Separate transport systems for biliary secretion of sulfated and unsulfated bile acids in the rat.

Biliary secretion of 3 alpha-sulfated bile acids has been studied in Wistar rats with an autosomal recessive defect in the hepatic transport of bilirubin. Liver function, established by measurement of various enzymes in plasma, by enzyme histochemical methods, and by electron microscopy, appeared to be normal in these rats. Serum levels of unconjugated, monoglucuronidated, and diglucuronidated bilirubin were 0.62, 1.62, and 6.16 mumol/liter, respectively, compared with 0.17, 0.08, and 0.02 mumol/liter in control rats. Biliary bilirubin secretion was strongly reduced in the mutant animals: 0.21 +/- 0.03 vs. 0.39 +/- 0.03 nmol/min per 100 g body wt in control rats. Despite normal biliary bile acid output, bile flow was markedly impaired in the mutant animals, due to a 53% reduction of the bile acid-independent fraction of bile flow. The transport maximum for biliary secretion of dibromosulphthalein (DBSP) was also drastically reduced (-53%). Biliary secretion of intravenously administered trace amounts of the 3 alpha-sulfate esters of 14C-labeled taurocholic acid (-14%), taurochenodeoxycholic acid (-39%), taurolithocholic acid (-73%), and glycolithocholic acid (-91%) was impaired in the jaundiced rats compared with controls, in contrast to the biliary secretion of the unsulfated parent compounds. Hepatic uptake of sulfated glycolithocholic acid was not affected in the jaundiced animals. Preadministration of DBSP (15 mumol/100 g body wt) to normal Wistar rats significantly impaired the biliary secretion of sulfated glycolithocholic acid, but did not affect taurocholic acid secretion. We conclude that separate transport systems in the rat liver exist for biliary secretion of sulfated and unsulfated bile acids; the sulfates probably share secretory pathways with the organic anions bilirubin and DBSP. The described genetic defect in hepatic transport function is associated with a reduced capacity to secrete sulfated bile acids into bile; this becomes more pronounced with a decreasing number of hydroxyl groups on the sulfated bile acid's molecule.

Dibromosulphophthalein: its pharmacokinetics and binding to hepatic cytosol proteins in rats with acute renal failure.

1. The pharmacokinetics, biliary excretion and binding of dibromosulphophthalein (DBSP) to plasma proteins and hepatic cytosol proteins have been studied in male rats with glycerol-induced acute renal failure (ARF). 2. The rate constants for hepatic uptake, efflux from liver to plasma and excretion into bile were all significantly decreased in rats with ARF. Furthermore, the plasma clearance of DBSP was also reduced. 3. The initial (0-10 min) and maximum biliary excretion rates of DBSP were both diminished in animals with ARF. The maximum excretion rate occurred between 5-10 min in control rats and 10-15 min in rats with ARF. However, there was no statistically significant change in the percentage dose recovered from bile after 30 min. 4. The plasma-protein binding of DBSP was decreased in rats with ARF and this change was due to a significant reduction in the association constant for the primary binding sites. 5. The binding of DBSP to ligandin (Y protein) was reduced by about 38% in rats with ARF but no change was noted in binding to Z protein. Reduced binding to ligandin was accompanied by decreased total liver glutathione S-transferase (GST) activity and a 36% reduction in the GST activity of ligandin. 6. The results support the contention that altered hepatic handling of cholephilic dyes in rats with ARF may be due to reduced binding to ligandin.

Mechanistic aspects of uptake and sinusoidal efflux of dibromosulfophthalein in the isolated perfused rat liver.

