Elevation of the serum bilirubin diconjugate fraction provides an early marker for cholestasis in the rat

Journal of Hepatology 1991; 27: 912-916 Printed in Denmark All rights reserved

Munksgaard

Copyright 0 European Association for the Study of the Liver 1997

’ Copenhagen

Journalof Hepatology ISSN 0168-8278

Elevation of the serum bilirubindiconjugate fraction provides an early marker for cholestasis in the rat Vital A. Mesa, Rita De Vos’ and Johan Fevery Laboratory of Hepatology, and ‘Histopathology and Cytology, Department of Medical Research, University of Leuven, Leuven, Belgium

Aims/Methods: During cholestasis, components normally excreted into bile, e.g. bilirubin, accumulate in liver cells and biliary passages. In order to assess the conjugation of bilirubin retained in the hepatocyte during cholestasis, we analyzed the pattern of bilirubin pigments in rat sermn and bile, using reversed phase alkaline methanolysis-HPLC. Cholestasis was induced by bile flow interruption for 1 to 2 h. Results: One hour after initiation of cholestasis, the serum concentration of total bilirubin rose 2-fold due to increases in bilirubin di-(BDC) and monoconjugate (BMC), while unconjugated bilirubin (UCB) decreased by 33%. As a result, the BDCYBMC ratio increased to 1.6720.20 vs 060~0.10 in controls (~~0.01) and the BMCAJCB ratio to 1.020.2 vs O.l+O.l QKO.01). After relief of biliary obstruction, vs 5.5kO.3 biliary output rose to 8.0~0.5 ~01 - min-’ - kg (p
trast, the biliary BMCKJCB ratio remained unchanged throughout. Increasing the duration of obstruction to 2 h led to a further increase in the sermn BMC/UCB ratio to 2.220.3 (p
M

findings strongly suggest that the slowing down of the intrahepatic transit of bilirubin pigments allows more efficient formation of BDC from the intermediate BMC (8,9). If this hypothesis is correct, the pattern of bilirubin pigments should be altered whenever the pigments are retained in the liver, regardless of the aetiology. We have analysed the effect of short-term (l-2 h) bile-duct mechanical obstruction on the pattern of bilirubin pigments in the rat serum and bile. The data obtained suggest that cholestasis favours a more efficient conjugation of bilirubin into BDC, the end-product of conjugation in rats.

excrete bilirubin in bile mainly as bilirubin mono-(BMC) and diconjugates (BDC), whereas several other species such as the sheep and guinea pig excrete only BMC (l-3). Formation of BDC from UCB presumably proceeds in two sequential steps (4) whereby the BMC formed initially is thereafter converted to BDC (5-7). The factors that modulate the relative formation rates of BDC and BMC, and hence the pattern of bilirubin conjugates excreted in bile remain ill defined. It has been shown that cholephilic organic anions that share the biliary excretory pathway of bilirubin conjugates significantly increase the BDCYBMC ratio in rat serum and bile (8,9). Similarly, the BDCYBMC ratio is higher in the TR-mutant rat characterized by a defective biliary transport of organic anions (10). Taken together, these AN AND RAT

Key words: Bilirubin diconjugate (BDC); Bilirubin monoconjugate (BMC); Cholestasis; Liver test; Ratio.

Materials

and Methods

Animals Received 9 December 1996; revised 27 May: accepted 4 June 1997

Correspondence: Prof J. Fevery, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Fax: 32-16-34 43 87.

