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Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates' protection of liver damage induced by chenodeoxycholic acid
LIVER,PANCREAS,AND DILIARYTRACT
DIGEST LIYER DIS 2000;32:318-28
Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates’ protection of liver damage induced by chenodeoxycholic acid F. Piazza M. Montagnanil c. Russo F. Azzarolil R. Aldini* E. Rodal A. Roda
fnml: Department of Pharmaoeuticel Sciences, I DepemmenC of lnternel Medicine and Gaatroerkwoicgy, 2 institute of chemical Sciences, Llnivaraiky of Bologne, Bologna, Italy.
Addrem .kr hf. A. Rods, D@a$timeni% di Soienze Fa~uticha, lJniversit& di Botogna, via Behnelom 6, 401?B Bologna, Italy. Fax: +39-051-343398. E-mail: r&@e/ma.onibo.it Submittad February 23, 1999. Revised December 2 I, 1999. Accepted Februtlry 22, 2DDff.
Background. Ursodeoxycholic acid has been widely used as a therapeutic agent in cholesterol gallstones and liver disease patients, but its mechanism of action is still under investigation. Aims. The protective effect of ursodeoxycholic acid, both free, taurine and glycine conjugated, against hepatotoxic bile acids such as chenodeoxycholic acid and its taurine amidate was studied in bile fistula rats and compared with the cholic and taurocholic acid effect. Methods. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid, taurocholic acid and cholic acid were infused iv over 1 hour (8 pmol/min/kg] together with an equimolar dose of either taurochenodeoxycholic acid or chenodeoxycholc acid. Bile flow, total and individual bile acid and biliary lactate dehydrogenase and alkaline phosphatase enzymes were measured. Results. Taurochenodeoxycholic acid and chenodeoxycholc acid caused cholestasis and liver damage associated with a decreased bile flow, total and individual bile acids secretion accompanied by a biliary leakage of lactate dehydrogenase and alkaline phosphatase enzymes. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid and taurocholic acid, on the contrary were choleretic, inducing an opposite effect on biliary parameters. Simultaneous infusion of taurochenodeoxycholic acid and the protective bile acid resulted in a functional and morphological improvement of the above parameters in the following order: glycine ursodeoxycholic acid > tauroursodeoxycholic acid > ursodeoxycholic acid followed by taurocholic acid: cholic acid was ineffective. Conclusions. The results show the protective effect of glycine ursodeoxycholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid. This may be due to a facilitated transport of the toxic bile acid into bile; conjugation with taurine is less effective than glycine. Finally the better protective effect of ursodeoxycholic acid and its amidates with respect to cholic acid and its taurine conjugated form seems to be related to their different lipophilicity and micellar forming capacity
Digest
Liver
Key words:
318
Ok 2000;32:318-28 bile acid
output;
bile flow;
UDCA
Introduction It has long since been known that bile acids (BAs), which are potent detergents, at millimolar concentrations are hepatotoxic ‘-4, as well as toxic on other tissues s-8; their toxicity is related to their lipid solubilizing ability 9, that increases with their hydrophobicity lo ll. Evidence has also emerged that BAs may be involved in liver disease, where they may play a role in the disruption of the canalicular plasma membrane ‘* acting also as free oxygen radical promoters 13. Ursodeoxycholic acid (UDCA), a rather hydrophilic BA that, unlike cholic acid (CA), presents poorer detergent capacity I4, has been shown to improve liver function in patients with cholestatic liver disease 15-19, primary biliary cirrhosis *O, cholestasis of pregnancy *’ and cholestatic liver disease in children ** 23. More recently, tauroursodeoxycholic acid (TUDCA) has been suggested as an alternative drug both for gallstone dissolution and the treatment of mild cholestatic liver disease 16-*0.Although the protective mechanism of UDCA and its conjugated forms has not yet been fully understood, in vivo 24and in vitro 2526 studies have shown that hepatotoxicity of hydrophobic BA is reduced by UDCA conjugates and that the effect seems to be at the canalicular membrane level. The therapeutic use of both UDCA and TUDCA in liver disease prompted the present investigation in order to gain further insight into the protective mechanism of UDCA and its amidates against the liver damage induced by chenodeoxycholic acid (CDCA) and taurochenodeoxycholic acid (TCDCA). TCDCA and CDCA have, therefore, been administered iv to bile fistula rats at increasing doses, in order to identify a maximum tolerated dose; TUDCA, glycoursodeoxycholic acid (GUDCA), UDCA, taurocholic acid (TCA), and cholic acid (CA) have also been co-administered iv. BAs with different physicochemical and biological properties, i.e., with different detergency, lipophilicity and also with different hepatic transport and absorption have been used Z7-29.For this purpose, unconjugated BAs were compared with amidated BAs and dihydroxy BAs with trihydroxy BAs. The aim of this study was to investigate the effect of UDCA and its amidates on TCDCA and CDCA uptake and secretion in bile fistula rat. Should this be the case, TUDCA efficacy may be due either to the reduction in the amount of TCDCA entering the liver cell or to the facilitation of its transport through the cell and secretion into bile. Studies have been performed with unconjugated BAs, since, due to their lipophilicity, they may partition differently among the components of the liver cell and may, therefore, share different pathways with respect
to conjugated idence time.
BAs, having also a different hepatic res-
Material and methods Chemicals All the chemicals used were of analytical grade. BAs were purchased from Sigma (St. Louis, MO, USA) and their purity assessed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) l6 17. All the BAs were more than 98% pure. UDCA was kindly supplied by Sanofi Synthelabo, Milan, Italy. Animal model BAs were administered iv to male Sprague Dowley rats (180-220 g bw). The rats were anaesthetized with pentobarbital ip at a dose of 50 mg per kg body weight and the bile duct was cannulated with PE-10 tubing (Clay-Adams, Parsippany, NJ, USA). The BAs studied were dissolved as sodium salts in 1 mVlO0 g body weight of saline solution (3% w/v bovine serum albumin, in saline solution), and the pH was brought to 7.4. The study was performed under a constant temperature of 37°C. After 1 hour baseline steady-state, the BAs were administered through the femoral vein at a slow infusion rate for 1 hour and the bile collected at intervals of 30 minutes for at least 2 hours. For the co-administration study, the two BAs were administered in the same volume of infusate. Experimental design The studied BAs included CDCA, TCDCA, UDCA, TUDCA, GUDCA, TCA, CA, acid. The experiments have been performed in the following order at the dosage listed below: Dose-response study TCDCA alone: administered doses: 4, 6, 8, 12, 16, 32 umol/min/kg TUDCA alone: administered doses: 4, 6, 8, 12, 16, 32 ymol/min/kg doses: 4, 6, 8, CDCA alone: administered umol/min/kg Co-administration study a) TCDCA + TUDCA 8 umol/min/kg bw TCDCA + GUDCA 8 ymol/min/kg bw TCDCA + UDCA 8 pmol/min/kg bw TCDCA + TCA 8 pmol/min/kg bw TCDCA + CA 8 pmol/min/kg bw b) CDCA + TUDCA 8 pmol/min/kg bw CDCA + GUDCA 8 pmol/min/kg bw CDCA + UDCA 8 umol/min/kg bw 319
UDCA prevention of liver damage induced by CDCA
The doses of the toxic BA, i.e., 8 pmol/min/kg bw for TCDCA and 8 urnol/min/kg bw for CDCA for the comparative study, were chosen from the dose-response study results. All animal studies were performed according to the guidelines of the University of Bologna Animal Studies Committee under the supervision of the Animal Welfare Veterinarian. Analytical methodology Bile flow was measured gravimetrically assuming the density of bile as one. Total biliary BA concentration was determined enzymatically by the 3o-hydroxysteroid-dehydrogenase assay (Stereognost 3a, Pho, Nycomed, AS, Torsov, Norway). The qualitative and quantitative BA composition was assessed by HPLC using an evaporative light scattering mass detector which allows quantification with the same sensitivity, both of the free and amidated BAs in bile 30. Briefly, Nova-Pack C-18 (Water) steel column (13.9 mm x 300 mm) particle size 4 pm, was used. The mobile phase is composed of a mixture of methanol/aqueous ammonium acetate 2 mM, pH=5.4, 65/35 v/v. The analysis was performed in an isocratic mode with a flow rate of 0.9 ml/min. BAs were isolated from bile or serum using a solid phase liquid extraction with C- 18 cartridge, as previously described 31. Biliary alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were determined by a standard kit (System Multi-Test C, Merck, Darmstadt, Germany) adapted for bile, using an autoanalyzer (Olympus, Eppendorf, Germany, ERIS analyzer 6176). Analyses of the enzymes were performed on the same day as the experiment. Tissue preparation and staining At the time of sacrifice, the liver was rapidly removed and small tissue cubes were cut from the left and the median lobes, attached to a piece of cork, quickly frozen at -75°C in a mixture of Freon 22/dry ice and stored at the same temperature in air-tight small vessels. The remaining liver was fixed in 10% buffered formalin, processed for paraffin embedding and stained with haematoxylin and eosin for morphological analysis. Frozen tissues were equilibrated overnight at -30°C before sectioning. Sections 8 u thick were cut at constant speed, deposited on Poly-L-Lysine (Sigma P8920) precoated glass coverslip and air dried at room temperature. Data expression Data were expressed as biliary concentration (mM) or enzymatic activity (IU/l), while total BA output as secretion rate (pmol/min/kg) taking into account the volume of bile excreted vs time and the rat weight.