Using the isolated perfused rat liver technique we examined whether the accumulation and sinusoidal efflux processes of the organic anion dibromosulfophthalein (DBSP) are dependent on the intracellular ATP content, chloride concentration in the perfusion medium as well as temperature of the medium and whether they are mediated by the same or by separate carrier mechanisms. The net sinusoidal efflux rate, being the resultant of sinusoidal efflux and re-uptake, was decreased more than 50% after lowering the medium temperature from 37 to 26 degrees indicating that the efflux process is carrier-mediated. The uptake rate was decreased only 18% after lowering the medium temperature to 26 degrees. Lowering of the hepatic ATP content for more than 80% clearly decreased the DBSP uptake rate but not the sinusoidal efflux rate. These observations indicate that these opposing transport steps probably are mediated by two separate carriers. Additional evidence for this hypothesis originated from the observation that sinusoidal efflux of DBSP was decreased about 30% whereas hepatic uptake of the substrate remained unaltered after replacing chloride in the perfusion medium with gluconate. In summary, we conclude that uptake and sinusoidal efflux of DBSP are mediated by two separate carrier systems that are influenced differently by ATP depletion, temperature lowering and presence of Cl-gradients.

Difference in hepatic uptake of tetra- and di-bromosulfophthalein in rat. Role of hydrophobicity, binding to plasma proteins and affinity for plasma membrane carrier protein.

The relative role of hydrophobicity, binding to plasma proteins and affinity for one of the plasma membrane transport proteins in the hepatic uptake of 3,4,5,6-tetra- (BSP) and 3,6-di- (DBSP) bromosulfophthalein was investigated in the rat. In terms of physicochemical characteristics, the two molecules show different pKa values and degrees of hydrophobicity, as determined from the n-octanol:water partition coefficient. In the intact animal, the plasma clearance and the plasma removal rate after a dose of 1.5 mumol/kg i.v. were significantly (P < 0.001) faster for BSP than DBSP, while no difference was found in the plasma distribution volume. The dissociation constant (Kd) of the high affinity binding sites of plasma proteins also differed for the two anions, being significantly lower for BSP than DBSP (0.95 +/- 0.02 vs 1.44 +/- 0.14 microM, P < 0.001). [35S]BSP uptake by liver plasma membrane vesicles was saturable with an apparent Km of 5.20 +/- 0.80 microM, and was competitively inhibited by DBSP (Ki 18.2 +/- 1.2 microM) indicating a common uptake system. The Kd value for binding of the organic anions to purified bilitranslocase, a plasma membrane protein involved in the electrogenic transport of pthaleins, was also significantly lower for BSP than DBSP (1.10 +/- 0.12 vs 3.02 +/- 0.27 microM, N = 3, P < 0.001), indicating a higher affinity of the former ligand for the carrier protein. No difference was observed in the capacity of the high affinity binding sites (32 +/- 3 vs 33 +/- 3 nmol/mg protein, BSP and DBSP, respectively). These data indicate that BSP and DBSP are two different cholephilic organic anions which share a common uptake mechanism, at least partly mediated by bilitranslocase. The greater affinity of BSP than DBSP for the carrier protein may account for the faster plasma disappearance rate of BSP observed in vivo, in spite of the higher plasma protein binding.

Hepatic drug transport in the rat. A comparison between isolated hepatocytes, the isolated perfused liver and the liver in vivo.

