Differential effect of ursodeoxycholate and its taurine conjugate on biliary transport maximum of bilirubin in the rat

Life Scicnccs, Vol. 57, No. 10, pp. 97X981,1995 wght 0 1995 E%evkr Science Ud Printed in the USA. All rights nservcd 0024-32Osp5 $9.50 + .OO

Pergamon 0024-3205(95)02032-E

DIFFERENTIAL EFFECT OF URSODEOXYCHOLATE AND ITS TAURINE CONJUGATE ON BILIARY TRANSPORT MAXIMUM OF BILIRUBIN IN THE RAT Enrique J. Sanchez Pozzi, Aldo D. Mottino, Alfonso Sisti and Marcel0 G. Roma* Instituto de Fisiologia Experimental. Facuhad de Ciencias Bioquimicas y Farmaceuticas. U.N.R. Suipacha 570,200O - Rosario, ARGENTINA

CONICET -

(Received in final form June 12, 1995)

Summary The effects of ursodeoxycholate and its taurine conjugate on biliary Tm of bilirubin were evaluated in rats. Ursodeoxycholate was administered at four different doses (4, 8, 12 or 16 umol per 100 g body wt i.v., followed by an iv. infusion of 0.3, 0.6, 0.9 or 1.2 pmol/min per 100 g body wt. respectively), whereas tauroursodeoxycholate was administered only at the maximal dose. A dose-dependent diminution of bilirubin Tm was observed during ursodeoxycholate administration, which ranged from no effect at the lowest dose to a virtual excretory blockage at the highest dose. This was associated with an increase in bilirubin concentrations in both plasma and liver as well as in the fractional amount of conjugated pigment in both sites, suggesting an impairment of bilirubin transfer at the canalicular level. Incomplete taurine conjugation of ursodeoxycholate well correlated with these effects. Unlike ursodeoxycholate, tauroursodeoxycholate had no inhibitory effect on bilirubin Tm, although a slight inhibition of bilirubin uptake and bihrubin conjugation became apparent. Taken together, these results suggest that ursodeoxycholate interferes with the hepatobiliary transport of bilirubin by impairing its transfer at the canalicular level and that incomplete taurine conjugation appears to be a key factor determining this effect. Key Wordr: bilirubin uptake, bilirubin diconjugate, bilirubin monoconjugate, biliary excretion, glycoursodeoxycholate, tauroursodeoxycholate, ursodeoxycholate glucuronide Ursodeoxycholate (UDC) is a bile salt widely used for the treatment of liver disorders, including cholelitbiasis (1) and a variety of other cholestatic liver diseases (2). In spite of extensive studies regarding the effect of this bile salt on bile formation (3, 4) and on its hepatoprotective properties in experimental pathologies (5, 6) limited information is available about its influence on the hepatic transport of endogenous compounds, particularly bilirubin. A previous study showed a dual effect of UDC on bilirubin transport depending on the dose administered (7). In fact, whereas a low dose appears to stimulate slightly biliary maximal bilirubin transport (Tm), a high dose inhibited this process. The mechanisms responsible for the latter unexpected harmful effect remains, however, unknown. Elucidation of the mechanisms involved in such a phenomenon may, however, be relevant for the understanding of the causes implicated in the lack of improvement of plasma bilirubin levels occurring in some patients during long-term ADC therapy despite the striking amelioration in other clinical and biochemical parameters (8-13). It could be hypothesized that the inhibitory effect induced by high loads of UDC may result from changes in hepatic UDC metabolism, compared with that occurring when low doses are administered. This hypothesis rests on the fact that the extent at which this bile salt is conjugated, as well as the kind of