From the biliary bile acid composition evaluated by HPLC, the BA secretion rate was calculated for each BA present in bile at different times during the study. In the dose-response study, data were expressed as mean maximum value reached during the study. Maximum bile acid output: Ms-BA umol/min/kg, maximum bile flow: Ms-V umol/min/kg, and maximum LDH and ALP activity: MA-LDH IU/l and MA-ALP IU/l. The histological findings were expressed as number of cells necrotic in the tissue preparation. Statistical analysis Each experimental group comprised six rats. Statistical analysis was performed using the analysis of variance (ANOVA) to demonstrate differences between the various groups studied. All values are expressed as the mean &SD; calculations were performed using the PROC ANOVA of the Statistical Analysis System (SAS). Results
Dose-response study Figure 1 shows maximum secretion rate of bile (MsV), total bile acids output (Ms-BA), and maximum activity of ALP (MA-ALP) and LDH (MA-LDH) in bile fistula rats during iv infusion of TCDCA or TUDCA at different doses. When administered at a dose less than 6 pmol/min/kg TCDCA was safe and almost normal biliary secretion was observed together with ALP and LDH values. A significant reduction in the Ms-V, Ms-BA with an increase in the excretion of ALP and LDH was observed when TCDCA was infused at a dose as high as 8 umol/min/kg and a significant increase in rat mortality was also observed (2/6 within the first hour). When administered at a dose of 12 prnol/min/kg TCDCA was highly cholestatic and caused death of the animals within the first hour of infusion. A 6 l.u-nol/min/kg dose was the highest TCDCA dose consistent with almost normal histological findings of the liver. The administration of TUDCA ranging from 4 to 32 pmol/min/kg was consistent with good health of the animals and no significant increase was observed in ALP and LDH activities in bile. TUDCA slightly increased bile flow while Ms-BA increased with doses up to 12 ymol/min/kg, dropping to baseline levels at TUDCA doses higher than 12 ymol/min/kg. The dose-response study of CDCA was not possible up to 8 ymol/min/kg dose, since it induced death of all animals, due to haemolysis.
Simultaneous administration of TCDCA with UDCA, GUDCA and TUDCA TUDCA, as well as the other BAs, were administered at an equimolar dose, i.e., 8 umol/min/kg, (8 ymol/min/kg TUDCA infusion proved to be the minimum effective dose). TCDCA infusion (Table I) induced a marked decrease in bile flow, slight decrease in BA output within the first hour, together with a high increase in ALP and LDH activity in bile. By the second hour, BA output was restored to baseline values. The administration of TUDCA alone increased bile flow and BA output, with no effect on ALP and LDH activity. Co-administration of TUDCA and TCDCA maintained, although to a lesser extent, the increase in bile flow and BA output; ALP and LDH secretion rates were almost within baseline values (Table I). UDCA co-administration increased bile flow to a similar extent as TUDCA, with less effect on BA output. The rise in enzyme activities in bile caused by TCDCA
was, in part, prevented by UDCA (~~0.01 compared with TCDCA data set). Similarly, GUDCA co-infusion significantly prevented ALP and LDH increases in bile (~~0.01 with respect to TCDCA data set), inducing an increase in bile flow and BA output (Table I). The effect of GUDCA on BAoutput was not so high as that of TUDCA, but it was more prolonged over time. Biliary bile acid analysis Bile secretion, total BA concentration and output in the co-infusion experiments together with BA composition and TCDCA/co-infused BA ratio are shown in Table II. In the TCDCA + UDCA experiments, UDCA secretion (as conjugated UDCA) was, respectively, 34 and 30 pmol/h versus a TCDCA secretion of, respectively, 48 and 26 ymol/h in the first and second hour, which accounted for 64% and, respectively, 74% recovery in bile of the administered BA. While in the first hour,
MICA prevention of liver damage induced by COCA
Ikbla I. Effect of TCDCA, UDCA, TUDCA infusion and UDCA, TUQCA, GUDCA equimolar co-infusion with TCDCA on bile flow, BA output, ALP and LDH activities in bile fistule rats.
~~M~~
7izi!r
Bile acid
I&
1st
40.5rt4.2
1.8rtO.4
16.5*5.8
14&4.01
2*
40.7*3.8
1.6~0.2
17.7k4.9
11.3k3.2
Id
32.Osc2.1'
1.0zt0.04'
176.7~21.2'
7OQ.O*45.3'
2nd
20.5k3.4'
1.7*0.8
225.Ozt40.6'
847.53t70.2'
1"
71.ck9.1
3.3~0.8
18.Ok3.8
6.5k2.4
2"d
7o.oka.2’
3.6il.O'
261t4.0
18.5rt4.1
let
60.2i5.3
7.1*1.8+
21.21t6.3
13.0a4.1
2nd
63.5i5.8'
3.1*0.4'
25.2zt6.9
22.2i3.8
1St
68.5k6.4'
4.5*1.2'
21.2ct5.1
14.Clt2.4
2nd
56.5il.9'
3.8scO.5'
25.8k4.8
36.5k3.5
1St
59.3k2.a
4.7zeo.9
67.3zt9.2
177.3m20.1"
2*
44.3k6.6
3.41to.5
143.8~15.2
260.01t25.4
1st
53.kl.6
7.OkO.8
39.2k7.8
49.81t9.9
2nd
62.0il.8
5.5*0.5""
59.5k8.4"
50.01t7.2
1"
57.5zt2.9"
6.0*1.1"
62k8.2"
2nd
54.5k3.0
6.6*1.4Q
Control *salineI
TCDCA3
UDCA3
TUDCA3
GUDCA3
UDCA+TCDCA4
TUDCA+TCDCA4 55.Osc8.4"
GUDCA+TCDCA4 82.5~6.6
76.5ztlS.l"
I: Date are expressed as mesn of 8re8 under the curve IAUCI, respective/y, at th8 1” and iM hour&D values hwm six animals for e8ch group. 2: D8t8 of the control group receiving only saline solution. ? infused 8t 8 pmoVmif@. $: w-infused 8t B+B pmovinin/kg. ? p
Ibbie II. Biliary concentration of total BAs, bile flow, BA output, BA composition and TCDCAko-infused BA ratio in the TCDCA + UDCA, GUDCA and TUDCA co-infusion experiments, respectively, at the first (I 1 and second f21 hour. Co-iufueed Lila acid
Tetel# Bile[r CORE.lmNl1
%TWl
%TM
%lfWA
c~Mt%l %llim
%lllMm
llalw
2i0.3
54.12t3.1
34.2i2.0
4.7rto.3
1.39
1”
9ozt5.2
0.98iO.2
88.2i12.3
5Ito.3
2nd
89k8.4
0.68iO.08
60.5*9.7
7.1*1.0
0.3zkO.03
43.Ok4.6
16.8i1.8
32.8k4.2
0.86
1 St
122rt9.4
1.04*0.3
126.8r23.4
7i0.6
3.1ztO.24
41.7zt3.5
-
48.2zt5.2
0.86
2nd
94ill
4.4zto.3
41.5t6.1
-
42.6k7.3
0.97
3.2kO.5
39.9k7.2
51.7h8.1
-
0.77
7.1i2.1
32.51t5.2
42.9~9.1
-
0.75
UDCA
GUDCA
T~~M~~
.o
0.87iO.05
B1.8z+z7.5
1st
143k20.1
0.90*0.3
128.7i19.6
2nd
105zt9.8
0.75*0,1
78.8rt6.1
11.5rt1.5 5.2*0.73
M
TUDCA Absolute
rates of secretion
tiplied by the BA output.
322
17.5rt3.3
of individual 8As during I* and 2@ h c8n be estimated from the mole fraction of the indivtiuel component Data are expressed 8s mesn k SD values fmm six enimals for e8ch group. TM%: t8ufvnMchofete.
lmmol
% +I001
muf-
E Piazza et al.
TCDCA was the predominant bile acid in bile, conjugated UDCA predominated in the second hour. Its appearance in bile in the first hour was mostly as taurine conjugated, then glycine conjugation prevailed over taurine conjugation, possibly due to a depletion of the taurine hepatic pool. In the TCDCA + GUDCA co-infusion experiments, GUDCA output was higher in the first hour (61 pmol/h) than in the second, when it was 35 pmol/h, in the presence of a TCDCA output of, respectively, 52 and 34 pmol/h, accounting, respectively, for 96% and 86% recovery in bile of these BAs. GUDCA percentage in bile was slightly higher than TCDCA, but both were maintained at almost similar constant values throughout the experiment. In the TCDCA + TUDCA experiments, TUDCA output in bile was the highest of all the bile acids in the first hour (66.5 pmol/h), then dropped to almost half the value in the second hour (33.1 pmol/h), which paralleled a similar but lower trend in TCDCA (respectively, 5 1.3 and 25.0 pmol/h); the percentage of TUDCA recovery in bile was almost lOO%, while TCDCA recovery was 76%. Therefore TCDCA % recovery in bile was in the following order for the co-infused therapeutic BA: GUDCA>GDCAsTUDCA.