The hepatic transport of three different drugs, the organic anion dibromosulphophthalein, the organic cation d-tubocurarine and the uncharged compound ouabain was studied in vivo in the isolated perfused rat liver and isolated hepatocytes. The respective clearances by uptake were determined for the various substrates and corrected for differences in hepatic blood flow and extracellular protein binding in the three liver preparations. The corrected uptake values in the intact organ, in vivo and in the isolated perfused liver were highly comparable; for dibromosulphophthalein a clearance of 2.1 ml/min per 10(6) hepatocytes was found in vivo, whereas in perfusion a value of 2.4 ml/min per 10(6) cells was calculated. For d-tubocurarine, the values were 34 x 10 (-4) and 55 x 10(-4) ml/min per 10(6) cells obtained in vivo and in the isolated perfused organ, respectively. With ouabain as the substrate, the in vivo clearance amounted to 5.1 x 10(-2), whereas in the isolated perfused liver a value of 4.8 x 10(-2) ml/min per 10(6) cells was calculated. The clearance by uptake obtained for dibromosulphophthalein was ouabain in the isolated hepatocytes appeared to be a factor of 2-3 lower than in the intact organ. In the case of d-tubocurarine however the clearance was identical to that in vivo and the isolated perfused liver. The rate of secretion from isolated hepatocytes was, for dibromosulphophthalein identical to, and for d-tubocurarine and ouabain lower than that in the intact organ, especially as compared with the in vivo preparation. It is concluded that transport function is well preserved in the isolated perfused liver and isolated hepatocytes. For certain substrates freshly isolated hepatocytes may exhibit a somewhat lower uptake and/or secretion rate, in spite of a good cell quality as judged by generally accepted criteria for cell viability. Whether this is due to changes in membrane composition (not detected by our viability tests) or a selection of a subpopulation of hepatocytes, is discussed.U

Secretion of the organic anion harmol sulfate from liver into blood. Evidence for a carrier-mediated mechanism.

In the liver, drugs with phenolic groups can be converted to sulfate or glucuronide conjugates and are then transported into bile or back into the bloodstream. The mechanism for transport of drugs and drug conjugates from the hepatocytes into the blood at the sinusoidal side of the cell are not well defined. In the case of carrier-mediated transport of these strongly polar conjugates, saturability of transport and mutual competition between structurally related compounds would be anticipated. This competitive aspect was investigated by using two organic anions, dibromosulfophthalein (DBSP) and harmol sulfate. The latter compound was generated by the hepatocytes from harmol, which was continuously infused into the rat in vivo and in isolated perfused rat livers. In addition we loaded the perfused rat livers with preformed harmol sulfate and studied its efflux rate to the perfusate as influenced by DBSP. In steady state, about 80% of harmol was sulfated and 20% was glucuronidated. Harmol sulfate was mainly excreted in the urine, the glucuronide was equally excreted in urine and bile. DBSP lowered the urinary excretion of harmol sulfate by about 30% which was due to a decrease in plasma concentration. However, renal clearance of harmol sulfate (3.2 +/- 0.2 ml/min) was unchanged. At the same time DBSP doubled the biliary clearance of harmol sulfate (1.0 +/- 0.1 and 2.2 +/- 0.2 ml/min in controls and DBSP studies respectively). DBSP decreased glucuronide excretion both in urine and bile. The increase in biliary output and decrease in urinary excretion of harmol sulfate is explained by competitive interaction between the organic anion DBSP and harmol sulfate at the sinusoidal level. Efflux experiments in single pass perfused isolated livers showed a clear decrease of harmol sulfate transport from liver into plasma by DBSP and provided evidence for such an inhibitory phenomenon (t 1/2 of efflux was 3.58 +/- 0.21 compared with 2.46 +/- 0.07 min for controls). These results indicate that organic anion transport from the hepatocyte into the blood stream is very likely carrier-mediated. A decrease in renal output of drug conjugates therefore may not only be due to a decrease in the conjugation process but also to a lower liver to blood transport rate which at the same time may produce a higher biliary output.

Impairment of hepatic glutathione S-transferase activity as a cause of reduced biliary sulfobromophthalein excretion in clofibrate-treated rats.

Administration of clofibrate reduced the maximal excretion rate of bile sulfobromophthalein (BSP) in rats but left that of phenol-3,6-dibromophthalein (DBSP) unchanged. This decrease in liver transport of BSP was due to reduced bile excretion of conjugated BSP. Hepatic uptake and storage of this dye were not impaired. Liver glutathione S-transferase activity in vitro, measured with BSP, 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2, 4-dinitrobenzene (CDNB) was significantly reduced. This alteration in liver conjugating activity was probably not related to a modification of the hepatic GSH pool, since the GSH level was unchanged or only increased slightly after clofibrate treatment. Detection of this inhibition required at least two daily doses of clofibrate. Inhibition was dose-related and lasted for several days after cessation of the drug. In clofibrate-treated rats, Lineweaver-Burk plots showed a reduced Vmax for both the BSP and GSH substrates. These results suggest that clofibrate decreases hepatobiliary transport of BSP by lowering glutathione S-transferase activity in the liver.