* Correspondence

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conjugation that this bile salt undergoes during its transit through the hepatocyte (indicated by the appearance in bile of conjugates other than that with taurine), were shown to depend largely on the dose administered (14-16). Therefore, in the present work, we tested this prediction by performing a formal dose-response study, aimed to correlate the magnitude of the inhibitory effect with changes in biliary UDC excretory products. In addition, the crucial role of taurine conjugation in preventing this inhibitory effect was assessed by comparing the effect on bilirubin transport induced by its taurine derivative, tauroursodeoxycholate (TUDC), which is neither deconjugated nor further metabolized during its transit throughout the hepatocyte (16). Materials and Methods Animals and treatments Adult male Wistar rats weighing 320-360 g were used. Before the experiments, the animals were maintained on a standard diet and water ad libitum, and housed in a temperature- (21-23°C) and humidity(45-50%) controlled room under a constant 12-h light-dark cycle. All animals received humane care as outlined in the “Guide ,for the Care and Use of Laboratory Animals” (NIH publication no. 86-23, revised 1985). Animals were anesthetized with a single dose of sodium pentobarbital (50 mg per kg body wt, ip.). Surgery procedure was essentially the same as previously reported by our laboratory (17). Briefly, surgical preparation included cannulation of femoral vein (for fluid infusion). carotid artery (for blood withdrawal), and common bile duct (for bile collection). Rectal temperature was maintained at 37.538.O”C with a heating lamp to prevent hypothermic alterations of bile flow. Experimental procedure Unconjugated bilirubin was dissolved in a minimal volume of 0. I mol/l NaOH, adjusted to pH 8.5 with 0.15 mol/l HCI and diluted with bovine serum albumin in saline (0.9% NaCI) to the required volume (final albumin concentration 3%). Bilirubin Tm studies were performed according to a procedure previously described by Van Steenbergen et al. (18). Briefly, after recovery from anesthesia, a baseline IO-min bile sample was collected. Then, a priming i.v. dose of bilirubin (3.42 pmol per 100 g body wt, dissolved in a volume of 1 ml) was injected, which was immediately followed by an infusion of 0.256 pmol/min per IO0 g body wt over a l-h period in a volume of 3.5 ml (control rats). Throughout the infusion period, six IO-mm bile samples were collected on ice and in the dark, and blood samples (100 pl) were collected through the carotid artery catheter at the midpoint of each bile collection period. In experiments aimed to assess the effect of the bile salts on bilirubin Tm, UDC (0.3, 0.6, 0.9 or 1.2 pmohmin per 100 g body wt) or TUDC (1.2 pmol/min per 100 g body wt) was dissolved conjointly with bilirubin and albumin (3%) in saline (final pH 8.5) and thus were administered. Previously, a single iv. dose of UDC (4, 8, I2 and 16 pmol per 100 g body wt) or TUDC (16 pmol per 100 g body wt) was coadministered with the priming dose of bilirubin to afford molar ratio of bile salts and bilirubin administration to be uniform throughout the experiment. Bile and blood samples were collected as described above. At the end of the experiment, rats were sacrificed by bleeding, and livers were perfused with ice-cold saline. Bile, serum and liver specimens were stored at -20°C until required. Analytical methods The volume of bile was determined gravimetrically, assuming the specific gravity of bile to be 1.0. Biliary excretion rates were calculated as the product of bile flow and biliary concentration of the corresponding solute. Total bilirubin concentrations in bile and plasma were determined by the method of Jendrassik et al. (19). To analyze the hepatic content of bilirubin, livers were previously homogenized with ice-cold saline (25%). Liver total pigment was determined as previously described (20) by a modification of the alkaline methanolysis procedure (21). For this purpose, an aliquot of the chloroform extract was diluted with methanol (final ratio 1: 1) and the absorbance of the resultant mixture was determined spectrophotometrically at 450 nm. The absorbance value was converted into pmol of total bilirubin assuming the same molar