912

Non-fasted male Wistar rats (280-300 g body weight), bred at the KU Leuven animal house were used throughout. They were maintained on a standard lab-

Cholestasis favours bilirubin conjugation

oratory diet and water ad libitum. Under ether anaesthesia, a jugular vein catheter and a 30-cm biliary cannula (inner diameter 0.7 and 0.3 mm, respectively, Biotrol, Paris, France) were inserted. Post-operatively, animals were put in restraining cages and kept in thermostatically-controlled chambers. When they had regained consciousness and a constant body temperature between 37.5 and 37.8”C, biliary obstruction was induced for a given period of time by elevation of the distal portion of the biliary cannula until hydrostatic pressure counteracted bile flow (20-21 cmH*O). When bile flow was re-established, the stagnant bile contained in the biliary cannula was discarded, and then the darker post-obstructive bile was collected in one 5-min sample, in the dark, into tared tubes cooled on ice. Sham-operated animals, whose bile flow was diverted for l-2 h served as controls. Preliminary experiments had shown that diversion of bile flow for 2 h did not alter the pattern of bilirubin pigments in either bile or serum. At the end of the experimental period, blood was collected at the bifurcation of the abdominal aorta of some of the animals, under light ether anaesthesia. Serum was promptly separated and stored at -20°C for use within 4 days. The liver was removed and pieces were taken for electron microscopy. Analytical methods Bile samples were analysed immediately after collection. Bile volume was determined gravimetrically, without correction for specific density. Concentrations of bilirubin, and its mono- and diconjugates were determined using reversed phase alkaline methanolysisHPLC (11). The hepatic bilirubin UDP-glucuronyl transferase activity was assayed according to Van Roy & Heirwegh (12), and the serum levels of total bile acids by means of an enzymatic fluorimetric method (Sterognost 3-alpha Flu, Nyegaard and Co, AS, Oslo, Norway), according to Mashige et al. (13). This method measures bile acids and other steroids with a 3-a-hydroxy group. The following liver enzymes were assayed using an automated method (SMAC Corp): alkaline phosphatase (EC 3.1.3. l), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) amino transferases. Protein was estimated using the method of Lowry et al. (14). Statistics Results are expressed as mean?SE. The significance of differences among the groups was analyzed by the Mann Whitney U-test, and p-values of 0.05 or less were considered statistically significant. Correlations were examined with Spearman’s rank correlation test.

Results Bilirubin pigments in rat serum and bile One hour after the onset of biliary obstruction, total serum bilirubin increased markedly due to elevation of the conjugated fraction. In contrast, the absolute amount of UCB decreased by 33% (Table 1). Concomitantly, the serum BDC/BMC and BMC/UCB ratios increased 3- and lo-fold above their respective control values. Prolongation of the obstruction to 2 h further increased the serum total bilirubin, while the absolute and the relative amounts of UCB dropped by 44 and 86%, respectively. The BMCYUCB ratio rose 22-fold, while the BDC/BMC ratio remained almost unaltered (Table 1). In an additional set of experiments, the serum BDCYBMC ratio was shown to increase more substantially when interruption of bile flow was prolonged to 72 h (2.2820.23, n=4). After relief of the biliary obstruction, bile flow increased slightly, while the biliary output of BDC increased by 93 and 145%, after 1 or 2 h obstruction (Table 2). BDC then comprised 78 to 80.3% of the total pigments, vs 59% in control bile. By contrast, biliary output of UCB and BMC did not change significantly

TABLE

1

Bihrubin

pigments

in the rat serum Control

Total pigment @M) UCB t$M) BMC @M) BDC t&M) BMCRJCB ratio BDCiBMC ratio

(n=9)

1.06+0.02 0.9kO.2 0.1+0.1 0.06+0.1 0.11+0.10 0.6OirO.10

OBl (n=8)

0B2 (n=8)

2.2+0.3* 0.620.2 0.6+0.2* 1.00+0.20* 1.00+0.20* 1.67?0.20*

3.5co.3* 0.5?0.2* 1.1*0.2* 1.90?0.20* 2.20?0.30* 1.73?0.21*

Blood was collected from rats whose bile flow was hampered for 0 (control), 1 (OB l), or 2 h (OB 2). Concentrations of bilirubin pigments were determined in serum by means of the alkaline methanolysis-HPLC procedure. Mean value&SE are given. *p
TABLE

2

Bilirubin output and relative amounts jugates in the post-obstructive bile Control Bilirubin output pmol . min-’ . kg UCB BMC BDC BMC/UCB ratio BDCYBMC ratio

