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PHARMACOKINETICS OF URSODEOXYCHOLIC ACID IN RAT ALDO RODA*, PATRIZIA HRELIA, MORENO PAOLINI, MILLER CALZOLARIW, BRUNELLA GRIGOLOt, PATRIZIA SIMONIt, RITA ALDINII and GIORGIO CANI'ELLI FORTI *Istituto di Chimica Analitica, Università di Messina, Messina, Italy ; Istituto di Farmacologia, tIstituto di Scienze Chimiche, Università di Bologna, Bologna ; +Laboratorio Analisi, Ospedale Civile - USL 15, Modena, Italy Received in final form 25 September 1990
SUMMARY The pharmacokinetic behaviour and metabolism of ursodeoxycholic acid (UDCA) have been studied in the rat . After oral administration of both 3H-labelled (4 PCi/kg body wt) and unlabelled (20 mg) UDCA, UDCA appeared in serum almost entirely in conjugated form (taurine conjugated); UDCA was present in bile mostly as taurine conjugated; the more relevant metabolite is 3a,6a,7ß-trihydroxycholanoic acid which represents 10% of the total bile acid pool. UDCA increased bile flow and selectively decreased biliary cholesterol secretion, while phospholipid secretion was unaffected . Faecal UDCA excretion was 15-20% while the urinary extraction was 1 .5% during 24 h . The data show that UDCA, when administered in high dose, is promptly secreted into bile almost entirely metabolized to tauroursodeoxycholic acid, where it (1) desaturates the cholesterol in bile, (2) exerts choleretic properties . KEY
wout>s : bile acids, ursodeoxycholic acid, gallstone dissolution . INTRODUCTION
From the results of clinical studies, the therapeutical effectiveness of and acid (3a,7ß-dihydroxycholanoic acid, UDCA) ursodeoxycholic chenodeoxycholic acid in desaturating the bile with respect to cholesterol [1] has been well documented . These naturally occurring bile acids present quite distinct pharmacokinetic behaviour, since : 1 . they are found almost entirely within the enterohepatic circulation ; Correspondence to : Prof. Aldo Roda, Istituto di Chimica Analitica, Università degli Studi di Messina, Via Salita Sperone, 31, 98010 S . Agata, Messina, Italy. 1043-6618/91/040327-09/$03 .00/0
© 1991 The Italian Pharmacological Society
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2. their serum levels under normal conditions are quite low, despite the efficient intestinal absorption [2], because of high hepatic clearance [3] ; 3. their elimination from the body is achieved almost entirely by the faecal route ; 4. after their oral ingestion, they undergo a series of biotransformations, both by liver and bacterial enzymes, which deeply modify their physicochemical and hence biological properties [4] . Systematic studies aimed at quantifying the bile acids' pharmacokinetic properties have not been thus performed, mainly due to analytical problems and to the difficulties in the choice of adequate animal models . In the present investigation, which is part of a broad programme of study about the quantitative structure-activity relationships of the bile acids [4], we have investigated the pharmacokinetic behaviour and the metabolism of ursodeoxycholic acid (UDCA) by using sensitive and accurate chemical and radioanalytical techniques, after the oral administration of this bile acid to the rat .
MATERIALS AND METHODS Serum level of UDCA over 24 h
Serum levels of UDCA over 24 h have been evaluated in 60 female SpragueDawley rats (250 ± 20 g body wt), allotted into 10 groups (6 animals each) . Nine groups were used for UDCA serum evaluation, one group was used as control. UDCA (20 mg + 3 .56 X 10 -5 mg of 3 H-labelled UDCA, i .e . 4,uCi/kg body wt) was orally administered and the blood was collected by cardiac puncture at 0 .5, 1 .0, 2 .0, 3 .0, 4 .0, 5 .0, 6 .0, 12 .0 and 24.0 h thereafter . The blood was centrifuged at 300 r .p .m. for 10 min, and serum was kept for analysis by both radioisotopic dilution technique and radioimmunoassay (RIA) [5] . For radioisotopic dilution technique, 200-400,ul of serum were counted after addition of Pico-fluor (Packard) as liquid scintillation cocktail, in a Packard 2425 liquid scintillation instrument. For the RIA analysis the serum samples were properly diluted (see below) . Bile secretion
Biliary excretion of UDCA has been evaluated over 6 h after administration of 20 mg + 3 .56 X 10 -5 mg/kg body wt of [3H]UDCA (i .e. 4 uCi/kg body wt) . Eight female Sprague-Dawley rats (250 ± 20 g body wt) were used . Eight additional female Sprague-Dawley rats (250 ± 20 g body wt) served as controls and were administered 20 mg cholic acid . Bile was collected over 6 h at 1-hour intervals as described earlier [6, 7] ; urethane was injected i .p . as a 15% solution in saline . The rats received between 1 .25 and 1 .5 mg/kg, as required . Urethane was chosen as the anaesthetic for these studies because of its long lasting action . After anaesthesia was achieved, the animals were restrained on a dissecting board and their temperature maintained at 36-37 °C with an infrared lamp 60 cm above the animal . A small abdominal incision was made and the common bile duct
