Reduced bile flow in rats during sulfobromophthalein infusion.

Male urethane-anesthetized Wistar rats with biliary fistulas were infused intravenously with sulfobromophthalein (BSP) or BSP-glutathione conjugate (BSP-GSH) (594 nmol/100 g/min) for 100 min. With BSP infusion the bile flow increased for 25 min and subsequently fell almost linearly. Biliary excretion of BSP-GSH correlated with the bile flow, whereas the hepatic concentration of this metabolite rose during infusion. The concentration of unconjugated BSP also increased in the liver. On the other hand, the infusion of BSP-GSH resulted in a long lasting choleresis accompanied by high biliary concentrations of BSP-GSH without a concurrent increase in the concentration of BSP-GSH in the liver. It is concluded that the increasing concentration of unconjugated BSP in the liver leads to an impaired excretion of BSP-GSH in the bile and concurrently to a reduction in bile flow. The conjugation with glutathione may be the rate-limiting step in the biliary excretion of BSP.     

The influence of cholic acid and dehydrocholic acid on the biliary excretion of unconjugated and conjugated sulfobromophthalein in rats.

Urethane anesthetized Wistar rats with biliary fistulas were infused during 100 min with sulfobromophthalein (BSP), the glutathione conjugate of sulfobromophthalein (BSP-GSH), cholic acid (CA) and dehydrocholic acid (DCA). The dyes (594 nmol/100 g/min) and the bile acids (1200 nmol/100 g/min) were infused separately, and in combination as well. When BSP was infused, CA and DCA increased the maximal excretion of total BSP (conjugated plus unconjugated) from 1400 to 4100 and 3300 nmol/100 g/10 min. The bile flow observed with BSP plus CA was not significantly different from that with BSP plus DCA. The biliary excretion of total BSP was higher throughout with CA than with DCA because CA increased the biliary concentration of PSP while DCA did not. The bile flow attained with CA alone was significantly lower than that with BSP plus CA. The current data provide arguments for abandoning the view that choleresis per se is the crucial determinant for BSP excretion. When BSP-GSH was infused instead of BSP, the excretion rate of the dye was not altered by the additional infusion of CA whereas it was significantly reduced by DCA. The maximal biliary concentration of BSP-GSH fell from 25.9 nmol/mul to 15.3 and 9.4 nmol/mul with CA and DCA, respectively. Both CA and DCA impaired the hepatic uptake of BSP and BSP-GSH. During the infusion with CA, BSP plus CA and BSP-GSH plus CA the biliary excretion rates of bile acids did not differ significantly from each other. This favours the view that the transfer for CA from the liver to bile is different from that for BSP and BSP-GSH. A fraction of bile fluid "independent of choleretics" (viz. of bile salts, BSP and BSP-GSH) is estimated and discussed in view of the different types of infusion.     

Studies on the decrease in bile flow produced by sulfobromophthalein

A reduction in bile flow was observed during BSP-infusion (1.875 mg/min) in urethane-anesthetized Wistar rats. The infusion times at which 50% diminution in bile flow occurred were almost equal in bile salt-depleted (biliary fistula during 5 hr) animals and in those given taurocholate (150 nmol/100 g/min) during the depletion; however, the infusion times differed significantly from those observed in non-depleted rats.While the overall excretion of the dye was mainly dependent on the BSP-GSH fraction in the bile, the reduction in bile flow was inversely related to the amount of BSP-GSH conjugate excreted and the hepatic GSH concentrations before the infusion. A marked fall in hepatic GSH was observed after the long-lasting urethane narcosis that occurred during bile salt depletion. Since the infusion of equimolar and higher amounts of BSP-GSH resulted in choleresis, it is concluded that the unconjugated BSP is responsible for the reduced bile flow during BSP infusion.It is suggested that for BSP-GSH, in contrast to BSP, the rate-limiting step in the transport from blood to bile is not its biliary excretion, but its uptake into the liver.     