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absorption coefftcient for unconjugated (UCB), monoconjugated (BMC) and diconjugated bihrubin (BDC) (21). Alkaline methanolysis was also applied to bile and serum samples. Ahquots of the chloroform extracts from bile, serum and liver homogenate were concentrated by evaporation under N2 at room temperature and applied to a thin-layer chromatography (TLC) plate precoated with silica gel to estimate the relative content of bilirubin species (running solvent: chloroform/methanol/acetic acid, 97:2: I). After TLC separation. UCB, BMC and BDC bands were immediately scraped off, eluted with chloroform-methanol (1: I), and measured spectrophotometrically at 450 nm. All procedures were carried out in red safety light and, when possible. under N2 to minimize pigment degradation. The recovery of the pigments after TLC analysis, when evaluated in samples representative of the different experimental procedures, was consistently over 90 %. Total bile salts in bile were measured by the 3a-hydroxysteroid dehydrogenase procedure (22). according to the modifications of Berthelot et al. (23) and after R-glucuronidase treatment as indicated previously (16). Composition of bihary bile salt pool was assessed by a bi-dimensional TLC analysis, which was a combination of two individual TLC methods previously described (24). Bile samples were promptly applied to TLC plates and developed with a first solvent system consisting of ethanol/ethyl acetate/ammonia (3:3:1), which allowed UDC glucuronide (UDC-gl) to be separated from unconjugated UDC and from Its chromatography using conjugates with taurine (TUDC) and glycine (GUDC). A second chIoroform/methanol/acetic acid/water (65:24: 15:9) as solvent system allowed UDC and UDC conjugates others than UDC-gl to be further separated from each other. This method, however, does not allow TUDC to be separated from other endogenous dihydroxylated bile salts, i.e. taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC). Measurement of the fractional amounts of these three bile salts was carried out using an alternative TLC method described by Batta et nl. (25) which allowed TUDC to be separated from the last two ones. After development, plates were sprayed with 20% (vol/vol) sulfuric acid and 3.5% phosphomolybdic acid, and heated at 120°C for 2 min. Quantification was carried out by direct densitometry at 600 mn using a Shimadzu CS-9000 densitometer (Shimadzu Corporation, Tokyo. Japan). Suitability of this procedure was checked with an alternative method, which involved TLC separation. extractton with ethanokwater (8: l), and subsequent bile salt quantitation using the 3a-hydroxysteroid dehydrogenase procedure (26). Previously, bile salt spots were identified by means of known standards or, in the case of UDC-gl, by the use of a specific spray for glucuronide residues (0.2% naphtoresorcinol rn ethanol:phosphoric acid 9: 1). Statistical analysis The results were expressed as mean f SD. Statistical analysis was performed using the NewmanKeuls multiple range test (27) which includes one-way analysis of variance (ANOVA). Any p value less than 0.05 was considered statistically significant. Chemicals Bilintbin, UDC, TUDC, bovine serum albumin, 3a-hydroxysteroid dehydrogenase. B-glucuronidase, phosphomolybdic acid. naphtoresorcinol and polyester sheets precoated with silica gel G 60 were purchased from Sigma Chemical Co. (St. Louis, MO). All the other reagents were of analytical grade. Results Bile sali excretion Table I illustrates the changes observed in bile flow and in biliary bile salt excretion rate and composition induced by UDC or TUDC administration in the last period of bile collection (50-60 mm). Bile flow and bile salt excretion remained virtually unchanged in control group throughout the experiment (data not shown). Administration of UDC induced a dose-dependent increase of both bile flow and total bile salt output. These changes were accompanied by a gradual replacement of endogenous bile salts for those corresponding to unconjugated UDC and its conjugates with taurine, glycine and glucuronic acid. As expected (14-16) the percentage of the taurine-conjugate dropped as the dose of UDC increased, with a concomitant increase in the proportion of the remaining UDC excretory products. TUDC induced essentially the same pattern of stimulation as UDC, but both bile flow and biliary output were higher than that obtained

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Table I Bile Flow, Biliary Bile Salt Output and Biliary Bile Salt Composition in Rats following Administration of UDC or TUDC UDC

Colltrols Infusion rate (~molhnin per 100 g body wt) Bile flow

&6)

0.3 (n = 4)

06 (n = 4) 2.88*0.19”

2.07 f 0.51

2.72 f 0.21”

47.9 * 9.3

116.6 + 15.1a

TC

61 f9

31 + 10”

TLX + TCDC

39 ?r 8 ND

GUDC

ND

4+2

UDC-gl

ND

ND

UJX

ND

5+3

26k6b

TUDC 0.9 (n = 5)

12 (n = 5)

1.2 (?I = 4)

3.30 + 0.23”bc

3.49 f0.41”bC

3 97 +0.38’

(kl/min per g liver wt) Bile salt output (mnol/ min per g liver wt)

169.3 k 16.1ab

237.4 f 32.7abc 249.6 + 33.1abc

361.6 + 5.8”