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was exposed and cannulated using polyethylene tubing (0 .58 i .d . x 0 .956 mm o.d . X 18 cm) with beveled end . The cannula was then secured with surgical thread immediately below ligation . When it was determined that the bile was flowing, the incision was closed and covered with gauze sponges saturated with saline . The bile was collected in 2 .0 ml polyethylene liquid nitrogen vials at room temperature . In the untreated controls, bile was collected immediately for six 1-hour intervals . In the animals receiving UDCA, after 30 min equilibration, bile was collected as described. ifty-one hundred 4ul of bile were counted for radioactivity, after addition of the scintillation cocktail. UDCA and its metabolites in serum and bile have been measured by chromatographic techniques, both by thin layer chromatography (TLC) [8] and high performance liquid chromatography (HPLC) [9]. The biliary lipid composition (bile acid, cholesterol, phospholipids) has been measured by enzymatic techniques [10-12] . F
Faecal excretion
The faecal excretion was evaluated over 24 h, after the administration of the same dose as described above . Eight female rats were used . Metabolic cages were used for complete stool collections . Faeces, pooled together for each animal, were homogenized, centrifuged and 200-400,ul aliquots were counted for radioactivity after digestion with 2 ml of Soluene 350 (Packard) and addition of 10 ml of Pico-fluor liquid scintillation cocktail (Packard). UDCA and its metabolites have been measured by TLC and HPLC [8, 9] . Urinary excretion
The urinary excretion was studied over 24 h after oral administration of the same dose as described above . Eight female rats, matched by weight, were used . Metabolic cages were used for complete urine collections . Urine, collected for 24 h was weighed, filtered through Millipore filters (0 .8 um) . Between 100 and 200,ul samples were counted for radioactivity, after addition of liquid scintillation cocktail . Evaluation of the data
All of the data were expressed as mean ± SE . For the RIA analysis of the serum samples, the results were expressed as % of the injected dose . Similarly, the percentage of the injected dose was considered the most appropriate expression of the results for all the radioanalytical analyses . Bile acid, cholesterol and phospholipid secretion rates were expressed as µcool/ min/kg body wt . Cholesterol and phospholipid secretion rates were plotted against bile acid secretion for all times, in order to obtain, if possible, a correlation between bile acid secretion and cholesterol or phospholipid secretion, in order to ascertain any differences between the UDCA and the cholic acid control group .
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Statistical differences between the groups were calculated using Wilcoxon's matched-pairs signed-ranks test . Analytical methods TLC. Thin-layer chromatography was performed on Silica Gel G-plates, using
propionic acid/isoamyl acetate/H20/n-propanol, 75/100/25/50 (v/v) as solvent system, in order to get the conjugation pattern of ursodeoxycholic acid in the biological fluids . HPLC. High performance liquid chromatography was performed following Van Berge Henegowen [9] . RIA. The radioimmunoassay was performed on properly diluted serum samples as already published . The sensitivity, accuracy and precision of the method, as well as the crossreactivity among the various classes of bile acids have been already reported [5]. Bile acids in bile have been measured by bioluminescence method, using immobilized 7a-hydroxysteroid dehydrogenase [10]. Phospholipids have been measured by enzymatic determination [12] as well as cholesterol [11], as previously published . Chemicals
UDCA was purchased from Calbiochem (La Jolla, CA, USA) ; (11,12-3H(N)) ursodeoxycholic (S .A . 37 .0 Ci/mmol) was purchased from NEN, Boston, USA. All other reagents were obtained from commercial sources and were of the highest purity available .
Table I Serum levels of UDCA at various time intervals (% of the administered dose), after administration of 20 mg UDCA+3 .56x10 -5 mg of [3H]UDCA/kg body wt Time
30 min 1 h 1 .5 h 2 h 3 h 4 h 6 h 12 h 24h
% of the administration radioactivity
% of the administration dose
0 .114 ± 0 .052 0 .125±0 .018 0 .063 ± 0 .009 0 .056±0 .006 0 .077±0 .010 0 .066 ± 0 .005 0 .034 ± 0 .002 0 .031 ± 0 .003 0 .025 ± 0 .001
0 .113 ± 0 .005 0 .108±0 .012 0 .056 ± 0 .006 0 .042 ± 0 .004 0 .056±0 .007 0 .049 ± 0 .003 0 .028 ± 0 .001 0 .025 ± 0 .002 0 .021 ± 0 .001
Nine groups of rats (6 animals each) were used for UDCA serum evaluation ; one group for control . The blood was collected by cardiac puncture at 0 .5, 1 .0, 2 .0, 3 .0, 4 .0, 5 .0, 6 .0, 12 .0, 24.0 h after oral administration of UDCA . The results are expressed as mean ± SE .