Biliary excretion of bromsulphthalein and glutathione conjugate of bromsulphthalein in rats pretreated with diethyl maleate

In rats diethyl maleate (DEM, 0.7 ml/kg i.p.) decreased the hepatic glutathione level to one tenth of the control value. Owing to the low glutathione level the conjugation of bromsulphthalein (BSP) with glutathione was markedly depressed. DEM-treated rats were given BSP and a glutathione conjugate of BSP (BSP-GSH) intravenously at various dose levels, and their biliary excretion and tissue concentrations were determined. No significant difference between the hepatic transport maxima for BSP (673 μg/min/kg) and for BSP-GSH (689 μg/min/kg) was found. BSP-GSH increased the biliary flow, BSP diminished it. Depending on the dose, 52–83 per cent of the BSP administered was taken up by the liver in 45 min, whereas the BSP-GSH predominantly appeared in extra-hepatic tissues. The half saturation doses for transport maxima were 75 mg/kg for BSP and 31 mg/ kg for BSP-GSH. After administration of these doses the hepatic concentration of BSP was approximately ten times as high as the hepatic concentration of BSP-GSH.     

Biliary excretion of drugs in the rat during liver regeneration

Alterations in rat liver function during liver regeneration were studied by measuring bile flow and the biliary excretion of bile salts, bromosulfophthalein (BSP), its glutathione conjugate (BSP-GSH), phenol-3,6-dibromophthalein disulfonate (DBSP) and tartrazine (TZ). Immediately after partial hepatectomy, bile flow and bile salt secretion, expressed per g of liver, were significantly increased. Thereafter bile flow remained high through the third day. The concentration of bile salts on the first day was significantly lower than that of controls and remained low through the fourth day, suggesting an increased osmotic potency. Plasma clearance of BSP was proportional to residual liver mass. The excretion of BSP-GSH after injection of BSP was decreased in parallel to the decrease in BSP-GSH transferase activity during liver regeneration. In contrast, the excretion of unconjugated BSP and unknown conjugates was actually increased. Biliary excretion of injected BSP-GSH was significantly decreased up to the third day postoperatively. These results suggest that some of the decreased BSP-GSH excretion after injection of BSP is attributable to the decreased activity of BSP-GSH transferase and to competition by unconjugated BSP. After partial hepatectomy the excretion of injected BSP-GSH and DBSP, expressed per g of liver, was about twice that of controls and thereafter rapidly returned to control levels. In contrast to the other dyes, the excretion of TZ decreased in proportion to liver mass following partial hepatectomy. On the second and third day, the marked sex differences seen in controls were not observed. These findings suggest that TZ may be transported by mechanisms different from those controlling the excretion of BSP and related compounds.     

The role of some pharmacokinetic factors in the biliary excretion of exogenous organic anions

The role of plasma and liver concentrations of exogenous organic anions and the significance of biotransformation of bromsulfphthalein (BSP) in the biliary excretion rate have been studied in male Sprague-Dawley rats weighing 200-250 g. No parallelism was found between plasma concentration and liver concentration after administration of the same dose of drugs to bile duct-ligated rats. A comparison of the values of hepatic concentration with the biliary excretion rate of drugs seemed to show a negative correlation between these parameters. Biotransformation of BSP (conjugation with glutathione) plays an important role in the biliary excretion of this drug, because the pharmacokinetic parameters of conjugated BSP are quite different from those of unconjugated BSP.     

Impairment of sulfobromophthalein biliary excretion and inhibition of glutathione S-transferase activity induced by perhexiline maleate in rats

The effect of the antianginal agent perhexiline maleate (160 mg/kg i.g., daily for 4 days) on the biliary excretion of sulfobromophthalein (BSP) and BSP-glutathione and the hepatic activity of glutathione S-transferases was investigated in Wistar rats. Perhexiline maleate caused a significant reduction in the maximal biliary excretion of BSP (-28%). The decrease corresponded to a lowered excretion of the conjugated dye whereas the excretion of the parent compound did not change significantly. Administration of the drug caused no effect on the maximal biliary excretion of infused BSP-glutathione. Liver glutathione concentrations were similar in control and treated rats. Perhexiline maleate significantly reduced liver glutathione S-transferase activities toward BSP (-25%), 3,4-dichloronitrobenzene (DCNB) (-21%) and 1-chloro-3,4-dinitrobenzene (DNCB) (-27%). Kinetic studies of the enzyme in liver cytosol showed that perhexiline maleate induced an uncompetitive inhibition for the BSP substrate with a reduced Vmax and Km. The results indicate that the reduction in glutathione S-transferase activity plays an important role as a factor determining the impairment in the hepatobiliary transport of BSP caused by perhexiline maleate.     