Tauromuricholate (TMCA), a usual BA in rat, accounted for about 7% in the UDCA experiments, 7-11.5% for GUDCA and 5-17% for TUDCA experiments. TCA, the predominant bile acid in rat, bile, accounted for a minimal percentage in the co-infusion experiments. Simultaneous administration of TCDCA with TCA and CA CA (8 pmol/min/kg, Table III) infusion increased bile flow and BAs output, with little effect on LDH activity in bile. CA co-administration with TCDCA reduced biliary ALP with respect to TCDCA administration. No effect was observed on bile flow and BA output with respect to TCDCA alone; LDH on the contrary, rose in bile. TCA (Table III) infusion similarly increased bile flow and BA output in bile, and as with CA infusion, little effect was observed on the enzymes in bile. Compared with the TUDCA co-infusion data set, TCA coinfusion with TCDCA increased bile flow and BA output although to a lesser extent than TUDCA co-infusion; on the contrary, TCA was unable to prevent enzyme increases in bile.
Table III. Effect of CA, TCA infusion and CA, TCA equimolar co-infusion with TCOCA on bile flow, BA output, ALP and LDH activities in bile fistula rats. Bus 8tweioe [ABCP
Bile acid
‘““(BA?tpnt
ALP IAUCI
L&H lAUCl
1st
40.5*4.2
1.8*0.4
16.!%5.8
14.ort4.0
2nd
40.7rt3.8
1.6i0.2
17.7*4.9
11.3i3.2
1 =t
32.0r2.1
1.0*0.04’
176.7*35.7+
700.0*85.4’
2nd
20.5rt3.4’
1.7*0.8
225.0*40.6+
847.5*70.2+
1°C
106.2*21.2’
79t3.4’
53.7ztl5.8
18.2zt5.1
Control (salineI
TCDCA3
TCA3 2”d
103*29.7+
6.5i2.2’
44i12.3
43.2k8.8’
1 St
114.7*31 .O’
6.B*l.Y
54.2*9.5’
39.5i12.8
2”d
126zt23.4’
7.9k1.6’
64.5il1.3’
11 9.5i31.4’
CA3 1st
51 .Bi4.0
5.1*1.3”
149ct24.6
500.2*42.6
2nd
44.Oi3.1”
6.6il
131*28.5
401.5rt35.8
1 St
30.7k6.3
2.OkO.5
2”d
9.5i1.5”’
0.7rtO.2
TCA+TCDCA4 .B*’
139.7i19.8
2478zt198.0””
CA+TCDCA4 88.5*19.5””
3325*212.3””
I: Date are expressed as meen of area under the curve IAUCI, respectively, et the 1” and P hour&D ve/ues from six enimels for each group. 2: Dece of the contml grump receiving only seline solution.? infused et 8 pmoYmit&g. ? co-infused et 8+8 pmo//mi&g. +: ~401 compared with control deee set; t: pcO.05 compared with control data set; *: pcO.01 compared with TCLJCA date set; “: p4 05 compared with TCDCA dete set.
323
UOCA prevention of liver damage induced by COCA
Simultaneous administration of CDCA with UDCA, GUDCA and TUDCA The iv infusion of CDCA induces less toxicity than TCDCA at the 8 pmol/min/kg dose (Table IV). At the same dose, only a slight increase in biliary LDH and
ALP was observed with respect to the control data set (~~0.05). On the contrary, at higher doses (>8 umol/minkg) the toxicity of CDCA was higher than that of TCDCA, due to the haemolysis and possibly to other systemic effects. The simultaneous administration of UDCA, GUDCA and TUDCA (8 pmol/min/kg) with CDCA showed similar effects and no significant differences have been found when bile flow was compared. When BA output was considered, co-infusion of CDCA with TUDCA and GUDCA increased the effect of the two protective BAs, while co-infusion with UDCA did not (Fig. 2A). In fact, during UDCA co-infusion, no substantial changes in BA output was observed with respect to UDCA infusion alone, whereas in the GUDCA and TUDCA co-infusion, BA output peaked as high as 7 and 10 pmoVmin/kg (area under the curve of: CDCA+TUDCA vs TUDCA, ~~0.05; CDCA+GUDCA vs GUDCA, ~~0.05). On the contrary, the ALP increase was not reverted by the co-infusion of the protective bile and the same holds
L Piazza et al.
CDCA+TUDCA
CDCA+UDCA
CDCA+GUDCA
w
80
Bo 4)
20
P 0
0 0
05
1
1.5
2
0
1
1.5
2
Tim fhours) I@. EIl. Effect of CCIW @l, LlDcioi IiIR GlJDGk N, lIKEA fDl, infueion and LfDEA, GUDCA, TfHXA, cc-ink&n with Coca fbl bn ALP end LDH secretion in trite ffetufe rate. W Data from the ccntrcl grcup receiving onfy saline soluticn. Mean&D veltm born six e&n& for each group.
for LDH in bile, which, in the three co-infusion experiments, reached the highest values towards the end of the study (Fig. 2B). A remarkable choleretic effect was observed, which continued throughout the study, with values ranging from loo-150 pmol/min/kg bw. Histological
>>>20 in the TCDCA group, ranging from 4/5 to >20 in the TCDCA + UDCA experiments and 5/10 in the TCDCA + GUDCA group. Therefore, on the basis of the reduction in number of necrotic hepatocytes, the protective effect of these bile acids was ranked as TUDCA > GUDCA >> UDCA.
findings
TCDCA induced acute necrosis in scattered individual hepatocytes randomly distributed in liver lobes. Nuclear changes with chromatin fragmentation and disintegration (karyorrhexis) were the most prominent feature of injured hepatocytes. Necrotic cells accumulated calcium in the cytoplasm as revealed by red alizarin staining. Animals treated with TUDCA, GUDCA or UDCA did not show liver injury. Furthermore, when cell injury was assessed in the different animals receiving simultaneously TCDCA and either TUDCA, GUDCA or UDCA, necrosis was decreased when compared to TCDCA administration. The hepatocyte damage evaluated as number of necrotic cells, was, respectively,
Discussion This investigation shows that the hepatic toxicity of TCDCA in rat is dose-dependent. Since the maximum secretion rate for TCDCA into bile is 2 umol/min/kg, at lower doses, i.e., below this value, most of the infused TCDCA is secreted into bile, while at higher doses (from 8 to 32 umol/min/kg doses) most of TCDCA is retained by the liver. This latter fact accounts for TCDCA hepatotoxicity; a similar effect has already been observed by infusion of TDCA, a BA with a detergency similar to TCDCA 32 and, although the exact mechanism is not yet well demonstrated, an increase in
325
UDCA prevention
of liver damage induced by COCA
such a detergent BA accumulation in liver cells may be the primary cause of its toxicity. The hepatic damage induced by TCDCA is different from that induced by BAs such as lithocholate 3334 or TCA 35 36 at high doses, which is mainly a primary cholestatic effect. TCDCA infusion has already been reported to cause severe biochemical abnormalities in rat bile, such as increases in biliary secretion of hepatic (ALP) and systemic (albumin and, possibly, LDH) proteins 37, the alterations of which were prevented by the simultaneous infusion of TUDCA. It was thus suggested that a possible enlargement of the paracellular pathways might occur for these biochemical abnormalities, though no morphological evidence could be provided 38 as to a leakage between plasma and bile. Damage in the canalicular membrane is also possible. This concept is supported by the increase in biliary ALP, an integral canalicular membrane enzyme, which is dependent on the dose of the toxic BA. For the present investigation, ALP secretion was also decreased by TUDCA co-administration, and to a much lesser degree by UDCA, partially in agreement with Heuman et al. 26 In the present investigation, both the ALP and LDH increase is highly dose-dependent. The minimal dose of TUDCA which completely normalizes biliary ALP and LDH activities, bile flow and the biliary secretion of BAs is 8 umol/min/kg. This effect is immediate and no histological signs of liver damage are present in the liver tissue. At the same dose, GUDCA shows a similar effect on biliary enzyme parameters while UDCA is much less potent although still able to partially prevent TCDCA induced toxicity. Liver injury, due to TCDCA, may still be present to some extent after the co-infusion with UDCA and its amidates, and their protective effect may be related to their different biliary secretion rates. In the co-infusion experiments, GUDCA induces the highest biliary secretion of TCDCA, and this effect is higher in the first than in the second hour. TUDCA output is the highest in the first hour and drops to almost half its value in the second hour; its effect on TCDCA output is less than for GUDCA, possibly on account of its too rapid passage through the hepatocyte, resulting in a very short hepatic residence time. The UDCA effect on the toxic bile acid is intermediate, due to its need for conjugation before excretion, the differences between the first and second hour are not so marked, thus maintaining a more constant clearance of the toxic bile acid throughout the two hours. These results show that the main effect of UDCA, TUDCA and even more of GUDCA is to promote the biliary TCDCA secretion and consequently to reduce 326
its toxicity by preventing its intrahepatic accumulation. The final result is that within 2 hours, respectively, 74%, 76%, 86% of TCDCA is secreted into the bile, while when TCDCA is infused alone, more than 50% is still retained in the liver with a large exposure of the liver cells to this detergent BA. The phenomenon is dose-related and this fact may explain differences between the different studies carried out under different experimental conditions. Should the hepatic toxicity be induced by a higher dose of TCDCA than that used here, the protective effect of TUDCA would not be easily observed since both the dose and the time might not be able to displace TCDCA from the liver. At the considered dose, UDCA and its amidates are able to reduce the intrahepatic concentration of TCDCA to such non-toxic levels as may occur in physiological conditions. It has previously been shown that the maximum secretion rate of TUDCA in the rat is 12 pmol/min/kg I4 39. It is possible that the prevention of TCDCA hepatic toxicity may be related to a high secretion rate of the hepatoprotective BA which, in turn, may lead to an increase in the secretion of TCDCA. TCA is still able to prevent, in some way, the hepatotoxicity but its effect is much lower than that of GUDCA and TUDCA; CA is ineffective or effective only to a very limited extent. These data suggest that TCDCA toxicity is prevented by hydrophilic BA such as UDCA and its amidates, which unlike TCA or CA present a poorer micellar forming capacity (higher CMC) despite a lower hydrophilicity l440. The final result is that for a given total BA concentration in the liver cells, the number and the size of micelles is lower, in the case of UDCA and its amidates, resulting in less detergency and membrane damage, thanks to the presence of a higher monomeric species concentration. The question as to whether BAs share a common biliary transport system, as occurs at the sinusoidal liver domain, has not been fully elucidated 41. Kitani et al, however, showed that a facilitated interaction may be involved at least by certain BAs, as far as the secretory step is concerned4*. Schubert et al. 43 showed that BAs partition into unilamellar phospholipid vescicles with high affinity and induce leakage of vesicles at concentrations below those required to solubilize phospholipids, UDCA being less damaging than CDCA, CA or DCA. Heuman et al. 44 postulated from in vitro studies that protection of conjugates of UDCA does not involve liver specific pathways of bile acid uptake, liver domain partition, transport or metabolism, since it is exerted also on human erythrocytes; at the same time, they showed that UDCA may interfere with solubiliza-
L Piazza et al.