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RESULTS Serum UDCA levels
Good agreement between the two methods (RIA and radioanalytical technique) was observed in all of the experiments ; therefore, either technique proved suitable for conducting pharmacokinetic studies and was considered for the evaluation of the results. After oral administration of UDCA, its serum levels are very low, with a maximum peak after an hour and a second one after 3 h (Table I) . The conjugation with taurine and glycine increased with time, reaching a maximum for taurine in the first hour, for glycine in the second hour (Fig . 1) .
Fig . 1 . Conjugation pattern of administered labelled unconjugated UDCA in serum during the first 2 h experiment . Serum samples were collected as described in the text . The glycine and taurine conjugation pattern was evaluated by TLC .
Biliary UDCA excretion
In the bile fistula rats, about 36% of the administered dose is secreted into bile within the first 6 h, the highest secretion rates (about 8%) being in the first and second hour (Table II) . UDCA is present in bile as taurine conjugated (80%), glycine conjugated (15%); only a small amount (5%) is unconjugated . The more relevant metabolite present in bile appeared to be 3 a,6 a,7ßtrihydroxycholanoic acid, which accounted for 10% of the total bile acid . UDCA increased bile flow (Fig. 2) ; cholesterol secretion into bile was significantly reduced (P< 0 .01) in the UDCA group, when UDCA was compared with the cholic acid control group, and was the lowest at highest ursodeoxycholic secretion rates (first 2 h). Phospholipid concentration in bile was unchanged compared to cholic acid administration (Fig . 2) .
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Table 11 Biliary excretion of UDCA (% of the administered dose) over 6 h after oral administration of 20 mg UDCA+3 .56X 10 -5 mg of [3H]UDCA/kg body wt Time
Bile flow (ml)
(h)
% UDCA
2 3 4 5 6
0 .48 0 .55 0 .57 0 .60 0 .59 0 .52
0 .03 0 .03 0 .04 0 .03 0 .03 0.02
8 .97±0 .42 8 .68±0 .63 5 .57±0 .36 5 .37±0 .37 4 .38±0 .39 3 .90±0 .36
8 .74±0.42 8 .45 ±0.63 5 .35±0.36 5 .15±0.37 4 .16±0.39 3 .69±0.36
Total
3 .31 ± 0 .14
36 .89 ± 1 .82
35 .56 ± 1 .83
1
± ± ± ± ± ±
Radioactivity
Eight rats were used . The bile flow was collected from the cannulated bile duct at hourly time intervals in preweighted tubes, after anaesthesia was achieved . The results are expressed as mean ± SE .
Y 1
E
200 -
0.4
3a7ß z 0 .3
150
C
i
o
3 100 o
0.2
50
m
0-1 I I 1 1 1 8 10 0 2 4 6 Bile acid secretion (µmol/min/kg)
0--1 0
1 1 I I 1 8 10 2 4 6 Bile acid secretion (µmol /min/kq)
P Y
0 .08 E ô i0.06 3a7a1Za
0
0.04
u0 N
Q 0
.02
O
O U
0-I 0
1 1 1 1 1 10 2 4 6 8 Bile acid secretion (µmol/min/kg)
Fig. 2 . Bile acids biliary secretion plotted versus bile flow, phospholipid and cholesterol secretion after treatment with 3a,7ß-dihydroxycholanoic acid (UDCA) . Cholic acid (3a, 7a, 12a) was used as control, as described in the text . Bile was collected from anaesthetized animals over 6 h, at hourly intervals .
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Urinary UDCA excretion
UDCA excretion in the 24-h urine was only 1 .5% of the administered dose . However, the excretion was more relevant between 0 and 12 h . Faecal UDCA excretion
UDCA excretion in stools (24-h) accounted for 15-20% of the administered dose (Table III) . The major metabolite was lithocholic acid (80%) .
Table III Urinary and faecal excretion of UDCA after oral administration of UDCA+3.56X 10-5 mg of [3 H]UDCA/kg body wt Time (h)
12 h 24h Total
Urinary volume (ml)
4.45 ± 3.20 ± 7 .65 ±
% Radioactivity
0 .42 0 .24 0 .58
Urinary
Urinary
1 .16 ± 0.04 0.37 ± 0.04 1 .53 ± 0.06
0.98 ± 0 .044 0.25 ± 0 .024 1.23 ± 0 .055
Faecal
24h The results are expressed as mean ±
% Excretion
18 .0±4.0
Faecal
18 .0±5 .0
SE .