5520.3 O.l?O.l 2.16kO.24 3.2420.24 21.6k4.5 1.5kO.2

(n=9)

of bilirubin

OBl (n=8) 8.00t0.4* O.l?O.l 1.55co.35 6.25+0.25* 15.5k4.0 4.0*0.3*

di- and monocon-

OB2 (n=8) 9.9t0.4* O.l?O.l 1.85kO.22 7.95+0.33* 18.5k4.0 4.3+0.3*

After cholestasis had been relieved, the content of the biliary cannula was discarded, and bile samples were then collected in the dark. Concentrations of total and individual bilirubin pigments were determined using reverse-phase HPLC. *p
913

V. A. Mesa et al. TABLE

3

Serum activities

of some liver enzymes Control

BA (mmolil) ALT (mu/ml) AST (mu/ml) AP (mu/ml)

(n=9)

822 3524 2323 16?4

and total bile acids OBl (n=8)

0B2 (n=S)

870?34* 1302 10* 79?8* 1724

965?45* 134*10* 82?8* 1524

Blood was collected from rats whose bile flow was stopped for 0 (control), 1 (OB l), or 2 h (OB 2). Enzyme activities and concentrations in total bile acids were assayed in serum as stated in the analytical methods section. Mean value&SE are given. *p
during the experimental period. As a consequence, the biliary BDC/BMC ratio increased almost 3-fold, while the BMC/UCB ratio remained unchanged. The absolute and relative amounts of BDC increased further, however slightly, when biliary obstruction was prolonged to 2 h (Table 2). To ascertain that the obse&d changes were not related to alterations in the hepatic UDP-glucuronyl transferase, enzyme activity was assayed in vitro. It averaged 300t20 nmoles of bilirubin conjugated per min per g of protein in both control (n=8) and biliary-obstructed rats (n=6).

among bilirubin pigments towards BDC, the end-product of bilirubin conjugation in rats (l-3,5). Studies in the rhesus monkey suggest that synthesis of BDC is a lower-capacity, higher-affinity step than formation of BMC from UCB (16). Should the same hold for rats, increasing the reaction time might conceivably favour formation of BDC. Indeed, rat liver homogenates or microsomal preparations synthetise relatively more BDC than BMC as the reaction time increases (17). Under physiological conditions, UCB is taken up by the liver for conjugation into BMC and then partly into BDC. A fraction of the conjugated pigments, in turn, undergoes deconjugation into BMC and UCB. Given the rapid equilibrium achieved between the hepatic and plasma bilirubin compartments, a small frac-

c 7

a

aerwll

UCB kepatocyte

A

tr

t

k~c

! . . . . . .._.. ;BD;

Other liver tests

One hour after the biliary obstruction, serum aminotransferases and levels of total bile acids rose 3- and lOO-fold above their respective control values (Table 3). In contrast, the activity of the alkaline phosphatase remained unchanged throughout. The enzymes assayed did not differ significantly among the l- and 2-h biliary-obstructed rats. Electron microscopical findings

Livers of control and l-h obstructed rats had normal ultrastructure. Changes indicative of cholestasis became obvious after 2 h of obstruction. In hepatocytes of zone 3 (centrilobular) the canaliculi were dilated, the number of their villi was reduced, and their lumina presented pronounced irregularity (not shown).