Bile salt composition (percent of total bile salts)

TUDC

14 *P

9 f 3ab

19*4*

9 -t 2”b

?f

41+5

38 + 3

36 f 3

5fl 8+2

lab

10 + Bab

I1 +2a

5 f 2”b

7 k 3”

28 * 2b”d

82 + 2

6+2

Sf lb

ND

9f2

12+4

33 f 3b

37 k 3bc

ND ND

Values correspond to the last bile-collection period (SO-SOmin) and are mean f SD. Bilirubin was systematically administered at a single i.v. dose of 3.42 pmol per 100 g body wt, followed by an i.v. infusion of 0.256 umollmin per 100 g body wt. UDC was coadministered at single i.v. doses of 4, 8, 12 or 16 umol per 100 g body wt, followed by i.v. infusions of 0.3, 0.6, 0.9 and 1.2 umollmin per 100 g body wt, respectively. TUDC was coadministered at a single i.v. dose of 16 umol per 100 g body wt, followed by an i.v. infusion of 1.2 umol/min per 100 g body wt. TDC, UDC, ursodeoxycholate; TUDC, tauroursodeoxycholate; TC, taurocholate; taurodeoxycholate; TCDC, taurochenodeoxycholate; GUDC, glycoursodeoxycholate; UDC-gl, ursodeoxycholate glucuronide. Other minor bile salts Tere not considered. ND: not detected. Significantly different from UDC (0.3 a Significantly different from controls (p.< 0.05). umollmin per 100 g body wt)-infused rats (p.c 0.05). ’ Significantly different from UDC (0.6 umollmin per 100 g body wt)-infused rats (p.< 0.05). d Significantly different from UDC (0.9 umollmin per 100 g body wt)-infused rats (p.< 0.05). when a similar dose of the unconjugated bile salt was administered. excreted without undergoing biotransformation

In addition, unlike UDC, TUDC was

Bilirubin excretion The effects of different doses of UDC on plasma concentration and biliary excretion of bilirubin are depicted in Fig. 1. In control rats, administration of unconjugated bilirubin caused a progressive increment of bilirubin excretion, which reached a maximum value at 30 min, remaining then stable until the end of the infusion period. Z’m condition was confirmed by plasma concentration data, since further increments of bilirubin plasma levels were recorded even though the excretory steady condition had already been reached. Administration of UDC caused a dose-dependent inhibition of bilirubin excretion and a concomitant increase of plasma bilirubin levels (Fig. 1). The changes observed in biliary excretion ranged from no effect at 0.3 umol/min per 100 g body wt to a virtual excretory blockage at an infusion rate of 1.2 umol/min per 100 g body wt. At those doses in which the inhibitory effect was apparent, bilirubin excretion began to decline soon after the highest output had been reached. Unlike UDC, TUDC had no inhibitory effect on biliary excretion of total bilirubin at infusion rates as high as 1.2 umol/min per 100 g body wt (Fig. 2). Instead, there was a trend toward an increase in plasma

Vol. 57, No. 10, 1995

c .g

z c P ;z

Ursodeoxycholate

1.2-

UDC (1.2) UDC (0.9) UDC (0.6) UDC (0.3) CONTROL

0.9 06-

0.3 E I h

977

50-

CONTROL UDC (0.3)

4o

UDC (0 6)

zg 5 s” 5 : ;, b 8 ;” gg

5

and Bilirubin Tm

3o 20-

= %

o0

10

20

30

40

50

UDC (0.9) UDC (1 2)

lo O0

60

10

Time (mm)

20

30

50

40

60

Time (mm)

Fig. 1

Sequential

changes in plasma bilirubin concentration and biliary bilirubin excretion in control rats without bile salt administration (0, n = 6) and in rats receiving UDC at single i.v. doses of 4, 8, 12 or 16 f_!mol per 100 g body wt, followed by i.v. infusions of 0.3 (0, n = 4), 0.6 (0, n = 4), 0.9 (0, n = 5) and 1.2 (A, n = 5) umollmin per 100 g body wt, respectively. Bilirubin was administered at a single i.v. dose of 3.42 nmol per 100 g body wt, followed by an i.v. infusion of 0.256 umollmin per 100 g body wt. Basal bilirubin outputs were 0.21 f 0.02, 0.23 f 0.04, 0.20 f 0.03, 0.21 f 0.03 and 0.20 f 0.02 nmollmin per g liver wt, respectively. Values are mean f SD. bilirubin concentration with controls.