Properties of the microsomal and cytosolic glutathione transferases involved in hexachloro-1:3-butadiene conjugation.

Hexachloro-1,3-butadiene (HCBD) is a substrate for the hepatic microsomal glutathione transferases and is metabolised at higher rates by these enzymes than their cytosolic counterparts. Conjugation reactions catalysed by the microsomal and cytosolic transferases have been studied and characterized using this substrate and 1-chloro-2,4-dinitrobenzene (CDNB). In rat liver microsomes the Km values for HCBD and CDNB were 0.91 and 0.012 mM and in cytosol 0.51 and 0.10 mM respectively. Vmax values for HCBD were 1.39 and 0.35 nmol conjugate formed/min/mg protein for microsomes and cytosol respectively. In microsomal systems HCBD was a potent competitive inhibitor of the metabolism of CDNB with a Ki value of approximately 10 microM. However, CDNB did not inhibit HCBD metabolism significantly. These data suggest that more than one microsomal enzyme is involved in HCBD metabolism. The microsomal membrane could be solubilized without significant inhibition of HCBD activity; however, some detergents did inhibit the conjugation reaction. Activity was also lost on treatment of microsomal membranes with trypsin indicating the enzyme is localized on the cytoplasmic surface of the endoplasmic reticulum. Pretreatment of the rats with Aroclor 1254, 3-methylcholanthrene or phenobarbital did not change the microsomal conjugation of HCBD or CDNB with glutathione. Of seven species investigated, a human liver sample showed the highest ratio of microsomal to cytosolic glutathione transferase activity for HCBD (in microsomes 40-fold higher specific activity than in cytosol). Glutathione conjugation appears to play a critical role in the toxicity and carcinogenicity of some halogenated hydrocarbons. These data substantiate the potentially important role for the microsomal glutathione transferase in catalysing these reactions.

Excretion of sulfobromophthalein in rats with iodomethane-induced depletion of hepatic glutathione.

Male urethane-anesthetized Wistar rats with biliary fistulas were infused for 60 min i.v. with sulfobromophthalein (BSP) or BSP-glutathione conjugate (BSP-GSH) at 594 nmol/100 g/min. Thirty minutes prior to the start of the infusion, 20 mg/kg iodomethane, dissolved in oliver oil, was given into the duodenum. The control received oil only. At the start of the infusion the hepatic concentration of GSH was 0.96 +/- 0.23 mg/g liver in the iodomethane-treated animals versus 1.93 +/- 0.13 mg/g liver in the control (P less than 0.001). When unconjugated BSP was infused, the excretion of total BSP (unconjugated plus conjugated) was markedly lower in the iodomethane-treated group than in the control. This difference was due solely to differences in biliary appearing conjugated BSP; the excretion of unconjugated BSP was identical in both groups. The different excretion patterns were paralleled by equal hepatic accumulation of total BSP in both groups. The ratio of unconjugated BSP/BSP-GSH in the liver was about twice as high after pretreatment with iodomethane than in the control group. When BSP-GSH instead of BSP was infused, the excretion rates of this dye were identical in both groups. The maximal transport capacity (Tm) was double that observed with infusion of unconjugated BSP in control animals. There is indirect evidence that BSP and BSP-GSH might have different excretion pathways.

Effect of nafenopin (SU-13,437) on liver function: influence on the hepatic transport of organic anions.