DISCUSSION UDCA is secreted in bile mainly as glycine and taurine conjugated ; the conjugation process occurs to facilitate the biliary excretion of UDCA by increasing its hydrophilicity and water solubility [13] . Since the pKa values of taurine and glycine conjugated UDCA are respectively about 1 and 3, and therefore lower than that of the unconjugated form [5], these molecules are completely ionized in the enterohepatic circulation, where the pH ranges from 5 .5 to 8 .0 [14] . The partial recovery in bile of the administered UDCA is probably due to the high dose administered, possibly exceeding the endogenous taurine and glycine pool ; in such conditions only a minor amount can be secreted in free form . Therefore, the kinetics of the appearance of UDCA in bile may be regulated by the hepatic availability as taurine and glycine. The choleretic effect [15] of administered UDCA may be due to a possible cholehepatic shunt pathway, by which the molecule secreted into bile undergoes passive absorption in the canaliculi . The cholehepatic shunt pathway, proposed by Yoon [16] for nor-UDCA, may also play an important role for UDCA, although not so relevant as for nor-UDCA . Unconjugated UDCA, secreted into bile, is absorbed passively into the
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cholangiocyte and transported into the basolateral spaces by unknown mechanisms . It then enters the periductural capillary plexus and is resecreted into the canaliculus . Bile flow is generated by the osmotic activity of the bile acids. The choleretic effect of unconjugated dihydroxy bile acid is greater than that of conjugated dihydroxy bile acid . The same is true for unconjugated cholate compared to its conjugates [13, 18], as well as for some radiocontrast agents [19] and polycyclic carboxylic acids . One explanation for this behaviour is that as a molecule, X, is conjugated, the molecular weight increases and the osmotic pressure for a given weight of X decreases . If it is hypothesized that bile flow is generated by the osmotic pressure of the bile acids, then these observations about choleretic effect are consistent with this hypothesis [20] . This repeated cycling explains both the apparent hypercholeresis and the poor recovery of unconjugated UDCA in bile . The low serum levels both rule out a cholestatic effect of UDCA and suggest an efficient hepatic uptake . In previous studies [5, 21], using the perfused rat liver, the hepatic uptake of labelled UDCA has been evaluated as about 55% of the injected dose, i .e . quite similar to the physiological bile acids . Serum UDCA levels, as evaluated by two highly sensitive techniques, proved substantially in agreement . The serum peak observed in the first hour parallels the biliary peak and may be related to the passive absorption of administered UDCA . Afterwards the serum peaks may reflect the dynamics of the enterohepatic circulation which the bile acid undergoes . About 90% of the serum peak is accounted for by unconjugated UDCA, while taurine and glycine UDCA conjugates make up the remainder, indicating that several forms of the drug pass through the liver. While a small amount of UDCA is extracted into the urine, a much larger amount (20-30%) is excreted in the stool . The faecal extraction of UDCA may be due to its physicochemical properties . When administered, UDCA is protonated and in this form it reaches the intestine . Protonated UDCA is very insoluble (about 10 -6 M) and it requires a relatively high pH for dissolution (CMpH=8 .7) . Under normal conditions, part of the UDCA remains precipitated, and unabsorbed and is, therefore, excreted in the stool .
ACKNOWLEDGEMENTS This work was supported jointly by MURST and by CNR (National Research Council for Italy) grant Bilateral Project Italy - USA (contract no. 89 .02887 .04) .
REFERENCES 1 . Hofmann AF. Pharmacology of chenodeoxycholic and ursodeoxycholic acid in man . In: Paumgartner G, Stiehl A, Gerok W, eds . Bile acid and cholesterol in health and disease . Lancaster (UK) : MTP Press Ltd, 1983 . 2. Wilson A. Intestinal transport of bile acids . Am JPhysiol 1981 ; 241 : G83-92 . 3 . Aldini R, Roda A, Morselli AM, Cappelleri G, Roda E, Barbara L . Hepatic bile acid
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uptake : effect of conjugation, hydroxyl and cheto groups and albumin binding . J Lipid Res 1982 ; 23 : 1167-72 . 4 . Hofmann AF, Roda A. Physicochemical properties of bile acids and their relationship to biological properties : an overview of the problem . J Lipid Res 1984; 25 : 1477-89 . 5 . Roda A, Roda E, Aldini R, et al. Development, validation, and application of single tube radioimmunoassay for cholic and chenodeoxycholic conjugated bile acids in human serum . Clin Chem 1977 ; 23 :2107 . 6 . Connor TH, Cantelli Forti G, Sitra P, Legator MS . Bile as a source of mutagenic metabolites produced in vivo and detected by Salmonella typhimurium . Environm Mutagen 1979 ; 1 : 269-76 . 7 . Cantelli Forti G, Trieff NM . Kinetics of uptake and biliary excretion of benzo(a)pyrene and mutagenic metabolites in isolated perfused rat liver . Teratogen Carcin Mut 1980 ; 1 : 269-82 . 8 . Beke R, De Weerdt GA, Parijs J, Huybrechts W, Barbier F. Separation of conjugated and unconjugated bile acids by TLC . Clin Chim Acta 1976 ; 70 : 197 . 9. Ruben AT, Van Berge Henegowen GP. A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system . Clin Chim Acta 1982 ; 119 : 41-50 . 