Studies on biliary excretion mechanisms of drugs—IV: Inhibitory studies on sulfobromophthalein and glucuronides in the rat

After the administration of 50μmoles sulfobromophthalein (BSP) or BSP glutathione conjugate (BSP-GSH) to rats, the maximal dye excretion was approximately 50 per cent higher for the latter as compared to the former. BSP or BSP-GSH and three glucuronides, thiamphenicol glucuronide (TPG). chloramphenicol glucuronide (CPG) and phenolphthalein glucuronide (PPG), depressed each other's bilary excretion. Competition between BSP or BSP-GSH and these glucuronides was not observed in the first phase of disappearance from blood. Transport of these drugs from blood to bile occurs rapidly. From these results, it can be inferred that in the transport of these drugs from blood to bile at least one common step is involved, but that this step is not the uptake from blood to liver. The binding affinity of these organic anions to hepatic cytoplasmic proteins was examined by Sephadex G-75 gel filtration and by ultraliltration. In the former experiment. BSP showed an apparent binding affinity to the cytosol fraction containing ligandin and Z protein. BSP-GSH showed a lower affinity than that of BSP. On the other hand, three glucuronides were not eluted with the fraction. In the latter experiment, 41 per cent of BSP was bound to the eytosol fraction, whereas only 10 per cent of BSP-GSH and PPG and none of CPG and TPG were bound. These results also suggest that the competition in biliary excretion observed between BSP or BSP-GSH and the glucuronides does not occur at the binding sites of ligandin or Z protein, and that binding to these proteins may not be required for the overall transport of these glucuronides from blood to bile.     

Pharmacokinetics, hepatic biotransformation and biliary and urinary excretion of bromosulfophthalein (BSP) in an experimental liver disease mimicking biliary cirrhosis

We studied the hepatic handling of bromosulfophthalein in healthy rabbits with hepatic coccidiosis 28 days after an experimental infection with sporulated oocysts of Eimeria stiedai, an experimental model of liver disease histopathologically resembling primary biliary cirrhosis in man. A pharmacokinetic study of the results was performed following a multicompartmental model with 7 transfer constants to describe the physiological disposition of the dye. The study showed that the plasma disappearance, distribution volume (Vi), hepatic biotransformation and the biliary and urinary elimination of conjugated (BSPc) and unconjugated (BSPu) bromosulfophthalein were markedly altered. Whereas Vi and urinary excretion of the dye were significantly increased, the hepatic clearance, biotransformation and biliary excretion of BSPc and BSPu were drastically reduced in infected rabbits. Satisfactory agreement was obtained between the experimental and estimated data, particularly those relating to biotransformation clearance and biliary and urinary excretion of the dye. These results demonstrate that severe liver disease in rabbits with histopathological liver alterations resembling several hepatic dysfunctions in man markedly reduce hepatic uptake, metabolism and biliary excretion of a xenobiotic such as BSP.     

Impairment of bromosulfophthalein hepatic transport and cholestasis induced by diethyl maleate in the rabbit

The present study was designed to investigate the effect of hepatic glutathione depletion induced by intraperitoneal administration of diethyl maleate (DEM) on the maximum biliary transport (Tm) and on the biliary excretion of bromosulfophthalein (BSP) in anaesthetized rabbits when the dye was perfused endovenously at doses exceeding Tm. The Tm of total BSP (BSPt) and that of conjugated BSP (BSPc) were significantly reduced after DEM administration whereas that of unconjugated BSP (BSPu) was markedly increased. A reduction in the biliary excretion of BSPt and BSPc, in the percentage of BSPc, in the cumulative excretion of BSPt and in the percent-dose recovery were also observed. However, no change in hepatic glutathione S-transferase activity was noted after DEM. The cholestasis observed following DEM administration coursed with falls in the biliary secretion of sodium, chloride and bicarbonate.     