tion of membranes by BAs, blocking the access of more toxic BAs to the membrane. However, Galle et al. 45 have indicated that the hepatoprotective effect of UDCA is not likely due to displacement of toxic BA by reduced ileal absorption or decreased synthesis, but have shown that it may be related to a direct effect on the hepatocyte due either to the interaction of UDCA and the toxic BA or to the actual displacement of toxic BA from the membrane binding sites. The present investigation shows that GUDCA, even more than TUDCA and UDCA, exerts a protective effect when co-infused with toxic BA (TCDCA and CDCA) by increasing the clearance of the toxic BA into bile, enhancing their maximum secretion rate. The effect is more striking for GUDCA than for TUDCA, possibly because of the more rapid hepatic clearance of the latter, due to its higher hydrophilicity. Since the increase in biliary secretion of BA occurred within a relatively short time, induction of the transcription of the recently cloned sister of P-glycoprotein (spgp) 46, identified as the major canalicular BAs export pump is unlikely. The protective effect of UDCA conjugates on the cellular membranes may reduce a short-term impairment of this and other transporter systems due to more hydrophobic BAs. A more constant and continuous effect is achieved by GUDCA, since the output in bile of the toxic BA is more constant and the total recovery of the toxic BA is higher. The CA effect is limited by its inefficacy to increase bile flow and BA output, while the effect of TCA is very limited when co-infused with toxic BAs. The more potent effect of UDCA, either free or conjugated, is related to its relatively higher CMC in comparison with CA and TCA I4 not unlike taurohyodeoxycholic acid or p muricolates 47. The final result is an effective localization in liver cells of a poor detergent solution, despite a similar total BA concentration; a UDCA rich solution presents a reduced number of BA micelles and a higher intramicellar monomeric concentration, with consequent reduced membrane damage.
Abbreviations ALP: alkaline phosphatase;AUC: area under curve; BA: bile acid; bw: body weight; CA: cholic acid; CDCA: chenodeoxycholic acid; Critical Micellar Concentration: ; GUDCA: glycoursodeoxycholic acid; HPLC: high-performance liquid chromatography; LDH: lactate dehydrogenase; TCA: taurocholic acid; TCDCA: taurochenodoexycholic acid; TLC: thin layer chromatography; TMCA: tauromuricholate; TUDCA: tauroursodeoxycholic acid; UDCA: ursodeoxycholic acid.
I
References ’ Blitzer BL, Boyer JL. Cellular mechanisms of bile formation. Gastroenterology 1982;82:346-57. z Palmer RH. Bile acids, liver injury and liver disease. Arch Intern Med 1972;130:606-17. 3 Oelberg DG, Lester R. Cellular mechanisms of cholestasis. Annu Rev Med 1986;37:297-317. 4 Scholmerich J, Becher M-S, Schmidt K, Schubert R, Kremer B, Feldhaus S, et al. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties. Studies on isolated hepatocytes and lipid membrane vesicles. Hepatology 1984;4:661-6. 5 Coleman R, Holdsworth G. Effects of detergents on erythrocyte membranes: different patterns of solubilization of the membrane proteins by dihydroxy and trihydroxy bile salts. Biochem Sot Trans 1975;3:747-8. 6 Duane WC, Wiegand DM. Mechanism by which bile salt disrupts the gastric mucosal barrier in the dog. J Clin Invest 1980;66: 1044-9. ’ Safaie Shirazi S, Denbesten L, Zike WL. Effect of bile salts on the ionic permeability of the esophageal mucosa and their role in the production of esophagitis. Gastroenterology 1975;68:728-33. 8 Mekhjian HS, Phillips SF. Perfusion of the canine colon with unconjugated bile acids: effect on water and electrolyte transport, morphology and bile acid absorption. Gastroenterology 1970;59: 120-3 1. 9 Salvioli G, Lugli R, Pradelli JM. Effects of bile salts on membranes. In: Calandra S, Carulli N, Salvioli G, editors. Liver and lipid metabolism. New York: Elsevier Science Publishers; 1984. p. 163-79. lo Attili AF, Angelic0 M, Cantafora A, Alvaro D, Capocaccia L. Bile acid-induced liver toxicity: relation to the hydrophobic-hydrophilic balance of bile acids. Med Hypotheses 1986;19:57-69. ” Yousef IM, Bamwell S, Gratton F, Tuchweber B, Weber A, Roy CC. Liver cell membrane solubilization may control maximum secretory rate of cholic acid in the rat. Am J Physiol 1987;252:g84-91. Iz Bamwell SG, Lowe PJ, Coleman R. Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-membrane enzymes, and the extent of cells damage, in isolated perfused rat livers. Biochem J 1983;216:107-11. I3 Roda A, Russo C, Pasini P, Piazza F, Feroci G, Kricka LJ, et al. Antioxidant properties of bile salts micelles evaluated with different chemiluminescent assays: a possible physiological role. J Biolumin Cemilumin 1998;13:327-37. ” Roda A, Hofmann AF, Mysels KJ. The influence of bile salt structure on self-association in aqueous solutions. J Biol Chem 1983;258:6362-70. ” Poupon R, Poupon RE, Calmus Y, Chritien Y, Ballet F, Damis F. Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis? Lancet 1987;1:834-6. I6 Kitani K. Ursodeoxycholic acid for cholestatic diseases. Lancet 1988;2:8601-49. ” Leuschner U, Fischer H, Kurtz W, Guldutuna S, Hubner K, Hellstem A, et al. Ursodeoxycholic acid in primary biliary cirrhosis: results of a controlled double-blind- trial. -Gastroenterology 1989;97: 1268-74. I8 Heathcote EJ, Lindor KD, Poupon R. Combined analysis of French, American and Canadian randomized controlled trials of ursodeoxycholic acid therapy in primary biliary cirrhosis. Gastroenterology 1995;108:A1088-95. ” Lindor KD, Therneau TH, Jorgensen RA, Malinchoc M, Dickson
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of liver damage induced by CDCA
ER. Effect of ursodeoxycholic acid on survival in patients with primary biliary cirrhosis. Gastroenterology 1996;llO: 15 15-8. 2o Poupon RE, Balkau B, Eschwtge E, Poupon R. UDCA-PBC Study Group. A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. N Engl J Med 1991;324: 1548-54. 21 Meng LJ, Reyes H, Axelson M, Palma J, Hemandez I, Ribalta J, et al. Progesterone metabolites and bile acids in serum of patients with intrahepatic cholestasis of pregnancy: effect of ursodeoxycholic acid therapy. Hepatology 1997;26: 1573-9. ” Poupon RE, Poupon R. Ursodeoxycholic acid therapy of chronic cholestatic conditions in adults and children. Pharmacol Ther 1995;66:1-15. 23 Colombo C, Battezzati PM. Hepatobiliary manifestations of cystic fibrosis. J Gastroenterol Hepatol 1996;8:748-54. 24 Leuschner U. Ursodeoxycholic acid therapy in primary biliary cirrhosis. Stand J Gastroenterol 1994;204(Suppl):40-6. 25 Schmucker DL, Ohta M, Kanai S, Sato Y, Kitani K. Hepatic injury induced by bile salts: correlation between biochemical and morphological events. Hepatology 1990;12: 1216-21. *6 Heuman DM, Mills AS, McCall J, Hylemon PB, Pandak WM, Vlachevic ZR. Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts. Gastroenterology 199 1; 100:203- 11. 27 Carrella M, Roda E. Evolving concepts in the pathophysiology of biliary lipid secretion. Ital J Gastroenterol hepatol 1999;31:643-8. ** Aldini R, Montagnani M, Roda A, Hrelia S, Biagi PL, Roda E. Intestinal absorption of bile acid in the rabbit: different transport rates in jejunum and ileum. Gastroenterology 1996;110:459-68. *9 Montagnani M, Aldini R, Roda A, Roda E. New insights in the physiology and molecular basis of the intestinal bile acid absorption. Ital J Gastroenterol Hepatol 1998;30:435-40. 3o Roda A, Cerre C, Simoni P, Polimeni C, Vaccari C, Pistillo A. Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative light-scattering mass detection. J Lip Res 1992;33:1393-402. Setchell KDR, Worthington J. A rapid method for the quantitative extraction of bile acids and their conjugates from serum using commercially available reversed-phase octadecylsilane bonded silica cartridges. Clin Chim Acta 1982;125:135-44. Heuman DM, Hartmann CB, Hylemon PB, Vlahcevic ZR. Structure-activity relationships in bile acid biofeedback: studies of taurochenodeoxycholate and taurodeoxycholate. Gastroenterology 1987;92: 1739-44. Javitt NB, Enerman S. Effect of sodium tauro-lithiocholate on bile flow and bile acid excretion. J Clin Invest 1968;47:1002-14.
