sulfate ester resulted in a significant increase in volume densities for the canalicular lumina and for the pericanalicular vacuoles accompanied by a decrease for the canalicular microvilli. However, these changes were significantly greater at 30 min than at 2 h after injection and more pronounced with the 3/3hydroxy-5cr-cholanic acid than with its sulfate ester. Volume densities for the “Golgi-rich areas” remained unchanged after the various treatments (Table 5).
Figure 3. Liver tissue from rats treated as in Figures 1 and 2. The biliary pole of the hepatocyte exhibits marked changes. The bile canaliculus is dilated and devoid of microvilli, and numerous pericanalicular vacuoles are observed. Also observed is the thickening of the pericanalicular web (X 9400).
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247
Figure 4. Hepatocytes from rats given 3/l-hydroxySa-cholanic acid and sacrificed 2 h after injection. A. One bile canaliculus appears moderately dilated and devoid of microvilli, while another canaliculus was relatively normal. B. Pericanalicular vacuoles are also noted (X 14,lM1).
Discussion The data obtained in this study show that injection of 3/?-hydroxy-5a-cholanic acid or its sulfate ester induces intrahepatic cholestasis in rats. The cholestasis produced was characterized by a significant reduction in bile flow and biliary lipids. Morphologically, the bile canaliculi were dilated; the microvilli disappeared; and numerous pericanalicular vacuoles and diverticuli were aiso noted. The location and morphology of pericanalicular vacuoles was similar to that observed in cholestatic experiments (19,22,23) and in choleretic studies (24). However, it is evident from the morphometric analysis that the volume densities for the pericanalicular vacuoles and diverticuli are considerably greater in the present experimental conditions. The origin and function of these vacuoles is not known; however,
Layden and Boyer (24) indicated that the pericanalicular vacuoles or diverticuli might be correlated with an increase in bile flow. Recently Boyer et al. (25) suggested that these cytoplasmic vacuoles may originate from the Golgi complex. Jones et al. (26) observed somewhat similar structures and suggested that they may be involved in the transport of certain bile substances through the liver cell and that these vacuoles may originate from the sinusoidal surface of the liver cell and then translocate to the bile canaliculi. The presence of cytoplasmic vacuoles and diverticuli (Figure 2) in these studies suggest that the pericanalicular vacuoles are not necessarily related to an increase in bile flow. Since these vacuoles surrounded the canaliculi which were devoid of microvilli (Figure 3), it appears possible that they may result from the dilatation and disappearance of the microvilli itself. Measurements of
248
VONK ET AL.
Table
5.
Volume
Densities
GASTROENTEROLOGY
of Pericanalicular
Vol. 86, No. 2
Structures in Liver from Rats Given 3/3-Hydroxy Sa-Cholanic Acid and its
Sulfated Ester 38-Hydroxy-5a-cholanic
acid
structures
Bile canaliculus lumina Microvilli Vacuoles and diverticuli Golgi apparatus
Albumin” 0.55 0.50 0.018 2.41
* f f f
0.09 0.04 0.010 0.17
30 min 1.02 0.11 1.22 2.58
f f f f
0.15s 0.02s 0.30s 0.22
120 0.89 0.18 0.89 2.48
acid
Time after injection
Time after injection Pericanalicular
3/%Sulfated&r-cholanic
min
* * 2 *
O.llb 0.04s 0.17 0.19
30 min 1.32 zt 0.21b 0.21 f 0.04b.c.d 0.83 f O.llb*” 2.37 f 0.20
120 min 0.93 0.40 0.20 2.23
f * f f
0.15s 0.07d 0.07b.d 0.15
a Values are from livers taken 30 min after injection. Values at 120 min were similar. Values represent the means f SEM. b Significant difference from control value p < 0.05. c Significant difference from value at 120 min p < 0.05. d Significant difference from corresponding 3 P-hydroxy-5a period p < 0.05.