Rats treated with hypolipidemic agent, nafenopin (SU-13, 437) exhibit a higher plasma retention and a markedly reduced biliary excretion of organic anions, such as sulfobromophthalein (BSP) and its dibromo analog (DPSP), indocyaninegreen (ICG), succinylsulfathiazole (SST) and polar metabolites of bilirubin and the carcinogens 7, 12-dimethylbenzanthracene (DMBA) and 3,4 benzpyrene (BP), despite an increase in liver mass and a profound choleresis. However, taurocholate is not affected in this manner, which supports the idea of a transport mechanism for taurocholate that differs from that of other organic anions. A pharmacokinetic study was made for DBSP in vivo. After nafenopin treatment, primary hepatic uptake (k12) and transport from liver into bile (k23) are reduced in vivo. Infusion studies indicate that biliary transport maximum (Tm) for DBSP is also decreased although the calculated hepatic storage (S) is only moderately affected. In the isolated perfused liver, hepatic clearance and biliary excretion of BSP are reduced by two-thirds. The time course of anion transport inhibition and the hepato-biliary disposition of 14C-nafenopin suggest a direct effect of the drug. The extra liver mass induced by nafenopin appears to be hypo- or nonfunctional with respect to hepatic transport of organic anions.

Formation of a metabolite of dibromosulfophthalein (DBSP) in man.

In bile specimens from postoperative patients with biliary drainage following cholecystectomy, in addition to unchanged dibromosulfophthalein (DBSP), a single polar metabolite of DBSP was found after i.v. injection of 5 mg/kg of the diagnostic dye. This metabolite, which has not previously been detected, was resistant to beta-glucuronidase and arylsulfatase and was remarkably stable in strongly acid and alkaline solutions. It exhibited the same spectrum and colour change interval as unchanged DBSP. Further studies of its identity revealed that it gave a ninhydrin-positive reaction and that its Rf-value on TLC could be restored by Raney-nickel reduction. Amino-acid analysis after reduction and acid hydrolysis showed an increase in glutamic acid and alanine that can be considered as splitting products of conjugated glutathione following these procedures. Estimation of the quantity of this possible glutathione conjugate indicates that it is formed less rapidly than the glutathione derivative of the tetrabromoanalogue BSP, and that it represents up to 25% of the total dye excreted in bile. The observed metabolism of DBSP in man may complicate its use in the study of hepatic transport function, and negates the previous assumption that, as in certain other animal species, the dye is excreted unchanged.

Compartmental analysis of the plasma clearance of tetrabromsulphthalein and dibromsulphthalein in the normal dog and in patients with liver disease.

A comparison has been made of the plasma clearances of the anionic dye analogues, tetrabromsulphthalein (BSP) and dibromsulphthalein (dBSP), analysed by means of a two-compartmental model. In the normal conscious dog all three transport rate constants and initial and steady state plasma clearances were greater with dBSP than BSP. On the other hand the equivalent liver volume (ELV) and storage capacity were lower with dBSP. In patients with various hepatic disorders dBSP values were greater than those obtained with BSP apart from rate constant b (liver to plasma). Patients with advanced liver impairment in general exhibited lower hepatic uptake, clearance and storage of both dyes compared to patients with mild forms of liver disease. The ratio of ELV values for BSP and dBSP appeared to be related to overall hepatic function, with ratios greater than 1 in the normal dogs and less than 1 in the patients.

Effects of taurocholate infusion on biliary excretion in isolated perfused rat livers. Decreased biliary excretion of dibromosulfophthalein in pregnancy.

