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Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

Pharmacological Research, Vol. 22, No . 3, 1990 297 EFFECT OF URSODEOXYCHOLIC ACID ADMINISTRATION ON BILE ACID COMPOSITION IN HAMSTER BILE M . MA...

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EFFECT OF URSODEOXYCHOLIC ACID ADMINISTRATION ON BILE ACID COMPOSITION IN HAMSTER BILE M . MAI BJKA, C . VESCINA, C . N. CARDUCCI, A . ALAYÖN, A. DIOS, E . SCARLATTO and A. MAMIANETTI Departamento de Quimica Analltica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 (1113) and Hospital Aeronâutico Central, Division Investigaciones, Ventura de la Vega 3697 (1437), Buenos Aires, Argentina Received in final form 13 October 1989

SUMMARY The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated . Male Golden Syrian hamsters were divided into five groups : a control group, two groups that received 0 . 5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days . After ether anaesthesia the gallbladder was removed and bile was immediately aspirated . Bile acids were determined by high performance liquid chromatography (HPLC) . Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups . The glyco/tauro ratio of 0 . 69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1 . UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives . A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives . KEY WORDS :

HPLC, bile acids, gallstone dissolution, hamster . INTRODUCTION

The two bile acids, chenodeoxycholic acid (3a,7a-dihydroxy-5ß cholanoic acid) (CDCA) and its 7ß-epimer, ursodeoxycholic acid (UDCA), are used in the treatment of cholesterol gallstones [1, 2] . UDCA has proven to be more useful than CDCA because the former acts at a lower dose and is not associated with any hepatotoxicity [3, 4] . 1043-6618/90/030297-09/$03 .00/0

© 1990 The Italian Pharmacological Society



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The administration of UDCA in gallstone patients produces marked changes in the biliary bile acid composition and suppresses the endogenous bile acid synthesis much less than CDCA [5, 6] ; lithocholic acid (LCA) rises moderately and UDCA becomes the major bile acid constituent [5, 7] . These changes are in relation to the administered dose [5, 8] . Marked species differences occur in the relative amounts of the bile acid derivatives found in bile [9] . The Golden Syrian hamster is used as a model animal of cholesterol gallstone formation because its biliary bile acid composition resembles that of human beings in the steroid moiety and, like humans, the hamster conjugates bile acids with both glycine and taurine amino acids [10-12] . When bile acid composition in hamster bile is assessed by HPLC [13] the percentage of UDCA is as small as it is in human beings [14] . The purpose of this work was to determine the modifications in the composition of bile acids in hamster bile produced by the administration of UDCA under different dosing schedules . High doses of UDCA were used to observe if the variations produced were related to those obtained with lower doses .

MATERIALS AND METHODS Experimental design

Male Golden Syrian hamsters (Centro Panamericano de Zoonosis, Buenos Aires, Argentina), each weighing 80-90 g at the end of the adaptation week were randomly divided into five groups and allocated in individual cages . The animals were fed a standard diet of Roden Chow (Purina, Labina, Bs .As .) throughout the experimental period . Group. I was the control group ; groups II and III received 0 . 5 g UDCA/100 g of standard diet for 30 and 60 days, respectively and groups IV and V were treated with 1 g UDCA/100 g of standard diet for 30 and 60 days, respectively . The actual amounts of UDCA received were 500-1000 mg/kg/day . The experimental diet was prepared by the method of Carella & Dietschy [15] and all the animals received standard diet and water ad libitum. The room temperature was 24°C and the lights in the vivarium were on from 7 a .m. to 7 p .m. Hamsters were fasted for 12 h before being sacrificed . The animals appeared healthy at the time of sacrifice (between 9 and 11 a.m.) . Ether was used as anaesthesia ; subsequently, the cystic duct was ligated and then the gallbladder was removed . Normal saline solution (50 µl) was injected and diluted bile was aspirated . Individual bile samples were processed immediately and bile acids were quantified by high performance liquid chromatography (HPLC) as previously described [13] . Experimental procedures

The sodium salts of taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA) and glycolithocholic acid (GLCA) were purchased from Sigma Chemical Co. (St Louis, MO, USA). The sodium salts of tauroursodeoxycholic acid