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34 Layden TJ, Boyer JL. Taurolithocholate-induced cholestasis: taurocholate, but not dehydrocholate, reverses cholestasis and bile canalicular membrane injury. Gastroenterology 1977;73: 120-8. 15 Kitani K, Kanai S. Tauroursodeoxycholate prevents taurocholate induced cholestasis. Life Sci 1982;30:515-23. 36 Kanai S, Kitani K. Glycoursodeoxycholate is effective as tauroursodeoxycholate in preventing the taurocholate induced cholestasis in the rat. Res Commun Chem Path01 Pharmacol 1983;42:423-30. ” Angelic0 M, Tisone G, Baiocchi L, Palmieri G, Pisani F, Negrini S. One-year pilot study on tauroursodeoxycholic acid as an adjuvant treatment after liver transplantation. Ital J Gastroenterol Hepatol 1999;31:462-8. 38 Miyai K, Price VM, Fisher HM. Bile acid metabolism in mammals. Ultrastructural studies on the intrahepatic cholestasis induced by lithocholic and chenodeoxycholic acids in the rat. Lab Invest 1971;24:292-302. j9 Kitani K, Kanai S. Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat. Life Sci 1981;29:269-75. 4o Roda A, Minute110 A, Angelotti MA, Fini A. Bile acid structureactivity relationship: evaluation of bile acid lipophilicity using loctanol/water partition coefficient and reverse phase HPLC. J Lipid Res 1990;31:1433-43. 4’ Kitani K, Kanai S. Interactions between different bile salts in the biliary excretion of the rat. Res Commun Chem Path01 Pharmaco1 1981;39:139-52. Q Kitani K, Ohata M, Kanai S. Tauroursodeoxycholate prevents biliary excretion induced by other bile salts inthe rat. Am J Physiol 1985;248:G407-17. 43 Schubert R, Jaroni H, Schoelmerich J, Schmidt KH. Studies on the mechanism of bile salt induced liposomal membrane damage. Digestion 1983;28:181-90. 44 Heuman DM, Pandak MW, Hyleman PB, Vlachevic Z. Conjugates of UDCA protect against cytotoxicity of more hydrophobic bile salts: in vitro studies in rat hepatocytes and human erythrocytes. Hepatology 1991;14:920-6. 45 Galle PR, Theilman L, Raedsch R, Otto G, Stiehl A. Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 1990;12:486-91. 46 Gerloff T, Stiger B, Hagenbuch B, Madon J, Landmann L, Roth J, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998;273: 10046-50. 47 Roda A, Piazza F, Baraldini M, Speroni E, Guerra MC, Cerre C, et al. Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat. Hepatology 1998;27:520-5.
DIGEST LIYER DIS 2000;32:318-28
Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates’ protection of liver damage induced by chenodeoxycholic acid F. Piazza M. Montagnanil c. Russo F. Azzarolil R. Aldini* E. Rodal A. Roda
fnml: Department of Pharmaoeuticel Sciences, I DepemmenC of lnternel Medicine and Gaatroerkwoicgy, 2 institute of chemical Sciences, Llnivaraiky of Bologne, Bologna, Italy.
Addrem .kr hf. A. Rods, D@a$timeni% di Soienze Fa~uticha, lJniversit& di Botogna, via Behnelom 6, 401?B Bologna, Italy. Fax: +39-051-343398. E-mail: r&@e/ma.onibo.it Submittad February 23, 1999. Revised December 2 I, 1999. Accepted Februtlry 22, 2DDff.
Background. Ursodeoxycholic acid has been widely used as a therapeutic agent in cholesterol gallstones and liver disease patients, but its mechanism of action is still under investigation. Aims. The protective effect of ursodeoxycholic acid, both free, taurine and glycine conjugated, against hepatotoxic bile acids such as chenodeoxycholic acid and its taurine amidate was studied in bile fistula rats and compared with the cholic and taurocholic acid effect. Methods. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid, taurocholic acid and cholic acid were infused iv over 1 hour (8 pmol/min/kg] together with an equimolar dose of either taurochenodeoxycholic acid or chenodeoxycholc acid. Bile flow, total and individual bile acid and biliary lactate dehydrogenase and alkaline phosphatase enzymes were measured. Results. Taurochenodeoxycholic acid and chenodeoxycholc acid caused cholestasis and liver damage associated with a decreased bile flow, total and individual bile acids secretion accompanied by a biliary leakage of lactate dehydrogenase and alkaline phosphatase enzymes. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid and taurocholic acid, on the contrary were choleretic, inducing an opposite effect on biliary parameters. Simultaneous infusion of taurochenodeoxycholic acid and the protective bile acid resulted in a functional and morphological improvement of the above parameters in the following order: glycine ursodeoxycholic acid > tauroursodeoxycholic acid > ursodeoxycholic acid followed by taurocholic acid: cholic acid was ineffective. Conclusions. The results show the protective effect of glycine ursodeoxycholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid. This may be due to a facilitated transport of the toxic bile acid into bile; conjugation with taurine is less effective than glycine. Finally the better protective effect of ursodeoxycholic acid and its amidates with respect to cholic acid and its taurine conjugated form seems to be related to their different lipophilicity and micellar forming capacity
Digest
Liver
Key words:
318
Ok 2000;32:318-28 bile acid
output;
bile flow;
UDCA
Introduction It has long since been known that bile acids (BAs), which are potent detergents, at millimolar concentrations are hepatotoxic ‘-4, as well as toxic on other tissues s-8; their toxicity is related to their lipid solubilizing ability 9, that increases with their hydrophobicity lo ll. Evidence has also emerged that BAs may be involved in liver disease, where they may play a role in the disruption of the canalicular plasma membrane ‘* acting also as free oxygen radical promoters 13. Ursodeoxycholic acid (UDCA), a rather hydrophilic BA that, unlike cholic acid (CA), presents poorer detergent capacity I4, has been shown to improve liver function in patients with cholestatic liver disease 15-19, primary biliary cirrhosis *O, cholestasis of pregnancy *’ and cholestatic liver disease in children ** 23. More recently, tauroursodeoxycholic acid (TUDCA) has been suggested as an alternative drug both for gallstone dissolution and the treatment of mild cholestatic liver disease 16-*0.Although the protective mechanism of UDCA and its conjugated forms has not yet been fully understood, in vivo 24and in vitro 2526 studies have shown that hepatotoxicity of hydrophobic BA is reduced by UDCA conjugates and that the effect seems to be at the canalicular membrane level. The therapeutic use of both UDCA and TUDCA in liver disease prompted the present investigation in order to gain further insight into the protective mechanism of UDCA and its amidates against the liver damage induced by chenodeoxycholic acid (CDCA) and taurochenodeoxycholic acid (TCDCA). TCDCA and CDCA have, therefore, been administered iv to bile fistula rats at increasing doses, in order to identify a maximum tolerated dose; TUDCA, glycoursodeoxycholic acid (GUDCA), UDCA, taurocholic acid (TCA), and cholic acid (CA) have also been co-administered iv. BAs with different physicochemical and biological properties, i.e., with different detergency, lipophilicity and also with different hepatic transport and absorption have been used Z7-29.For this purpose, unconjugated BAs were compared with amidated BAs and dihydroxy BAs with trihydroxy BAs. The aim of this study was to investigate the effect of UDCA and its amidates on TCDCA and CDCA uptake and secretion in bile fistula rat. Should this be the case, TUDCA efficacy may be due either to the reduction in the amount of TCDCA entering the liver cell or to the facilitation of its transport through the cell and secretion into bile. Studies have been performed with unconjugated BAs, since, due to their lipophilicity, they may partition differently among the components of the liver cell and may, therefore, share different pathways with respect
to conjugated idence time.