in the treated group, but the differences were not statistically

significant,

compared

Bilirubin metabolism Table II summarizes the effects of UDC and its taurine conjugate on bilirubin levels and composition in plasma, liver and bile by the time of the sacrifice. As indicated above, plasma bilirubin levels increased as the dose of UDC augmented. Direct assessment of the different plasma bilirubin fractions showed that such an increment was mainly accounted for by conjugated pigment, as indicated by the dose-dependent enhancement of the conjugated-to-total bilirubin ratio. Liver content of total bilirubin also increased in a dose-dependent manner. Again, no effect was observed at the infusion rate of 0.3 umohmin per 100 g body wt. Hepatic accumulation of the pigment was concomitant with a marked increase in both the fractional amounts of conjugated bilirubin and the BDC-to-BMC ratio. Unlike what happened in liver. BDC-to-BMC ratio decreased in bile, compared with controls, A dissociation of this parameter between liver and bile

TUDC (1,2) CONTROL

0

10

20

30

40

Time (min)

50

0

60

Fig. 2

10

20

30

40

50

60

Time (mm)

Sequential changes in plasma bilirubin concentration and biliary bilirubin excretion in control rats without bile salt administration (*, n = 6) and in rats receiving TUDC (0, n = 4) at a single i.v. dose of 16 f.imol per 100 g body wt. followed by an i.v. infusion of 1.2 umol/min per 100 g body wt. Bilirubin was administered at a single i.v. dose of 3.42 umol per 100 g body wt. followed by an i.v. infusion of 0.256 pmollmin per 100 g body wt. Basal bilirubin outputs were 0.21 f 0.02 and 0.24 f 0.02 nmollmin per g liver wt in control and TUDC-infused rats, respectively. Values are mean f SD.

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Table II. Plasma,

Liver and Bile Levels and Composition

of Bilirubins TUDC

in Rats following

Controls Infusion rate (~molhnin per 100 g body wt)

__ (it = 6)

Administration

of UDC or

UDC

TUDC

0.3 (n = 4)

0.6 (n = 4)

0.9 (?I = 5)

1.2 (!I = 5)

1.2 (II = 4)

Plasma (55 min) Bilirubin concentration (mM)

0.68 + 0. IO

0.77 f 0.07

0.89 f 0.02”b

1.04 k 0.06”bc

1.10 k0.03”bc

0.77 f 0.12

Conjugated-to-total ratio

0.14 * 0.03

0. I 1 f 0.02

0.22 AZ0.05”

0.26 Y+O.O+’

0.30 + 0.08””

0 IO * 0.05

0.70 kO.15

0.70 * 0.09

0.56 f0.18

0.69 f 0.1 I

0.80 f 0.22

0 5 I f 0.25’

0.66 f 0. IO

0.60 + 0.02

0.81 +O.l I”

1.03 f o.04’b

I I6 + 0.02”“’

0.66 ?r 0.06

0.47 k 0.09

0.39 f 0.03

0.59 * 0.07”b

0.61 f 0.07”b

0.56 k 0.02”b

0.3

0.60+0.13

0.53 f 0. IO

0.58kO.15

0.86 + 0.13abc

0.93 + 0.2 lvbc

0 39 + 0.05”

Bilirubin output (nmol/min per g liver wt)

47.1 + 4.4

47.2 f 2 8

36.0 + 4.9’b

6.9 + 3.5”“”

2.3 f 0.7”“C”

48. I t 3.4

BDC-to-BMC ratio

0.67 + 0.08

0.55 f 0 09&

0.62 + 0.06

0 5I

0.51 k 0.07”

0.56 i 0.09”

bilinlbin

BDC-to-BMC ratio Liver Bilirubin content (pm01 per g liver wt) Conjugated-to-total ratio

bilirubin

BDC-to-BMC ratio

I f o.08a

Bile (SO-60 min)

f 0.07”