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(TUDCA) and glycoursodeoxycholic acid (GUDCA) were the generous gift of Tokyo Tanabe Co. (Tokyo, Japan) . Glyco and tauro conjugates of 3a,12a-dihydroxy-7-keto-5ß-cholanoic acid (7ketodeoxycholic acid) (7-KDCA) ; 3a-hydroxy-7-keto-5ß-cholanoic acid (7ketolithocholic acid) (7-KLCA) and 3-keto-7a-hydroxy-5ß-cholanoic acid were prepared by incubation of respective tri- and dihydroxylated bile acid standard solutions with 3a- or 7a-hydroxysteroid dehydrogenase (Sigma Chemical Co.) and ß-nicotinamide adenine dinucleotide (Sigma Chemical Co .) for 1 h at 37°C in 0 . 1 M pyrophosphate buffer (pH 9-5) . Following incubation, the reaction mixtures were passed through a Sep-Pak C 1s cartridge for purification [16] . Dexamethasone (Sigma Chemical Co.) was used as internal standard (I .S .). All reagents used were HPLC grade . HPLC of biliary bile acids. The equipment used was a Varian 5020 (Palo Alto, CA, USA) liquid chromatograph with a VUV 10 detector and a data processor CDSL 111 L . A reversed phase column, Micropack MCH-5 (Varian) 30 cm x 4 mm i.d ., particle size 5 µm was used . The mobile phase consisted of two solvents : A was 0 . 3% ammonium dihydrogen phosphate, pH 7 . 5 and B was acetonitrile. Gradient elution profile: solvent B increased from 25% to 35% during the first 19 min, to 37% during the next 11 min, and then to 40% during the next 5 min . Flow rate : 0 . 7 ml/min. Temperature : 32°C . Injection volume: 10µl. Detection was performed at 208 nm and 0.05 aufs . A reference methanolic solution of bile acid standards, containing 100-200 µg/ ml of each, was used and bile samples were conditioned as described previously [13] . Although the use of this column produced the shortening of certain retention times, there was no decrease in resolution . A reference methanolic solution of bile acids was run daily . For statistical evaluation the Student's test was used to evaluate differences between groups .

RESULTS A typical chromatogram of a control hamster bile is shown in Fig . 1 . CA, CDCA, DCA and 7-KDCA were the predominant bile acids comprising CA and CDCA conjugates ca. 90% of total bile acids . Figure 2 illustrates the biliary bile acid composition of a hamster bile of an UDCA fed animal (1 g UDCAl100 g of standard diet for 60 days) . The predominant bile acids in group V were UDCA, CDCA and LCA conjugates . Table I summarizes mean values of individual bile acids determined by HPLC in all animals studies . Total bile acids decreased compared to the control in all treated groups. TUDCA and GUDCA increased with UDCA doses, whereas TCA and GCA showed a significant decrease (P< 0 . 001) together with lower values of the 7KDCA derivatives . In all treated groups GCDCA was higher than in controls (P< 0 . 001) but TCDCA decreased only in groups IV and V (P< 0 . 005) . However, the 3-keto derivatives of CDCA, glyco and tauro 7a-hydroxy-3-keto-5ß-cholanoic acids, increased in all treated groups, especially the glyco conjugate (peaks 4 and 5



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Fig. 2 . Bile acid composition of a representative hamster bile of group V. Peaks : 1,2,3,6,7,8, unknowns ; 4 and 5, 3-keto derivatives of CDCA (4, tauro 7a-hydroxy-3-keto-5ß-cholanoic acid ; 5, glyco 7a-hydroxy-3-keto-5ß-cholanoic acid) ; I .S., internal standard, dexamethasone . Operating conditions and abbreviations as described in the text . in Fig. 2) . The DCA conjugates decreased to trace values and proportionally to the lower levels of CA conjugates produced by UDCA treatment . Significant increases (P< 0 . 001) of TLCA and GLCA were also observed (Table I) . The mean glyco/tauro (G/T) ratios obtained for total and individual bile acids are shown in Table II . In all treated groups, the mean G/T ratio was significantly



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Table I Total (mg/ml) and individual bile acids (%) in control and treated groups Bile acid