10 . Roda A, Kricka J, De Luca M, et al. Bioluminescence measurement of primary bile acids using immobilized 7a-hydroxysteroid dehydrogenase : application to serum bile acids . J Lipid Res 1982 ; 23 :1354-61 . 11 . Roda A, Festi D, Sama C, et al. Enzymatic determination of cholesterol in bile. Clin Chim Acta 1975 ; 64 : 337-41 . 12 . Roda A, Geminiani S, Rossi R, et al. Enzymatic determination of phospholipids in bile . Lab J Res Med 1981 ; 2 : 119-22 . 13 . O'Maille ERL, Richard TG . Possible explanations for the differences in the excretory characteristics between conjugated and free bile acids . J Physiol (Lond) 1977 ; 265 : 855-66 . 14 . Fini A, Roda A. Chemical properties of bile acids, Part II : pKa values in water and aqueous methanol of some hydroxy bile acids. Eur J Med Chem 1982 ; 5 : 467-70 . 15 . Renner EL, Lake JR, Cragoe EJ, Van Dyke R, Scharschmidt BF. Ursodeoxycholic acid choleresis : relationship to biliary HCO3 and effect of Na'-H' exchange inhibitors . Am J Physiol 254 (Gastroint Liver Physiol 17) 1988 ;G :232-41 . 16 . Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbach JH . Effect of side chain shortening on the physiological properties of bile acids : hepatic transport and effect on biliary secretion of 23-norursodeoxycholate in rodents . Gastroenterology 1986 ; 90 :837-52 . 17 . Klaassen CD . Comparison of the choleretic properties of bile acids . Eur J Pharmacol 1973 ;23 :270-5 . 18 . Paumgartner G, Santer K, Schwartz HP, Herz R. Hepatic excretory transport maximum for free and conjugated cholate in the rat . In : Paumgartner G, Preising R, eds . The liver, quantitative aspects of structure and function . Basel: Karger, 1973 : 337-44 . 19 . Feld GK, Loeb PM, Berk RN, Wheeler HO . The choleretic effect of iodipamide . J Clin Invest 1975 ; 55 : 528-35 . 20 . Barnhart JL, Combes B . Characterization of SC 2644-induced choleresis in the dog . Evidence for canalicular bicarbonate secretion . J Pharmacol Exp Ther 1978 ; 206 : 190-7 . 21 . Aldini R, Roda A, Simoni P, Lenzi PL, Roda E . Hepatic bile acid uptake : structure-activity relationship . Hepatology(in press) .
Pharmacological Research, Vol. 23, No. 4, 1991
PHARMACOKINETICS OF URSODEOXYCHOLIC ACID IN RAT ALDO RODA*, PATRIZIA HRELIA, MORENO PAOLINI, MILLER CALZOLARIW, BRUNELLA GRIGOLOt, PATRIZIA SIMONIt, RITA ALDINII and GIORGIO CANI'ELLI FORTI *Istituto di Chimica Analitica, Università di Messina, Messina, Italy ; Istituto di Farmacologia, tIstituto di Scienze Chimiche, Università di Bologna, Bologna ; +Laboratorio Analisi, Ospedale Civile - USL 15, Modena, Italy Received in final form 25 September 1990
SUMMARY The pharmacokinetic behaviour and metabolism of ursodeoxycholic acid (UDCA) have been studied in the rat . After oral administration of both 3H-labelled (4 PCi/kg body wt) and unlabelled (20 mg) UDCA, UDCA appeared in serum almost entirely in conjugated form (taurine conjugated); UDCA was present in bile mostly as taurine conjugated; the more relevant metabolite is 3a,6a,7ß-trihydroxycholanoic acid which represents 10% of the total bile acid pool. UDCA increased bile flow and selectively decreased biliary cholesterol secretion, while phospholipid secretion was unaffected . Faecal UDCA excretion was 15-20% while the urinary extraction was 1 .5% during 24 h . The data show that UDCA, when administered in high dose, is promptly secreted into bile almost entirely metabolized to tauroursodeoxycholic acid, where it (1) desaturates the cholesterol in bile, (2) exerts choleretic properties . KEY
wout>s : bile acids, ursodeoxycholic acid, gallstone dissolution . INTRODUCTION
From the results of clinical studies, the therapeutical effectiveness of and acid (3a,7ß-dihydroxycholanoic acid, UDCA) ursodeoxycholic chenodeoxycholic acid in desaturating the bile with respect to cholesterol [1] has been well documented . These naturally occurring bile acids present quite distinct pharmacokinetic behaviour, since : 1 . they are found almost entirely within the enterohepatic circulation ; Correspondence to : Prof. Aldo Roda, Istituto di Chimica Analitica, Università degli Studi di Messina, Via Salita Sperone, 31, 98010 S . Agata, Messina, Italy. 1043-6618/91/040327-09/$03 .00/0
© 1991 The Italian Pharmacological Society
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Pharmacological Research, hol. 23, No . 4, 1991
2. their serum levels under normal conditions are quite low, despite the efficient intestinal absorption [2], because of high hepatic clearance [3] ; 3. their elimination from the body is achieved almost entirely by the faecal route ; 4. after their oral ingestion, they undergo a series of biotransformations, both by liver and bacterial enzymes, which deeply modify their physicochemical and hence biological properties [4] . Systematic studies aimed at quantifying the bile acids' pharmacokinetic properties have not been thus performed, mainly due to analytical problems and to the difficulties in the choice of adequate animal models . In the present investigation, which is part of a broad programme of study about the quantitative structure-activity relationships of the bile acids [4], we have investigated the pharmacokinetic behaviour and the metabolism of ursodeoxycholic acid (UDCA) by using sensitive and accurate chemical and radioanalytical techniques, after the oral administration of this bile acid to the rat .