membrane perimeters for the normal canaliculi and for the canaliculi and pericanalicular vacuoles in control and experimental groups were virtually similar (average: 12 pm). Therefore the pericanalicular vacuoles may represent an inversion of the microvilli from the bile canalicular lumen into the underlying cell cytoplasm. The inversion of the microvilli could result from loss of the canalicular tone due to an alteration of the microfilamentous network. There is evidence in this study to suggest alteration in the filamental network since there occurred a moderate accumulation of amorphous and filamentous material in pericanalicular areas resembling that previously reported in experimental cholestasis (23). The possibility that the bile acid resulted in incorporation of new membrane from intracellular membranes at the maturing face of the Golgi complex is unlikely since Golgi volume densities of the bile acid-treated rats were not significantly different from controls. The data obtained in these studies suggest that the allomonohydroxy bile acid is a more potent cholestatic agent than the 5/3-analogue. The dose of allo bile acid used in these studies is about one-fourth of the 5/3-dose required to produce a similar decrease of bile flow (3,17). Furthermore, the sulfate ester of the monohydroxy-5P-cholanic acid protected the rat liver from the cholestatic effect of the nonsulfated compound (19), presumably due to its conjugation with taurine which is more soluble than the parent compound (27). However, despite that sulfate allomonohydroxy bile acids, conjugated with taurine, the cholestatic effect remained unchanged. This may indicate that the physical properties of sulfate allo bile acids are not different from their parent compound. This is true with regard to their melting point which was 221-224’C for both forms of the bile acid (unpublished data). However, more information will be needed regarding their Kraft point and their solubility at body temperature. Secondly, the data on the sulfated compound suggest that sulfation of the bile acid in its own merit is not necessarily a protective
mechanism against its toxic effect and support previous data obtained by this laboratory (19) and by Carey et al. (27). Thirdly there is morphologic evidence showing that the bile canalicular membrane of the hepatocyte is markedly altered, and that the pericanalicular vacuoles in this model may be an inversion of the microvilli into the hepatocyte which may result from a dilatation of the canalicular membrane. To our knowledge this is the first study on the biologic effect of one of the allo bile acids. Because this compound is not reported to occur in human bile, its significance to human cholestasis is not clear. However, allo bile acids are a major metabolite of cholestanol in mammals such as rats and gerbils (1). Furthermore, the formation of cholestanol from cholesterol in the body is well documented, and allo bile acids were reported in cerebrotendinous xanthomatosis patients (28) and in the urine from rats with ligated bile ducts (1). These findings suggest that through the diet or in some diseases, mammalian liver can synthesize allo bile acids. Because of the high cholestatic potential of these compounds compared with their 5/?-analogues, it might be of importance to investigate their role in human cholestasis. This could be of significance in neonatal cholestasis, because the bile acid synthesis is not as developed as in the adult (29-31).
References 1. Elliott WH. In: Taylor W, ed. The allo bile acids in the hepatobiliary system. New York: Plenum Publishing Co., 1978:48981. 2. Haslewood GAD. Bile Salts. London: Methuen and Co., 1967. 3. Kakis G, Yousef IM. Pathogenesis of lithocholate and taurolithocholate induced intrahepatic cholestasis in rats. Gastroenterology 1978;75:595-607. 4. Kakis G, Yousef IM. Mechanism of cholic acid protection in lithocholate induced intrahepatic cholestasis in rats. Gastroenterology 1980;78:1402-11. 5. Kakis G, Phillips MJ, Yousef IM. The respective roles of mem-
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20. Weibel ER, Staubli W, Gnagi HR. et al. Correlated morphometric and biochemical studies of the liver cell. I. Morphometric model, stereologic methods and normal morphometric data for rat liver. J Cell Biol 1969:42:68-91. 21. Bartlett GR. Phosphorous assay in column chromatography. J Biol Chem 1959;234:466-8. 22. Jones AL, Schmucker DL, Mooney JS et al. Morphometric analysis of rat hepatocyte after total biliary obstruction. Gastroenterology 1976;71:1050-60. 23. Tuchweber B, Begin G, Badonnel MC, et al. Localization of actin protein in hepatocytes during experimental cholestasis. In: Preisig R, Bircher J, eds. The Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor, 1979:35-42. 24. Layden TJ, Boyer JL. Influence of bile acids on bile canalicular membrane morphology and the lobular gradient in canalicular size. Lab Invest 1978;39:110-19. 25. Boyer JL, Itabashi M, Hruban 2. Formation of pericanalicular vacuoles during sodium dehycholate choleresis: a mechanism for bile transport. In: Preisig R, Bircher J, eds. Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor. 1979:163-78. 26. Jones AL, Renston RH, Schmucker DL, et a.1.A morphological evaluation of the pericanalicular cytoplasm in the rat hepatocyte: demonstrating possible components of the bile secretory apparatus. In: Preisig R, Bircher J, eds. Liver, quantitative aspects of structure and function. Aulendorf, Germany: Editio Cantor. 1979:63-70. 27. Carey MC, Shu-fung J Wu, Watkins JB. Solution properties of sulfated monohydroxy bile salts. Relative insolubility of glycolithocholate sulfate. Biochim Biophys Acta 1979;575:16-26. 28. Salem G, Mosbach EH. The metabolism of sterols and bile acids in cerebrotendinous xanthomatosis. In: Nair PP, Kritchevsky D, eds. The bile acids: Vol. 3 Pathophysiology. New York: Plenum Press, 1967:115-53. 29. Li JR, Dinh DM, Marai L, et al. Sterol and bile acid metabolism during development. 2. Identification of 3P-hydroxy-5cholenoic acid in newborn and fecal guinea pig. Steroid 1977;30:815-26. 30. Eyssen H, Paumgartner G, Compenolle, et al. Trihydroxycoprastanic trahepatic bile
induodenal
duct
fluid of two children
anomalies.
Biochem
with
Biophys
in-
Acta
1972;273:212-21. 31. Hanson RF, Isenburg JN, Williams GC, et al. The metabolism of 3o, 7a, 12a-trihydroxy-5/I-cholestane-26-oic acid in two siblings with cholestasis due to intrahepatic An apparent inborn error of cholic acid vest 1975;56:577-87.
bile duct synthesis.
anomalies. J Clin In-