I

II

III

IV

V

TBA TUDCA GUDCA TCA GCA TCDCA GCDCA TDCA GDCA TLCA GLCA n

45 . 2±4 . 4 0 . 2±0 . 1

32 .5±4 . 61 3 . 9±0 . 8* 5-9±1-6* 1 . 6±0 . 3* 2 . 4±0 . 8* 27 .2±2 . 9 40 . 8 ± 2-4* 0-1±0-1t 0-4±0-3t 10 . 4±1 . 4* 7 . 2±0 . 8* 5

31 . 9±4 . 31 4 . 4±1 . 4l 7 . 1 ±1-9* 0 . 3±0 . 2* 3 . 8±0 . 9* 21 . 9±4 .0 44 . 8 ± 3-6* 0-4±0-2t Tracest 10 . 8±1 . 5* 6 . 6±1 .0* 4

31 . 8±2 . 31 8 . 4±2 . 8t 19 . 2 ± 3 . 1* 2 . 4±0 . 6* 3 . 2±0 . 9* 11 . 0±2 . 11 43-4±3-5* 0-1±0-1t 0-6±0-3t 6 . 2±2 . 11 5 . 5±1 .51 5

34 . 2±1 . 61 7 . 0±2 . 8t 14-9 ± 5 . 4t 2 . 9±0 . 8* 3 . 2±0 . 4* 11 . 8±2 .21 41 . 5 ± 5 . 2* Tracest Traces* 10 . 0±1 . 8* 8 . 6±1 . 8* 5

37 . 3±3 . 8 23 . 7±3 . 2 20 . 4±2 .5 12-2 ±I-9 2.4± 0.5 1-7 ±0-3 1 . 7±0 . 3 0 . 3±0 . 2 8

Group 1 : control group ; group II : 0 . 5 g UDCA'100 g standard diet-30 days ; group III : 0 . 5 g UDCA/100 g standard diet-60 days ; group IV: 1 . 0 g UDCA/100 g standard diet-30 days ; group V: 1 . 0 g UDCA/100 g standard diet-60 days . Values are mean±sE ; TBA, total bile acids ; *P<0-001 ; tP< 0 . 01 ; 1P< 0 . 05 (treated groups versus control group) ; n, number of hamsters .

Table II CA/CDCA ratio and G/T ratios of total and individual bile acids I CA/CDCA G/T

II

III

2-02+0-24 0-06±0-01* 0-06±0-02* 0 . 69±0 . 16 1 . 37±0 .161 1 . 81±0 . 42+ GUDCA/TUDCA 1-47 ± 0 . 13 1-88 ± 0-28 GCA/TCA 0-75±0-19 2-40±1-08 7-04±2-48 GUDCA/TUDCA 0 . 66 ± 0 . 13 1-58±0-19t 2-41±0-73t GDCA/TDCA 1-15±0-50 GLCA/TLCA 0-13±0-07 0-72±0-09t 0-63±0-11t n 8 5 4

IV

V

0 . 11±0 . 04 2 . 72±0 . 37* 2 . 90±0 . 63 1 . 80 ±0-70 4 . 34±0 . 54* 3 . 48 ± 1 . 84 1 . 05±0 . 301 5

0 . 12±0 . 02 2 . 22±0 . 21* 2 . 14±0 . 31 1 . 32 ± 0 . 20 3 . 83±0 . 57* 0 .87±0 . 11* 5

Group I : control group ; group II: 0 . 5 g UDCA/100 g standard diet-30 days ; group III: 0 . 5 g UDCA/100 g standard diet-60 days ; group IV: 1 . 0 g UDCA/100 g standard diet-30 days; group V: 1 . 0 g UDCA/100 g standard diet-60 days . Values are mean ±sE . CA/CDCA, cholic/cheno ; G/T, glyco/tauro ; *P< 0 . 001, t P< 0 1P< 0 . 05 (treated groups versus control groups) ; n, number of hamsters . higher than in the control group, reaching values > 1 for individual bile acids except for LCA values which remained < 1 . In controls the mean G/T ratio for LCA was lower than the mean G/T ratio for the other individual bile acids . In Table II, CA/CDCA ratios are also shown . A significant decrease of CA/ CDCA ratio was observed in all treated animals .