BAs, having also a different hepatic res-
Material and methods Chemicals All the chemicals used were of analytical grade. BAs were purchased from Sigma (St. Louis, MO, USA) and their purity assessed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) l6 17. All the BAs were more than 98% pure. UDCA was kindly supplied by Sanofi Synthelabo, Milan, Italy. Animal model BAs were administered iv to male Sprague Dowley rats (180-220 g bw). The rats were anaesthetized with pentobarbital ip at a dose of 50 mg per kg body weight and the bile duct was cannulated with PE-10 tubing (Clay-Adams, Parsippany, NJ, USA). The BAs studied were dissolved as sodium salts in 1 mVlO0 g body weight of saline solution (3% w/v bovine serum albumin, in saline solution), and the pH was brought to 7.4. The study was performed under a constant temperature of 37°C. After 1 hour baseline steady-state, the BAs were administered through the femoral vein at a slow infusion rate for 1 hour and the bile collected at intervals of 30 minutes for at least 2 hours. For the co-administration study, the two BAs were administered in the same volume of infusate. Experimental design The studied BAs included CDCA, TCDCA, UDCA, TUDCA, GUDCA, TCA, CA, acid. The experiments have been performed in the following order at the dosage listed below: Dose-response study TCDCA alone: administered doses: 4, 6, 8, 12, 16, 32 umol/min/kg TUDCA alone: administered doses: 4, 6, 8, 12, 16, 32 ymol/min/kg doses: 4, 6, 8, CDCA alone: administered umol/min/kg Co-administration study a) TCDCA + TUDCA 8 umol/min/kg bw TCDCA + GUDCA 8 ymol/min/kg bw TCDCA + UDCA 8 pmol/min/kg bw TCDCA + TCA 8 pmol/min/kg bw TCDCA + CA 8 pmol/min/kg bw b) CDCA + TUDCA 8 pmol/min/kg bw CDCA + GUDCA 8 pmol/min/kg bw CDCA + UDCA 8 umol/min/kg bw 319
UDCA prevention of liver damage induced by CDCA
The doses of the toxic BA, i.e., 8 pmol/min/kg bw for TCDCA and 8 urnol/min/kg bw for CDCA for the comparative study, were chosen from the dose-response study results. All animal studies were performed according to the guidelines of the University of Bologna Animal Studies Committee under the supervision of the Animal Welfare Veterinarian. Analytical methodology Bile flow was measured gravimetrically assuming the density of bile as one. Total biliary BA concentration was determined enzymatically by the 3o-hydroxysteroid-dehydrogenase assay (Stereognost 3a, Pho, Nycomed, AS, Torsov, Norway). The qualitative and quantitative BA composition was assessed by HPLC using an evaporative light scattering mass detector which allows quantification with the same sensitivity, both of the free and amidated BAs in bile 30. Briefly, Nova-Pack C-18 (Water) steel column (13.9 mm x 300 mm) particle size 4 pm, was used. The mobile phase is composed of a mixture of methanol/aqueous ammonium acetate 2 mM, pH=5.4, 65/35 v/v. The analysis was performed in an isocratic mode with a flow rate of 0.9 ml/min. BAs were isolated from bile or serum using a solid phase liquid extraction with C- 18 cartridge, as previously described 31. Biliary alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were determined by a standard kit (System Multi-Test C, Merck, Darmstadt, Germany) adapted for bile, using an autoanalyzer (Olympus, Eppendorf, Germany, ERIS analyzer 6176). Analyses of the enzymes were performed on the same day as the experiment. Tissue preparation and staining At the time of sacrifice, the liver was rapidly removed and small tissue cubes were cut from the left and the median lobes, attached to a piece of cork, quickly frozen at -75°C in a mixture of Freon 22/dry ice and stored at the same temperature in air-tight small vessels. The remaining liver was fixed in 10% buffered formalin, processed for paraffin embedding and stained with haematoxylin and eosin for morphological analysis. Frozen tissues were equilibrated overnight at -30°C before sectioning. Sections 8 u thick were cut at constant speed, deposited on Poly-L-Lysine (Sigma P8920) precoated glass coverslip and air dried at room temperature. Data expression Data were expressed as biliary concentration (mM) or enzymatic activity (IU/l), while total BA output as secretion rate (pmol/min/kg) taking into account the volume of bile excreted vs time and the rat weight.
From the biliary bile acid composition evaluated by HPLC, the BA secretion rate was calculated for each BA present in bile at different times during the study. In the dose-response study, data were expressed as mean maximum value reached during the study. Maximum bile acid output: Ms-BA umol/min/kg, maximum bile flow: Ms-V umol/min/kg, and maximum LDH and ALP activity: MA-LDH IU/l and MA-ALP IU/l. The histological findings were expressed as number of cells necrotic in the tissue preparation. Statistical analysis Each experimental group comprised six rats. Statistical analysis was performed using the analysis of variance (ANOVA) to demonstrate differences between the various groups studied. All values are expressed as the mean &SD; calculations were performed using the PROC ANOVA of the Statistical Analysis System (SAS). Results
Dose-response study Figure 1 shows maximum secretion rate of bile (MsV), total bile acids output (Ms-BA), and maximum activity of ALP (MA-ALP) and LDH (MA-LDH) in bile fistula rats during iv infusion of TCDCA or TUDCA at different doses. When administered at a dose less than 6 pmol/min/kg TCDCA was safe and almost normal biliary secretion was observed together with ALP and LDH values. A significant reduction in the Ms-V, Ms-BA with an increase in the excretion of ALP and LDH was observed when TCDCA was infused at a dose as high as 8 umol/min/kg and a significant increase in rat mortality was also observed (2/6 within the first hour). When administered at a dose of 12 prnol/min/kg TCDCA was highly cholestatic and caused death of the animals within the first hour of infusion. A 6 l.u-nol/min/kg dose was the highest TCDCA dose consistent with almost normal histological findings of the liver. The administration of TUDCA ranging from 4 to 32 pmol/min/kg was consistent with good health of the animals and no significant increase was observed in ALP and LDH activities in bile. TUDCA slightly increased bile flow while Ms-BA increased with doses up to 12 ymol/min/kg, dropping to baseline levels at TUDCA doses higher than 12 ymol/min/kg. The dose-response study of CDCA was not possible up to 8 ymol/min/kg dose, since it induced death of all animals, due to haemolysis.
Simultaneous administration of TCDCA with UDCA, GUDCA and TUDCA TUDCA, as well as the other BAs, were administered at an equimolar dose, i.e., 8 umol/min/kg, (8 ymol/min/kg TUDCA infusion proved to be the minimum effective dose). TCDCA infusion (Table I) induced a marked decrease in bile flow, slight decrease in BA output within the first hour, together with a high increase in ALP and LDH activity in bile. By the second hour, BA output was restored to baseline values. The administration of TUDCA alone increased bile flow and BA output, with no effect on ALP and LDH activity. Co-administration of TUDCA and TCDCA maintained, although to a lesser extent, the increase in bile flow and BA output; ALP and LDH secretion rates were almost within baseline values (Table I). UDCA co-administration increased bile flow to a similar extent as TUDCA, with less effect on BA output. The rise in enzyme activities in bile caused by TCDCA
was, in part, prevented by UDCA (~~0.01 compared with TCDCA data set). Similarly, GUDCA co-infusion significantly prevented ALP and LDH increases in bile (~~0.01 with respect to TCDCA data set), inducing an increase in bile flow and BA output (Table I). The effect of GUDCA on BAoutput was not so high as that of TUDCA, but it was more prolonged over time. Biliary bile acid analysis Bile secretion, total BA concentration and output in the co-infusion experiments together with BA composition and TCDCA/co-infused BA ratio are shown in Table II. In the TCDCA + UDCA experiments, UDCA secretion (as conjugated UDCA) was, respectively, 34 and 30 pmol/h versus a TCDCA secretion of, respectively, 48 and 26 ymol/h in the first and second hour, which accounted for 64% and, respectively, 74% recovery in bile of the administered BA. While in the first hour,
MICA prevention of liver damage induced by COCA
Ikbla I. Effect of TCDCA, UDCA, TUDCA infusion and UDCA, TUQCA, GUDCA equimolar co-infusion with TCDCA on bile flow, BA output, ALP and LDH activities in bile fistule rats.
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25.2zt6.9
22.2i3.8
1St
68.5k6.4'
4.5*1.2'
21.2ct5.1
14.Clt2.4
2nd
56.5il.9'
3.8scO.5'
25.8k4.8
36.5k3.5
1St
59.3k2.a
4.7zeo.9
67.3zt9.2
177.3m20.1"
2*
44.3k6.6
3.41to.5
143.8~15.2
260.01t25.4
1st
53.kl.6
7.OkO.8
39.2k7.8
49.81t9.9
2nd
62.0il.8
5.5*0.5""
59.5k8.4"
50.01t7.2
1"
57.5zt2.9"
6.0*1.1"
62k8.2"
2nd
54.5k3.0
6.6*1.4Q
Control *salineI
TCDCA3
UDCA3
TUDCA3
GUDCA3
UDCA+TCDCA4
TUDCA+TCDCA4 55.Osc8.4"
GUDCA+TCDCA4 82.5~6.6
76.5ztlS.l"
I: Date are expressed as mesn of 8re8 under the curve IAUCI, respective/y, at th8 1” and iM hour&D values hwm six animals for e8ch group. 2: D8t8 of the control group receiving only saline solution. ? infused 8t 8 pmoVmif@. $: w-infused 8t B+B pmovinin/kg. ? p
Ibbie II. Biliary concentration of total BAs, bile flow, BA output, BA composition and TCDCAko-infused BA ratio in the TCDCA + UDCA, GUDCA and TUDCA co-infusion experiments, respectively, at the first (I 1 and second f21 hour. Co-iufueed Lila acid
Tetel# Bile[r CORE.lmNl1
%TWl
%TM
%lfWA
c~Mt%l %llim
%lllMm
llalw
2i0.3
54.12t3.1
34.2i2.0
4.7rto.3
1.39
1”
9ozt5.2
0.98iO.2
88.2i12.3
5Ito.3
2nd
89k8.4
0.68iO.08
60.5*9.7
7.1*1.0
0.3zkO.03
43.Ok4.6
16.8i1.8
32.8k4.2
0.86
1 St
122rt9.4
1.04*0.3
126.8r23.4
7i0.6
3.1ztO.24
41.7zt3.5
-
48.2zt5.2
0.86
2nd
94ill
4.4zto.3
41.5t6.1
-
42.6k7.3
0.97
3.2kO.5
39.9k7.2
51.7h8.1
-
0.77
7.1i2.1
32.51t5.2
42.9~9.1
-
0.75
UDCA
GUDCA
T~~M~~
.o
0.87iO.05
B1.8z+z7.5
1st
143k20.1
0.90*0.3
128.7i19.6
2nd
105zt9.8
0.75*0,1
78.8rt6.1
11.5rt1.5 5.2*0.73
M
TUDCA Absolute
rates of secretion
tiplied by the BA output.