MATERIALS AND METHODS Serum level of UDCA over 24 h
Serum levels of UDCA over 24 h have been evaluated in 60 female SpragueDawley rats (250 ± 20 g body wt), allotted into 10 groups (6 animals each) . Nine groups were used for UDCA serum evaluation, one group was used as control. UDCA (20 mg + 3 .56 X 10 -5 mg of 3 H-labelled UDCA, i .e . 4,uCi/kg body wt) was orally administered and the blood was collected by cardiac puncture at 0 .5, 1 .0, 2 .0, 3 .0, 4 .0, 5 .0, 6 .0, 12 .0 and 24.0 h thereafter . The blood was centrifuged at 300 r .p .m. for 10 min, and serum was kept for analysis by both radioisotopic dilution technique and radioimmunoassay (RIA) [5] . For radioisotopic dilution technique, 200-400,ul of serum were counted after addition of Pico-fluor (Packard) as liquid scintillation cocktail, in a Packard 2425 liquid scintillation instrument. For the RIA analysis the serum samples were properly diluted (see below) . Bile secretion
Biliary excretion of UDCA has been evaluated over 6 h after administration of 20 mg + 3 .56 X 10 -5 mg/kg body wt of [3H]UDCA (i .e. 4 uCi/kg body wt) . Eight female Sprague-Dawley rats (250 ± 20 g body wt) were used . Eight additional female Sprague-Dawley rats (250 ± 20 g body wt) served as controls and were administered 20 mg cholic acid . Bile was collected over 6 h at 1-hour intervals as described earlier [6, 7] ; urethane was injected i .p . as a 15% solution in saline . The rats received between 1 .25 and 1 .5 mg/kg, as required . Urethane was chosen as the anaesthetic for these studies because of its long lasting action . After anaesthesia was achieved, the animals were restrained on a dissecting board and their temperature maintained at 36-37 °C with an infrared lamp 60 cm above the animal . A small abdominal incision was made and the common bile duct
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329
was exposed and cannulated using polyethylene tubing (0 .58 i .d . x 0 .956 mm o.d . X 18 cm) with beveled end . The cannula was then secured with surgical thread immediately below ligation . When it was determined that the bile was flowing, the incision was closed and covered with gauze sponges saturated with saline . The bile was collected in 2 .0 ml polyethylene liquid nitrogen vials at room temperature . In the untreated controls, bile was collected immediately for six 1-hour intervals . In the animals receiving UDCA, after 30 min equilibration, bile was collected as described. ifty-one hundred 4ul of bile were counted for radioactivity, after addition of the scintillation cocktail. UDCA and its metabolites in serum and bile have been measured by chromatographic techniques, both by thin layer chromatography (TLC) [8] and high performance liquid chromatography (HPLC) [9]. The biliary lipid composition (bile acid, cholesterol, phospholipids) has been measured by enzymatic techniques [10-12] . F
Faecal excretion
The faecal excretion was evaluated over 24 h, after the administration of the same dose as described above . Eight female rats were used . Metabolic cages were used for complete stool collections . Faeces, pooled together for each animal, were homogenized, centrifuged and 200-400,ul aliquots were counted for radioactivity after digestion with 2 ml of Soluene 350 (Packard) and addition of 10 ml of Pico-fluor liquid scintillation cocktail (Packard). UDCA and its metabolites have been measured by TLC and HPLC [8, 9] . Urinary excretion
The urinary excretion was studied over 24 h after oral administration of the same dose as described above . Eight female rats, matched by weight, were used . Metabolic cages were used for complete urine collections . Urine, collected for 24 h was weighed, filtered through Millipore filters (0 .8 um) . Between 100 and 200,ul samples were counted for radioactivity, after addition of liquid scintillation cocktail . Evaluation of the data
All of the data were expressed as mean ± SE . For the RIA analysis of the serum samples, the results were expressed as % of the injected dose . Similarly, the percentage of the injected dose was considered the most appropriate expression of the results for all the radioanalytical analyses . Bile acid, cholesterol and phospholipid secretion rates were expressed as µcool/ min/kg body wt . Cholesterol and phospholipid secretion rates were plotted against bile acid secretion for all times, in order to obtain, if possible, a correlation between bile acid secretion and cholesterol or phospholipid secretion, in order to ascertain any differences between the UDCA and the cholic acid control group .