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DISCUSSION In control animals the value for the CA/CDCA ratio (2 . 02 ± 0 Table II) was similar to those found in previous investigations in hamsters [17-19], but this ratio decreased significantly in the treated animals . The therapeutic dose of UDCA in humans causes little or no effect on bile acid synthesis, which is in contrast to the results obtained with the administration of CDCA [6] . In our experience, CA decreased significantly, which would demonstrate the depletion of CA synthesis as has been described previously [20, 21] . During urso treatment, it seems that CDCA level increased because of a likely transformation of administered UDCA into CDCA [7, 22] . 7-KDCA glyco and tauro conjugates also diminished simultaneously with the decrease of CA level (Figs 1 and 2) . The total biliary bile acids decreased significantly in all groups in comparison to the control (Table II) and this result may be correlated with the hydrocholeretic effect of UDCA [7, 23, 24] . UDCA conjugates increased proportionally to the dose administered to hamsters, similar to observations made in humans [8, 22, 25, 26] . The percentage observed (27 . 6%) (Table I), however, did not correlate well with results reported in hamsters by other investigators [11, 27] . In these studies, Handelsman et al. [27], supplying 20 mg of UDCA/kg/day during 2 weeks, found 33 . 8% UDCA and Pearlman et al. [11], giving the same amount of UDCA during 12 weeks, obtained 73 . 8% UDCA . But, in both experiments the diet also contained cholesterol and ethynyloestradiol, and the latter could diminish hepatic 7 a-hydroxylase activity [28], thereby decreasing the synthesis of bile acids . Thus, this lack of agreement with our findings may be due to different experimental design . Furthermore, the total CDCA level was between 53 and 68% in all treated groups which was higher than the values found in previous works [11, 27] . More recently, Bellentani et al [29], giving 180-300 mg/TUDCA/kg/body wt during 2 weeks, found 33 .3% CDCA and 16 . 9% UDCA. The increased CDCA in UDCA fed animals probably arose via the transformation of UDCA to CDCA . We are unable to hypothesize the mechanism of this transformation (keto reduction or direct inversion) . UDCA is converted to CDCA by intestinal bacterial transformation through 7-KLCA [11, 30-32] which can be partially absorbed in the intestinal tract and then may be transformed to CDCA by hepatic 7a-hydroxylase [33] . On the other hand, Emerman & Javitt [34] demonstrated that CDCA can be synthesized from LCA in hamsters . The analysis of keto bile acids in control hamsters showed that 7-KDCA, the predominant keto bile acid, came from the major bile acid, CA, in agreement with Bergman et al. [35] . Both were decreased by UDCA treatment . During our studies, a moderate increase in keto derivatives of CDCA was also observed, especially of the glyco and tauro 7a-hydroxy-3-keto-5ß-cholanoic acids, simultaneously with an increase of CDCA (Fig . 2) . LCA conjugates increased significantly in all treated animals and they may be the result of 7-bacterial dehydroxylation of either UDCA or CDCA [32, 36] . In our experience, the administration of a higher dose of UDCA than was used in previous studies [11, 29], could produce more metabolism to LCA and, as a



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consequence, a higher percentage of this bile acid . Furthermore, alterations in liver tissue were observed by light microscopy in all treated animals . The correlation between the increase of LCA conjugates produced by UDCA treatment and liver damage should require further investigation . We observed that G/T ratio (0 . 69, Table II) in the control animals differed from that of 2 . 13 observed by Kuroki et al. [18] and 1 . 5 reported by Berry-Lortsch & Sable-Amplis [37] . This difference may be due to the variability of animals and/or to their diet . In agreement with the work of Yoon et al. [38] we found that TCA was the major endogenous bile acid . In our studies, the GLCA/TLCA ratio remained less than 1 in contrast to G/T ratio of the other individual bile acids . This could be due to intestinal bacterial deconjugation and further hepatic conjugation with taurine [39, 40] . It is also known that the extent of the relative conjugation may depend on enzymatic affinity and the available amounts of glycine and taurine amino acids [41 ] . In conclusion, the high dose of UDCA administered to the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives .

ACKNOWLEDGEMENTS This work was supported in part by a grant from Consejo Nacional de Investigaciones Cientificas y Técnicas . The authors wish to thank Mr M . Bergamalli without whose generous help these studies would not have been possible and Tokyo Tanabe (Tokyo, Japan) for the supply of ursodeoxycholic acid . We are also grateful to biochemist A . Rojas and Mr F . Pedrouzo for their technical assistance and to Segunda Câtedra de Patologia, Facultad de Medicina, Universidad de Buenos Aires, for the use of the vivarium and to Dr J . Muse for his English revision .

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27 . Handelsman B, Bonorris G, Marks JW, Schoenfield LJ . Enrichment of bile with tauroursodeoxycholic acid and biliary cholesterol saturation in hamsters . Am J Physiol 1982 ; 243 : G424-7 . 28 . Bonorris G, Coyne MJ, Chung A, Schoenfield LJ . Mechanism of estrogen-induced saturated bile in the hamster . JLab Clin Med 1977 ; 90 : 963-70 . 29 . Bellentani S, Bosisio E, Pecorari M, et al. Effect of tauroursodeoxycholate feeding, with or without taurine supplementation on hepatic bile acids and cholesterol metabolism in the hamster. Pharmac Res Commun 1987 ; 19/5 : 327-39 . 30 . Higashi S, Setoguchi T, Katsuki T . Interconversion between chenodeoxycholic acid and ursodeoxycholic acid in anaerobic cultures of intestinal microorganisms and reduction of 7-ketolithocholic acid to both bile acids . Acta Hepatol Japan 1978 ; 19 : 803 (abst .) . In : Ikawa S, Kawasaki H, Kaibara N, eds . Advances in bile acid and bile alcohol research in Japan. Yonago, Japan : Tottori University, 1984 : 234 . 31 . Macdonald IA, Hutchinson DM, Forrest TP. Formation of urso- and ursodeoxycholic acids from primary bile acids by Clostridium absonum . J Lipid Res 1981 ; 22 : 458-66 . 32 . Macdonald IA, Bokkenheuser VW, Winter J, McLernon AM, Mosbach EH . Degradation of steroids in the human gut (Review) . J Lipid Res 1983 ; 24 : 675-700 . 33 . Fromm H, Carlson GL, Hofmann AF, Farivar S, Amin P. Metabolism in man of 7ketolithocholic : precursor of cheno- and ursodeoxycholic acids . Am J Physiol 1980 ; 239 : G 161-6 . 34 . Emerman S, Javitt NB . Metabolism of taurolithocholic acid in the hamster . J Biol Chern 1967 ; 242(4) :661-4 . 35 . Bergman F, van der Linder W, Sjövall J . Biliary bile acids and hepatic ultra-structure in hamsters fed gallstone-inducing and -dissolving diets . Acta Physiol Scand 1968 ; 74 : 480-91 . 36 . White BA, Fricke RJ, Hylemon PB . 7ß-Dehydroxylation of ursodeoxycholic acid by whole cells and cell extracts of the intestinal anaerobic bacterium Eubacterium species V.P.I . 12708 . JLipid Res 1982 ; 23 : 145-53 . 37 . Berry-Lortsch E, Sable-Amplis R . Qualitative and quantitative changes in biliary secretion induced by apple consumption in hamsters . Nutr Rep Int 1981 ; 23 : 505-16 . 38 . Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbach JH . Effect of side-chain shortening on the physiologic properties of bile acids : hepatic transport and effect on biliary secretion of 23-norursodeoxycholate in rodents . Gastroenterology 1986 ; 90 : 837-52 . 39 . Hofmann AF. Chemistry and enterohepatic circulation of bile acids . Hepatology 1984 ; 4 : 4S-14S . 40 . Kuipers F, Heslinga H, Havinga R, Vonk RL . Intestinal absorption of lithocholic acid sulfates in the rat: inhibitory effects of calcium . Am J Physiol 1986 ; 251 : G189-94 . 41 . Vessey DA . The biochemical basis for the conjugation of bile acids with either glycine or taurine . Biochem J 1978 ; 174 : 621-6 .