322
17.5rt3.3
of individual 8As during I* and 2@ h c8n be estimated from the mole fraction of the indivtiuel component Data are expressed 8s mesn k SD values fmm six enimals for e8ch group. TM%: t8ufvnMchofete.
lmmol
% +I001
muf-
E Piazza et al.
TCDCA was the predominant bile acid in bile, conjugated UDCA predominated in the second hour. Its appearance in bile in the first hour was mostly as taurine conjugated, then glycine conjugation prevailed over taurine conjugation, possibly due to a depletion of the taurine hepatic pool. In the TCDCA + GUDCA co-infusion experiments, GUDCA output was higher in the first hour (61 pmol/h) than in the second, when it was 35 pmol/h, in the presence of a TCDCA output of, respectively, 52 and 34 pmol/h, accounting, respectively, for 96% and 86% recovery in bile of these BAs. GUDCA percentage in bile was slightly higher than TCDCA, but both were maintained at almost similar constant values throughout the experiment. In the TCDCA + TUDCA experiments, TUDCA output in bile was the highest of all the bile acids in the first hour (66.5 pmol/h), then dropped to almost half the value in the second hour (33.1 pmol/h), which paralleled a similar but lower trend in TCDCA (respectively, 5 1.3 and 25.0 pmol/h); the percentage of TUDCA recovery in bile was almost lOO%, while TCDCA recovery was 76%. Therefore TCDCA % recovery in bile was in the following order for the co-infused therapeutic BA: GUDCA>GDCAsTUDCA.
Tauromuricholate (TMCA), a usual BA in rat, accounted for about 7% in the UDCA experiments, 7-11.5% for GUDCA and 5-17% for TUDCA experiments. TCA, the predominant bile acid in rat, bile, accounted for a minimal percentage in the co-infusion experiments. Simultaneous administration of TCDCA with TCA and CA CA (8 pmol/min/kg, Table III) infusion increased bile flow and BAs output, with little effect on LDH activity in bile. CA co-administration with TCDCA reduced biliary ALP with respect to TCDCA administration. No effect was observed on bile flow and BA output with respect to TCDCA alone; LDH on the contrary, rose in bile. TCA (Table III) infusion similarly increased bile flow and BA output in bile, and as with CA infusion, little effect was observed on the enzymes in bile. Compared with the TUDCA co-infusion data set, TCA coinfusion with TCDCA increased bile flow and BA output although to a lesser extent than TUDCA co-infusion; on the contrary, TCA was unable to prevent enzyme increases in bile.
Table III. Effect of CA, TCA infusion and CA, TCA equimolar co-infusion with TCOCA on bile flow, BA output, ALP and LDH activities in bile fistula rats. Bus 8tweioe [ABCP
Bile acid
‘““(BA?tpnt
ALP IAUCI
L&H lAUCl
1st
40.5*4.2
1.8*0.4
16.!%5.8
14.ort4.0
2nd
40.7rt3.8
1.6i0.2
17.7*4.9
11.3i3.2
1 =t
32.0r2.1
1.0*0.04’
176.7*35.7+
700.0*85.4’
2nd
20.5rt3.4’
1.7*0.8
225.0*40.6+
847.5*70.2+
1°C
106.2*21.2’
79t3.4’
53.7ztl5.8
18.2zt5.1
Control (salineI
TCDCA3
TCA3 2”d
103*29.7+
6.5i2.2’
44i12.3
43.2k8.8’
1 St
114.7*31 .O’
6.B*l.Y
54.2*9.5’
39.5i12.8
2”d
126zt23.4’
7.9k1.6’
64.5il1.3’
11 9.5i31.4’
CA3 1st
51 .Bi4.0
5.1*1.3”
149ct24.6
500.2*42.6
2nd
44.Oi3.1”
6.6il
131*28.5
401.5rt35.8
1 St
30.7k6.3
2.OkO.5
2”d
9.5i1.5”’
0.7rtO.2
TCA+TCDCA4 .B*’
139.7i19.8
2478zt198.0””
CA+TCDCA4 88.5*19.5””
3325*212.3””
I: Date are expressed as meen of area under the curve IAUCI, respectively, et the 1” and P hour&D ve/ues from six enimels for each group. 2: Dece of the contml grump receiving only seline solution.? infused et 8 pmoYmit&g. ? co-infused et 8+8 pmo//mi&g. +: ~401 compared with control deee set; t: pcO.05 compared with control data set; *: pcO.01 compared with TCLJCA date set; “: p4 05 compared with TCDCA dete set.
323
UOCA prevention of liver damage induced by COCA
Simultaneous administration of CDCA with UDCA, GUDCA and TUDCA The iv infusion of CDCA induces less toxicity than TCDCA at the 8 pmol/min/kg dose (Table IV). At the same dose, only a slight increase in biliary LDH and
ALP was observed with respect to the control data set (~~0.05). On the contrary, at higher doses (>8 umol/minkg) the toxicity of CDCA was higher than that of TCDCA, due to the haemolysis and possibly to other systemic effects. The simultaneous administration of UDCA, GUDCA and TUDCA (8 pmol/min/kg) with CDCA showed similar effects and no significant differences have been found when bile flow was compared. When BA output was considered, co-infusion of CDCA with TUDCA and GUDCA increased the effect of the two protective BAs, while co-infusion with UDCA did not (Fig. 2A). In fact, during UDCA co-infusion, no substantial changes in BA output was observed with respect to UDCA infusion alone, whereas in the GUDCA and TUDCA co-infusion, BA output peaked as high as 7 and 10 pmoVmin/kg (area under the curve of: CDCA+TUDCA vs TUDCA, ~~0.05; CDCA+GUDCA vs GUDCA, ~~0.05). On the contrary, the ALP increase was not reverted by the co-infusion of the protective bile and the same holds
L Piazza et al.
CDCA+TUDCA
CDCA+UDCA
CDCA+GUDCA
w
80
Bo 4)
20
P 0
0 0
05
1
1.5
2
0
1
1.5
2
Tim fhours) I@. EIl. Effect of CCIW @l, LlDcioi IiIR GlJDGk N, lIKEA fDl, infueion and LfDEA, GUDCA, TfHXA, cc-ink&n with Coca fbl bn ALP end LDH secretion in trite ffetufe rate. W Data from the ccntrcl grcup receiving onfy saline soluticn. Mean&D veltm born six e&n& for each group.
for LDH in bile, which, in the three co-infusion experiments, reached the highest values towards the end of the study (Fig. 2B). A remarkable choleretic effect was observed, which continued throughout the study, with values ranging from loo-150 pmol/min/kg bw. Histological
>>>20 in the TCDCA group, ranging from 4/5 to >20 in the TCDCA + UDCA experiments and 5/10 in the TCDCA + GUDCA group. Therefore, on the basis of the reduction in number of necrotic hepatocytes, the protective effect of these bile acids was ranked as TUDCA > GUDCA >> UDCA.