Values correspond to data obtained by the end of the experiment and are mean f Bilirubin, UDC and TUDC were administered as indicfted in Table I. a Significantly different from controls (p.< 0.05). Significantly different from UDC pmol/min per 100 g body wt)-infused rats (p.< 0.05). ’ Significantly different from UDC umollmin per 100 g body wt)-infused rats (p.< 0.05). d Significantly different from UDC pmollmin per 100 g body wt)-infused rats (p.c 0.05) became apparent at the two highest doses employed, where proportion of the diconjugates decreased despite BDC-to-BMC ratio in liver increased.

SD. (0.3 (0.6 (0.9

excreted into bile

TUDC had a rather different effect on bihrubin metabolism, compared with UDC. Whereas hepatic bilirubin content was not affected by TUDC, a significant decrease in both conjugated-to-total bilirubin ratio and BDC-to-BMC ratio was observed. This last parameter also decreased in bile in this experimental group. Discussion The results of the present study clearly showed that UDC, but not its taurine conjugate, impairs bilirubin transport in the rat. Our results agree well with the preliminary observation by Gahin et al. (7) in that high doses of this bile salt are required to induce this inhibitory effect. Instead, our data differed from theirs in that no stimulation of bilirubin transport could be assessed even when a relatively low dose of UDC was administered. Our protocol of UDC administration was, however, somewhat different from that employed by these authors. Whereas a priming i.v. dose of UDC (conjointly with that of bilirubin) was administered by us, Gal&r et al. preloaded the hepatocyte with the bile salt previous to bilirubin administration by means of an i.v. infusion. Acute preloading of this bile salt as employed here appears to be critical in accounting for this difference since in ancillary experiments in which the initial bolus was omitted and UDC was infused at the doses of 0.3 pmol/min per 100 g body wt, a significant increase of bilirubin Tm could be observed (+12%, p cO.025; data not shown). Taking into account our goal, however, we preferred to adopt the first approach because it has the advantage of allowing molar administration ratio of bile salts, bilirubin and bovine serum albumin to be uniform throughout the experiment. Since a displacement of the binding of bilirubin to albumin by the bile acids is expected to occur in the administration mixture (28) this