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Statistical differences between the groups were calculated using Wilcoxon's matched-pairs signed-ranks test . Analytical methods TLC. Thin-layer chromatography was performed on Silica Gel G-plates, using
propionic acid/isoamyl acetate/H20/n-propanol, 75/100/25/50 (v/v) as solvent system, in order to get the conjugation pattern of ursodeoxycholic acid in the biological fluids . HPLC. High performance liquid chromatography was performed following Van Berge Henegowen [9] . RIA. The radioimmunoassay was performed on properly diluted serum samples as already published . The sensitivity, accuracy and precision of the method, as well as the crossreactivity among the various classes of bile acids have been already reported [5]. Bile acids in bile have been measured by bioluminescence method, using immobilized 7a-hydroxysteroid dehydrogenase [10]. Phospholipids have been measured by enzymatic determination [12] as well as cholesterol [11], as previously published . Chemicals
UDCA was purchased from Calbiochem (La Jolla, CA, USA) ; (11,12-3H(N)) ursodeoxycholic (S .A . 37 .0 Ci/mmol) was purchased from NEN, Boston, USA. All other reagents were obtained from commercial sources and were of the highest purity available .
Table I Serum levels of UDCA at various time intervals (% of the administered dose), after administration of 20 mg UDCA+3 .56x10 -5 mg of [3H]UDCA/kg body wt Time
30 min 1 h 1 .5 h 2 h 3 h 4 h 6 h 12 h 24h
% of the administration radioactivity
% of the administration dose
0 .114 ± 0 .052 0 .125±0 .018 0 .063 ± 0 .009 0 .056±0 .006 0 .077±0 .010 0 .066 ± 0 .005 0 .034 ± 0 .002 0 .031 ± 0 .003 0 .025 ± 0 .001
0 .113 ± 0 .005 0 .108±0 .012 0 .056 ± 0 .006 0 .042 ± 0 .004 0 .056±0 .007 0 .049 ± 0 .003 0 .028 ± 0 .001 0 .025 ± 0 .002 0 .021 ± 0 .001
Nine groups of rats (6 animals each) were used for UDCA serum evaluation ; one group for control . The blood was collected by cardiac puncture at 0 .5, 1 .0, 2 .0, 3 .0, 4 .0, 5 .0, 6 .0, 12 .0, 24.0 h after oral administration of UDCA . The results are expressed as mean ± SE .
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RESULTS Serum UDCA levels
Good agreement between the two methods (RIA and radioanalytical technique) was observed in all of the experiments ; therefore, either technique proved suitable for conducting pharmacokinetic studies and was considered for the evaluation of the results. After oral administration of UDCA, its serum levels are very low, with a maximum peak after an hour and a second one after 3 h (Table I) . The conjugation with taurine and glycine increased with time, reaching a maximum for taurine in the first hour, for glycine in the second hour (Fig . 1) .
Fig . 1 . Conjugation pattern of administered labelled unconjugated UDCA in serum during the first 2 h experiment . Serum samples were collected as described in the text . The glycine and taurine conjugation pattern was evaluated by TLC .
Biliary UDCA excretion
In the bile fistula rats, about 36% of the administered dose is secreted into bile within the first 6 h, the highest secretion rates (about 8%) being in the first and second hour (Table II) . UDCA is present in bile as taurine conjugated (80%), glycine conjugated (15%); only a small amount (5%) is unconjugated . The more relevant metabolite present in bile appeared to be 3 a,6 a,7ßtrihydroxycholanoic acid, which accounted for 10% of the total bile acid . UDCA increased bile flow (Fig. 2) ; cholesterol secretion into bile was significantly reduced (P< 0 .01) in the UDCA group, when UDCA was compared with the cholic acid control group, and was the lowest at highest ursodeoxycholic secretion rates (first 2 h). Phospholipid concentration in bile was unchanged compared to cholic acid administration (Fig . 2) .
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Table 11 Biliary excretion of UDCA (% of the administered dose) over 6 h after oral administration of 20 mg UDCA+3 .56X 10 -5 mg of [3H]UDCA/kg body wt Time
Bile flow (ml)
(h)
% UDCA
2 3 4 5 6
0 .48 0 .55 0 .57 0 .60 0 .59 0 .52
0 .03 0 .03 0 .04 0 .03 0 .03 0.02
8 .97±0 .42 8 .68±0 .63 5 .57±0 .36 5 .37±0 .37 4 .38±0 .39 3 .90±0 .36
8 .74±0.42 8 .45 ±0.63 5 .35±0.36 5 .15±0.37 4 .16±0.39 3 .69±0.36
Total
3 .31 ± 0 .14
36 .89 ± 1 .82
35 .56 ± 1 .83
1
± ± ± ± ± ±
Radioactivity
Eight rats were used . The bile flow was collected from the cannulated bile duct at hourly time intervals in preweighted tubes, after anaesthesia was achieved . The results are expressed as mean ± SE .