findings
TCDCA induced acute necrosis in scattered individual hepatocytes randomly distributed in liver lobes. Nuclear changes with chromatin fragmentation and disintegration (karyorrhexis) were the most prominent feature of injured hepatocytes. Necrotic cells accumulated calcium in the cytoplasm as revealed by red alizarin staining. Animals treated with TUDCA, GUDCA or UDCA did not show liver injury. Furthermore, when cell injury was assessed in the different animals receiving simultaneously TCDCA and either TUDCA, GUDCA or UDCA, necrosis was decreased when compared to TCDCA administration. The hepatocyte damage evaluated as number of necrotic cells, was, respectively,
Discussion This investigation shows that the hepatic toxicity of TCDCA in rat is dose-dependent. Since the maximum secretion rate for TCDCA into bile is 2 umol/min/kg, at lower doses, i.e., below this value, most of the infused TCDCA is secreted into bile, while at higher doses (from 8 to 32 umol/min/kg doses) most of TCDCA is retained by the liver. This latter fact accounts for TCDCA hepatotoxicity; a similar effect has already been observed by infusion of TDCA, a BA with a detergency similar to TCDCA 32 and, although the exact mechanism is not yet well demonstrated, an increase in
325
UDCA prevention
of liver damage induced by COCA
such a detergent BA accumulation in liver cells may be the primary cause of its toxicity. The hepatic damage induced by TCDCA is different from that induced by BAs such as lithocholate 3334 or TCA 35 36 at high doses, which is mainly a primary cholestatic effect. TCDCA infusion has already been reported to cause severe biochemical abnormalities in rat bile, such as increases in biliary secretion of hepatic (ALP) and systemic (albumin and, possibly, LDH) proteins 37, the alterations of which were prevented by the simultaneous infusion of TUDCA. It was thus suggested that a possible enlargement of the paracellular pathways might occur for these biochemical abnormalities, though no morphological evidence could be provided 38 as to a leakage between plasma and bile. Damage in the canalicular membrane is also possible. This concept is supported by the increase in biliary ALP, an integral canalicular membrane enzyme, which is dependent on the dose of the toxic BA. For the present investigation, ALP secretion was also decreased by TUDCA co-administration, and to a much lesser degree by UDCA, partially in agreement with Heuman et al. 26 In the present investigation, both the ALP and LDH increase is highly dose-dependent. The minimal dose of TUDCA which completely normalizes biliary ALP and LDH activities, bile flow and the biliary secretion of BAs is 8 umol/min/kg. This effect is immediate and no histological signs of liver damage are present in the liver tissue. At the same dose, GUDCA shows a similar effect on biliary enzyme parameters while UDCA is much less potent although still able to partially prevent TCDCA induced toxicity. Liver injury, due to TCDCA, may still be present to some extent after the co-infusion with UDCA and its amidates, and their protective effect may be related to their different biliary secretion rates. In the co-infusion experiments, GUDCA induces the highest biliary secretion of TCDCA, and this effect is higher in the first than in the second hour. TUDCA output is the highest in the first hour and drops to almost half its value in the second hour; its effect on TCDCA output is less than for GUDCA, possibly on account of its too rapid passage through the hepatocyte, resulting in a very short hepatic residence time. The UDCA effect on the toxic bile acid is intermediate, due to its need for conjugation before excretion, the differences between the first and second hour are not so marked, thus maintaining a more constant clearance of the toxic bile acid throughout the two hours. These results show that the main effect of UDCA, TUDCA and even more of GUDCA is to promote the biliary TCDCA secretion and consequently to reduce 326
its toxicity by preventing its intrahepatic accumulation. The final result is that within 2 hours, respectively, 74%, 76%, 86% of TCDCA is secreted into the bile, while when TCDCA is infused alone, more than 50% is still retained in the liver with a large exposure of the liver cells to this detergent BA. The phenomenon is dose-related and this fact may explain differences between the different studies carried out under different experimental conditions. Should the hepatic toxicity be induced by a higher dose of TCDCA than that used here, the protective effect of TUDCA would not be easily observed since both the dose and the time might not be able to displace TCDCA from the liver. At the considered dose, UDCA and its amidates are able to reduce the intrahepatic concentration of TCDCA to such non-toxic levels as may occur in physiological conditions. It has previously been shown that the maximum secretion rate of TUDCA in the rat is 12 pmol/min/kg I4 39. It is possible that the prevention of TCDCA hepatic toxicity may be related to a high secretion rate of the hepatoprotective BA which, in turn, may lead to an increase in the secretion of TCDCA. TCA is still able to prevent, in some way, the hepatotoxicity but its effect is much lower than that of GUDCA and TUDCA; CA is ineffective or effective only to a very limited extent. These data suggest that TCDCA toxicity is prevented by hydrophilic BA such as UDCA and its amidates, which unlike TCA or CA present a poorer micellar forming capacity (higher CMC) despite a lower hydrophilicity l440. The final result is that for a given total BA concentration in the liver cells, the number and the size of micelles is lower, in the case of UDCA and its amidates, resulting in less detergency and membrane damage, thanks to the presence of a higher monomeric species concentration. The question as to whether BAs share a common biliary transport system, as occurs at the sinusoidal liver domain, has not been fully elucidated 41. Kitani et al, however, showed that a facilitated interaction may be involved at least by certain BAs, as far as the secretory step is concerned4*. Schubert et al. 43 showed that BAs partition into unilamellar phospholipid vescicles with high affinity and induce leakage of vesicles at concentrations below those required to solubilize phospholipids, UDCA being less damaging than CDCA, CA or DCA. Heuman et al. 44 postulated from in vitro studies that protection of conjugates of UDCA does not involve liver specific pathways of bile acid uptake, liver domain partition, transport or metabolism, since it is exerted also on human erythrocytes; at the same time, they showed that UDCA may interfere with solubiliza-
L Piazza et al.
tion of membranes by BAs, blocking the access of more toxic BAs to the membrane. However, Galle et al. 45 have indicated that the hepatoprotective effect of UDCA is not likely due to displacement of toxic BA by reduced ileal absorption or decreased synthesis, but have shown that it may be related to a direct effect on the hepatocyte due either to the interaction of UDCA and the toxic BA or to the actual displacement of toxic BA from the membrane binding sites. The present investigation shows that GUDCA, even more than TUDCA and UDCA, exerts a protective effect when co-infused with toxic BA (TCDCA and CDCA) by increasing the clearance of the toxic BA into bile, enhancing their maximum secretion rate. The effect is more striking for GUDCA than for TUDCA, possibly because of the more rapid hepatic clearance of the latter, due to its higher hydrophilicity. Since the increase in biliary secretion of BA occurred within a relatively short time, induction of the transcription of the recently cloned sister of P-glycoprotein (spgp) 46, identified as the major canalicular BAs export pump is unlikely. The protective effect of UDCA conjugates on the cellular membranes may reduce a short-term impairment of this and other transporter systems due to more hydrophobic BAs. A more constant and continuous effect is achieved by GUDCA, since the output in bile of the toxic BA is more constant and the total recovery of the toxic BA is higher. The CA effect is limited by its inefficacy to increase bile flow and BA output, while the effect of TCA is very limited when co-infused with toxic BAs. The more potent effect of UDCA, either free or conjugated, is related to its relatively higher CMC in comparison with CA and TCA I4 not unlike taurohyodeoxycholic acid or p muricolates 47. The final result is an effective localization in liver cells of a poor detergent solution, despite a similar total BA concentration; a UDCA rich solution presents a reduced number of BA micelles and a higher intramicellar monomeric concentration, with consequent reduced membrane damage.
Abbreviations ALP: alkaline phosphatase;AUC: area under curve; BA: bile acid; bw: body weight; CA: cholic acid; CDCA: chenodeoxycholic acid; Critical Micellar Concentration: ; GUDCA: glycoursodeoxycholic acid; HPLC: high-performance liquid chromatography; LDH: lactate dehydrogenase; TCA: taurocholic acid; TCDCA: taurochenodoexycholic acid; TLC: thin layer chromatography; TMCA: tauromuricholate; TUDCA: tauroursodeoxycholic acid; UDCA: ursodeoxycholic acid.
I
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321
UDCA pnuention
of liver damage induced by CDCA
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328
34 Layden TJ, Boyer JL. Taurolithocholate-induced cholestasis: taurocholate, but not dehydrocholate, reverses cholestasis and bile canalicular membrane injury. Gastroenterology 1977;73: 120-8. 15 Kitani K, Kanai S. Tauroursodeoxycholate prevents taurocholate induced cholestasis. Life Sci 1982;30:515-23. 36 Kanai S, Kitani K. Glycoursodeoxycholate is effective as tauroursodeoxycholate in preventing the taurocholate induced cholestasis in the rat. Res Commun Chem Path01 Pharmacol 1983;42:423-30. ” Angelic0 M, Tisone G, Baiocchi L, Palmieri G, Pisani F, Negrini S. One-year pilot study on tauroursodeoxycholic acid as an adjuvant treatment after liver transplantation. Ital J Gastroenterol Hepatol 1999;31:462-8. 38 Miyai K, Price VM, Fisher HM. Bile acid metabolism in mammals. Ultrastructural studies on the intrahepatic cholestasis induced by lithocholic and chenodeoxycholic acids in the rat. Lab Invest 1971;24:292-302. j9 Kitani K, Kanai S. Biliary transport maximum of tauroursodeoxycholate is twice as high as that of taurocholate in the rat. Life Sci 1981;29:269-75. 4o Roda A, Minute110 A, Angelotti MA, Fini A. Bile acid structureactivity relationship: evaluation of bile acid lipophilicity using loctanol/water partition coefficient and reverse phase HPLC. J Lipid Res 1990;31:1433-43. 4’ Kitani K, Kanai S. Interactions between different bile salts in the biliary excretion of the rat. Res Commun Chem Path01 Pharmaco1 1981;39:139-52. Q Kitani K, Ohata M, Kanai S. Tauroursodeoxycholate prevents biliary excretion induced by other bile salts inthe rat. Am J Physiol 1985;248:G407-17. 43 Schubert R, Jaroni H, Schoelmerich J, Schmidt KH. Studies on the mechanism of bile salt induced liposomal membrane damage. Digestion 1983;28:181-90. 44 Heuman DM, Pandak MW, Hyleman PB, Vlachevic Z. Conjugates of UDCA protect against cytotoxicity of more hydrophobic bile salts: in vitro studies in rat hepatocytes and human erythrocytes. Hepatology 1991;14:920-6. 45 Galle PR, Theilman L, Raedsch R, Otto G, Stiehl A. Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 1990;12:486-91. 46 Gerloff T, Stiger B, Hagenbuch B, Madon J, Landmann L, Roth J, et al. The sister of P-glycoprotein represents the canalicular bile salt export pump of mammalian liver. J Biol Chem 1998;273: 10046-50. 47 Roda A, Piazza F, Baraldini M, Speroni E, Guerra MC, Cerre C, et al. Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat. Hepatology 1998;27:520-5.