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approach ensures the administration of a constant unbound bilirubin fraction over the experiment. This is important since this fraction is believed to govern hepatic uptake of non-bile-salt organic anions (29). Our results that biliary excretion was inhibited with a concomitant enhancement of the hepatic content and intrahepatic conjugation of the pigment (see Table II), clearly suggests that a blockage of its transfer at the canalicular level occurs during UDC administration. Indeed, bilirubinostasis induced by different causes, including coadministration of competitors for canalicular carriers (30) transitory cholestasis (3 I), colchicine administration (32) or genetic defect of bilirubin excretion (33) resulted in a more efficient conjugation of the pigment. This finding, however, do not exclude the possibility that, in addition, UDC may partially inhibit bilirubin conjugating enzyme activity, as should be expected to occur based on preliminary studies in viva (7) and in vitro (8, 34). In line with this possibility, TUDC, which was shown to have a similar inhibitory effect to that of UDC when evaluated in vitro (34) induced a significant decrease in both the fractional amount of bilirubin conjugates and the BDC-to-BMC ratio in the liver (see Table II). This alteration, which suggests an inhibition of in vivo bilirubin conjugation, was however not enough to affect biliary bilirubin output at 7’111. This concurs with the previous observation that different situations conducing to a moderate impaimrent of bihrubin conjugation, capable of modifying the conjugation pattern of the pigment, do not decrease bilirubin Tm (18). Based on these considerations, it is reasonable to suppose that an inhibitory effect on conjugating activity was also induced by UDC, but the parallel increment in the efficiency for bilirubin conjugation induced by its intrahepatic retention could have masked this effect. Reversion of the polarity for bilirubin excretion induced by its excretory blockage contributes to explain the dose-dependent increment in plasma. The finding that conjugated rather than non-conjugated bilirubin mainly contributed to this increment (see Table II), strongly supports this view. In addition, both bile salts apparently tended to interfere with hepatic uptake, as suggested by the somewhat higher value of plasma concentration in absence of excretory impairment, i.e. in UDC- and TUDC-infused rats at rates of 0.3 and 1.2 umol/min per 100 g body wt, respectively. In line with this view, enhancements of plasma bilirubin levels in these circumstances were fully produced at the expense of unconjugated bilirubin, thus suggesting a blockage of its transfer at the sinusoidal level. Interestingly, a similar trend was previously reported for sulfobromophthalein (35, 36) an organic anion that shares the same uptake system with bilirubin (37) and confirmed by in vitro competition studies (38). Interference with bihrubin uptake, however, was not enough to decrease biliary excretion of the pigment. Thus, such an effect, if present. appears to affect minimally the overall hepatic transport of the pigment. at least under Tm conditions. The pattern of bilirubin conjugates in bile did not reflect that of the liver since BDC-to-BMC ratio decreased rather than increased during UDC administration (see Table II). A possible explanation for this unexpected observation is that UDC may impair preferentially BDC canalicular excretion, Indirect support for this view comes from the study of Ricci el al. (39) who found that glucagon administration leads to a preferential excretion of BMC, thus suggesting that BMC and BDC excretion may be influenced in an independent manner by modulating agents. Confirmation of this hypothesis awaits, however. more information regarding the nature of this process. A relevant result of the present study was the finding that whereas UDC impaired biliary bilirubin output, its taurine conjugate had no inhibitory effect on this parameter. A similar differential effect has been reported for sulfobromophthalein after infusion of high doses of these bile salts (35). In that study. the authors reported that following an initial stimulatory effect, UDC but not TUDC induces a subsequent inhibition of sulfobromophthalein excretion (35). Reevaluated at light of the present results. this impaimrent may be interpreted as an subsequent blockage of the transport system common to both sulfobromophthalein and bilirubin. which is elicited when UDC fails to be conjugated with taurine. Differences in bihary composition of bile salts during UDC and TUDC administration may help to find the possible mediators of the inhibitory effect induced by the former. TUDC was neither deconjugated nor glucuronidated during its transit throughout the hepatocyte (see Table I). On the other hand, UDC failed

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to be conjugated with taurine as the dose augmented. Instead, alternative excretory products appeared in bile, namely UDCgl, GUDC and unconjugated UDC, in agreement with previous studies (16, 24). Among them, UDC-gl and unconjugated UDC are likely involved in the observed effect, as suggested on the basis of previous studies. Indeed, UDCgl is expected to compete with bilirubin glucuronides since these compounds appear to share the same canalicular transport system (24, 40). On the other hand, unconjugated UDC, but not its taurine derivative, inhibited P-glycoprotein-mediated drug transport in canalicular membrane vesicles (41). A similar inhibitory effect, which may involve either a direct effect on the transport protein or a disturbing effect on membrane environment, may account for our results. Interestingly, free UDC was suggested to insert deeper than TUDC within membranes (42). Consequently, this difference in membrane disturbing properties between both bile salts may be important in determining their dissimilar effects on membrane transport systems. In conclusion, this study provides evidence that UDC, but not TUDC, interferes with the hepatobiliary transport of bilirubin by impairing its transfer at the canalicular level. Incomplete taurine conjugation, which occurs when high doses are administered, appears to be a key factor in the inhibitory effect induced by the former. Although other additional effects of these bile salts on both hepatic uptake and intrahepatic conjugation were also apparent, they do not seem to have more than a marginal importance in the overall transport of the pigment. Our way of UDC administration (an acute i.v infusion preceded by a high priming dose) is totally different from what is adopted in patients, who receive UDC orally in a much smaller dosage. Therefore, caution may be exercised in extrapolating any clinical inference from the present data. Nevertheless, we believe that our results may be relevant to the understanding and prevention of potential undesirable effects of UDC on bilirubin depuration during therapy with this bile salt. Acknowledgments This study was supported by a Research Grant from Consejo National de lnvestigaciones Cientificas y TCcnicas (CONICET), Argentina. We thank Dr. Ralil A. Marinelli and Dr. Emilio A. Rodriguez Garay for their critical review of the manuscript. References I. 2. 3. 4. 5.

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