A crossover experimental design was used to examine the effects of saline (SAL) vs. taurocholate (TC) infusion on hepatic excretory function in isolated perfused livers from pregnant (19-21 days gestation) and nonpregnant female rats. Bile flow, bile acid concentration, bile acid secretory rate, dibromosulfophthalein (DBSP) concentration in bile, and DBSP secretory rates were determined in livers infused continuously with DBSP and initially with SAL (1 ml/hr, 45 min), followed by TC (60 mumol/hr, 1 ml/hr; 75 min) or initially with TC (45 min) followed by SAL (75 min). The order of infusion (SAL-TC vs. TC-SAL) had no significant effect. TC infusion significantly increased all measures in livers from both nonpregnant and pregnant rats. Two-way analysis of variance followed by the Tukey-Kramer test showed that bile flow (microliter/min/g liver) and DBSP concentration in bile (mumol/ml) were significantly decreased during SAL infusions in pregnancy. These two measures plus bile acid and DBSP secretory rates (nmol/min/g liver) were also significantly decreased during TC infusion in pregnancy. Pregnancy had no effect on bile acid concentration in the presence of SAL or TC infusions. When bile flow, bile acid, and DBSP secretory rates were calculated per whole liver, only the DBSP secretory rate was significantly decreased in pregnancy. These data indicate that bile flow and bile acid secretion do not increase in proportion to the increase in liver weight in pregnancy so that these measures are decreased when expressed per g liver. Pregnancy appears to have a real inhibitory effect on DBSP since its secretion is depressed when activity is expressed per g liver or per whole organ.

Influence of retrograde volume and short time cholestasis on the biliary reabsorption of sulfobromophthalein sodium (BSP) and phenol 3,6 dibromphthalein disulfonate (DBSP) from the rat biliary tree after retrograde intrabiliary injection (RII).

The retrograde intrabiliary injection (RII) technique was used to study the reabsorption of equimolar doses (1,2 mumol) of sulfobromophthalein sodium (BSP) and its analogue phenol 3,6 dibromphthalein disulfonate (DBSP) from the rat biliary tree under the experimental conditions established in this study i.e. variation of retrograde volumes injected and of short time cholestasis. RII was performed with a microliter syringe joined to a cannula positioned in the proximal third of the common bile duct. This technique guaranteed accurate administration of microliter quantities, also free bile flow could be reinitiated 3 - 5 sec after RII. Bile samples were collected in 2, 5 min intervals up to the 5th min, then in 5 min intervals up to the 15th min, and the following 15 min up to the 30th min and analysed for compounds administered by RII. No significant differences in the biliary reabsorption of BSP and DBSP after RII could be detected. Increasing retrograde volumes of 20, 40, 60, 110, and 160 mul lead to decreasing percent recoveries in the bile in the first 2,5 min after RII (88,0 +/- 3,8 - 15,6 +/- 3,1) when bile flow was started at once after RII. An inverse correlation between retrograde volume and percent recoveries was found. Increasing duration of cholestasis (bile flow was started at once, 0,5 min, 3 min, 6 min, and 30 min after RII) results in decreasing percent recoveries of BSP and DBSP in the first 2,5 min after free bile flow was reinitiated (52,7 +/- 3,6 - 20,0 +/- 1,2). A time dependent proportional effect could be detected. Furthermore in the case of BSP concomitant with increasing cholestasis increasing amounts of BSP - conjugates were found in the bile supporting the idea of a continuing ductular - hepatocellular circulation even during complete obstruction of the common bile duct.

Urinary excretion of dye in dogs infused with BSP or its glutathione conjugate.

Renal clearance of BSP compounds was investigated in dogs during infusion of sulfobromophthalein (BSP) or its glutathione conjugate (BSP-GSH). Conjugated BSP compounds are more readily excreted into urine than unconjugated BSP. Dye clearance into urine was much less than simultaneously measured inulin clearance. This suggests that protein binding of BSP compounds significantly retards the glomerular filtration of the dye. BSP was found to bind more avidly to albumin than BSP-GSH. The ratio of dye clearance to inulin clearance remained relatively constant over a broad range of plasma concentrations of dye. The data support but do not prove glomerular filtration of non-protein-bound dye as the major mechanism accounting of urinary elimination of BSP compounds in the dog.