Discussion The present study has documented a rapid and significant increase in BDC and a lesser increase in BMC during short-term cholestasis, while UCB decreased markedly. As a result, serum BDC/BMC and BMC/ UCB ratios rose significantly. This is reminiscent of the observations made in dye-infused rats (8,9), the TR-mutant rat (10) and in man with the Dubin-Johnson syndrome (15). Taken together, these data suggest a more efficient conjugation of bilirubin when retained in the hepatocyte. This causes a shift of the equilibrium 914

bile

v

v

eemn

iepatocyte

bile

Fig. I. Schematic representation of the dynamic equilibrium among bilirubin pigments, a) in the normal state, and b) during cholestasis. a) In the normal state, UCB is taken up by the liver to become conjugated by glucuronyl transferase (GT) into BMC and then BDC. A minor fraction of the conjugated pigments, in turn, undergoes deconjugation into BMC and UCB. Given the rapid equilibrium achieved between the hepatic and plasma bilirubin compartments, a fraction of UCB, BMC and BDC refluxes into plasma. b) During cholestasis, BMC and BDC are not excreted; they reflux into serum, more so for BDC than BMC. In the hepatocyte, BMC builds up in the immediate vicinity of the conjugating enzyme system, allowing more BDC to be formed. As UCB becomes more efJiciently conjugated, its re$ux decreases and equilibrium among the bilirubin pigments is shifted towards BDC.

Cholestasis favours bilirubin conjugation

tion of UCB (18), BMC and BDC (19) refluxes into plasma (Fig. la). Whenever biliary excretion of bilirubin conjugates is delayed (Fig. lb), more BMC builds up in the vicinity of the conjugating enzyme system, allowing more BDC to be formed. As a result, equilibrium among the bilirubin pigments is shifted towards BDC, as borne out by the marked elevation of the serum BMCXJCB and BDC/BMC ratios. The fact that the decrease in the serum concentration of UCB was concomitant with the increase in the BDC/BMC ratio supports this hypothesis (Table 1). Indeed, the serum level of UCB depends directly on the pigment formation rate, and the hydrolysis rate of bilirubin already conjugated (18,20). It is inversely related to the hepatic uptake and conjugation of the pigment (20). It seems unlikely that bilirubin formation and/or its hepatic uptake have been altered significantly in the present experimental design. Since bile flow was completely prevented, bile contents, e.g. bilirubin pigments and bile acids, refluxed into serum (Table 1). Elevation of the serum concentration of bilirubins, however, is not specific to cholestasis. What seems to be more specific is the elevation of the BMCYUCB and BDC/BMC ratios, which also occurs in dye-infused rats (8,9), in the TR-mutant rat (lo), and in the Dubin-Johnson syndrome (15). We therefore suggest that such a shift in equilibrium among bilirubin pigments towards BDC provides an early marker for cholestasis in the rat. Enhancement of the serum-conjugated bilirubin fraction has already been shown to be a sensitive marker for chronic lowgrade cholestasis in man (21). The origin of the minute amounts of UCB found in bile is unknown. It has been shown recently that interaction of bile salts with UCB increases the aqueous solubility of this pigment, more so as the concentration of bile salts increases (22). Conceivably, such an increased aqeuous solubility might favour UCB excretion into the post-obstructive bile. Indeed, the serum level of bile acids increased sharply during biliary obstruction (Table 3) in agreement with reports by other authors (23). However, as observed previously (8,9), UCB was linearly related to BMC, in both control (r,=0.97) and post-obstructive bile (r,=0.96) QKO.01, n=28 in each group). This suggests that UCB found in bile derives, at least in part, from BMC by hydrolysis. Furthermore, the fact that UCB was linearly related to BMC also explains why the biliary BMCKJCB ratio remained unchanged throughout (Table 2). Elevation of the serum activity of aminotransferases, generally viewed as indicative of cell necrosis, occurred in rats during acute biliary obstruction. Similarly, peak

activities above 300 IU have been described in man with acute biliary tract obstructions (24), and have been ascribed to an increased cell permeability. The increase in serum activity of alkaline phosphatase results from induction of the hepatic enzyme activity (25,26). This suggests that elevation in the serum activity of this enzyme is a feature of chronic rather than acute cholestasis (24) and therefore explains our finding of unchanged activity under the present experimental design (Table 3). In summary, an increase in the BDC/BMC ratio is a rapid consequence of cholestasis, presumably as a result of a more efficient conjugation of bilirubin. Elevation of this ratio provides an early marker for cholestasis.

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