Y 1
E
200 -
0.4
3a7ß z 0 .3
150
C
i
o
3 100 o
0.2
50
m
0-1 I I 1 1 1 8 10 0 2 4 6 Bile acid secretion (µmol/min/kg)
0--1 0
1 1 I I 1 8 10 2 4 6 Bile acid secretion (µmol /min/kq)
P Y
0 .08 E ô i0.06 3a7a1Za
0
0.04
u0 N
Q 0
.02
O
O U
0-I 0
1 1 1 1 1 10 2 4 6 8 Bile acid secretion (µmol/min/kg)
Fig. 2 . Bile acids biliary secretion plotted versus bile flow, phospholipid and cholesterol secretion after treatment with 3a,7ß-dihydroxycholanoic acid (UDCA) . Cholic acid (3a, 7a, 12a) was used as control, as described in the text . Bile was collected from anaesthetized animals over 6 h, at hourly intervals .
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Urinary UDCA excretion
UDCA excretion in the 24-h urine was only 1 .5% of the administered dose . However, the excretion was more relevant between 0 and 12 h . Faecal UDCA excretion
UDCA excretion in stools (24-h) accounted for 15-20% of the administered dose (Table III) . The major metabolite was lithocholic acid (80%) .
Table III Urinary and faecal excretion of UDCA after oral administration of UDCA+3.56X 10-5 mg of [3 H]UDCA/kg body wt Time (h)
12 h 24h Total
Urinary volume (ml)
4.45 ± 3.20 ± 7 .65 ±
% Radioactivity
0 .42 0 .24 0 .58
Urinary
Urinary
1 .16 ± 0.04 0.37 ± 0.04 1 .53 ± 0.06
0.98 ± 0 .044 0.25 ± 0 .024 1.23 ± 0 .055
Faecal
24h The results are expressed as mean ±
% Excretion
18 .0±4.0
Faecal
18 .0±5 .0
SE .
DISCUSSION UDCA is secreted in bile mainly as glycine and taurine conjugated ; the conjugation process occurs to facilitate the biliary excretion of UDCA by increasing its hydrophilicity and water solubility [13] . Since the pKa values of taurine and glycine conjugated UDCA are respectively about 1 and 3, and therefore lower than that of the unconjugated form [5], these molecules are completely ionized in the enterohepatic circulation, where the pH ranges from 5 .5 to 8 .0 [14] . The partial recovery in bile of the administered UDCA is probably due to the high dose administered, possibly exceeding the endogenous taurine and glycine pool ; in such conditions only a minor amount can be secreted in free form . Therefore, the kinetics of the appearance of UDCA in bile may be regulated by the hepatic availability as taurine and glycine. The choleretic effect [15] of administered UDCA may be due to a possible cholehepatic shunt pathway, by which the molecule secreted into bile undergoes passive absorption in the canaliculi . The cholehepatic shunt pathway, proposed by Yoon [16] for nor-UDCA, may also play an important role for UDCA, although not so relevant as for nor-UDCA . Unconjugated UDCA, secreted into bile, is absorbed passively into the
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cholangiocyte and transported into the basolateral spaces by unknown mechanisms . It then enters the periductural capillary plexus and is resecreted into the canaliculus . Bile flow is generated by the osmotic activity of the bile acids. The choleretic effect of unconjugated dihydroxy bile acid is greater than that of conjugated dihydroxy bile acid . The same is true for unconjugated cholate compared to its conjugates [13, 18], as well as for some radiocontrast agents [19] and polycyclic carboxylic acids . One explanation for this behaviour is that as a molecule, X, is conjugated, the molecular weight increases and the osmotic pressure for a given weight of X decreases . If it is hypothesized that bile flow is generated by the osmotic pressure of the bile acids, then these observations about choleretic effect are consistent with this hypothesis [20] . This repeated cycling explains both the apparent hypercholeresis and the poor recovery of unconjugated UDCA in bile . The low serum levels both rule out a cholestatic effect of UDCA and suggest an efficient hepatic uptake . In previous studies [5, 21], using the perfused rat liver, the hepatic uptake of labelled UDCA has been evaluated as about 55% of the injected dose, i .e . quite similar to the physiological bile acids . Serum UDCA levels, as evaluated by two highly sensitive techniques, proved substantially in agreement . The serum peak observed in the first hour parallels the biliary peak and may be related to the passive absorption of administered UDCA . Afterwards the serum peaks may reflect the dynamics of the enterohepatic circulation which the bile acid undergoes . About 90% of the serum peak is accounted for by unconjugated UDCA, while taurine and glycine UDCA conjugates make up the remainder, indicating that several forms of the drug pass through the liver. While a small amount of UDCA is extracted into the urine, a much larger amount (20-30%) is excreted in the stool . The faecal extraction of UDCA may be due to its physicochemical properties . When administered, UDCA is protonated and in this form it reaches the intestine . Protonated UDCA is very insoluble (about 10 -6 M) and it requires a relatively high pH for dissolution (CMpH=8 .7) . Under normal conditions, part of the UDCA remains precipitated, and unabsorbed and is, therefore, excreted in the stool .
ACKNOWLEDGEMENTS This work was supported jointly by MURST and by CNR (National Research Council for Italy) grant Bilateral Project Italy - USA (contract no. 89 .02887 .04) .
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