Impairment in the hepatic clearance of (35S)-bromosulphophthalein in paracetamol-intoxicated rats.

1 The overall functional capacity of the liver was evaluated using [35S]-bromosulphophthalein (BSP, 100 mg/kg, i.v.) in biliary fistulated adult rats pretreated orally with different doses of paracetamol (APAP) for varying time intervals. 2 The maximal hepatic damage occurred between 12-18 h after single doses of APAP (0.5 or 1 g/kg); hepatic excretory function returned to control levels by 48-72 hours. 3 Administration of either 0.5 or 1 g/kg APAP 18 h before BSP caused a dose-dependent inhibition of the choleretic effect of BSP and of the 60 min cumulative excretion of the dye, but conversely, produced a significant increase in the liver and plasma concentrations of 35S. 4 Following acute (0.25 g/kg), or subacute (0.5 g/kg, twice daily for 7 days) treatment with APAP, the total excretion of 35S in bile and the retention of 35S in the liver or plasma remained essentially the same as that for the controls. 5 In rats given single doses of 1 g/kg APAP, the hepatic uptake of the dye was significantly increased during the early stages of intoxication, while the opposite effect was observed at late periods. 6 The bile flow appeared to be inversely related to the excretion of unchanged BSP, and directly related to the excretion of the major BSP conjugate in bile. 7 The hepatic clearance of BSP was more rapid in rats treated subacutely with 0.5 or 1 g/kg APAP, than in those treated acutely with equal doses, suggesting that the intensity of APAP-induced hepatotoxicity became less severe after the repeated administration of this drug. 8 It is concluded that the hepatic uptake, metabolism and excretion of BSP are reversibly impaired following APAP-induced liver injury.     

Metabolism and biliary excretion of sulfobromophthalein in vitamin a deficiency

Vitamin A deficiency in rats significantly increased hepatic glutathione S-aryltransferase activities measured in vitro against 1,2-dichloro-4-nitrobenzene (DCNB) and sulfobromophthalein (BSP). These changes were accompanied by a decrease in hepatic glutathione reductase activity in the deficient animals, but there were no alterations in either oxidized glutathione (GSSG) levels or the ratio of reduced glutathione (GSH) to GSSG in the liver. The relation between plasma BSP clearance, biliary BSP excretion, and the enzymatic conjugation rate of the dye were examined. Plasma clearance, hepatic uptake and hepatic storage of BSP were reduced in vitamin A deficient animals. Biliary concentration, excretion rate and cumulative excretion of BSP, on the other hand, were increased in these animals. The increases in these variables were due predominantly to increased formation of conjugated BSP. It is concluded, therefore, that the rate of the enzymatic conjugation of BSP with GSH is a rate-limiting step in the biliary excretion of the dye.     

INFLUENCE OF BILE ACIDS ON THE BILIARY TRANSPORT MAXIMUM OF PHENOLSULPHONPHTHALEIN IN THE RAT

1. The effect of changes in bile acid secretion induced by cholestyramine treatment or taurocholate infusion on the biliary transport maximum (Tm) of phenolsulfonphthalein (PSP) was studied in Wistar rats. 2. Five hours after oral administration of cholestyramine (1.5 g/kg bodyweight) the biliary output of bile acids decreased to 51% and bile flow to 76% of control values. The percentage of conjugated and unconjugated PSP excreted into bile and the Tm of the dye were not significantly modified by cholestyramine pretreatment. 3. Administration of sodium taurocholate at increasing rates (60-480 nmol/100 g bodyweight per min) enhanced bile flow and the biliary output of bile acids in a linear dose-related fashion. The Tm of PSP increased progressively until a maximum of 29% above the control values was reached at a taurocholate dose of 240 nmol/100 g bodyweight per min). The enhancement corresponded mainly to the unconjugated dye, the excretion of conjugated PSP not being significantly modified by the infusion of the bile acid. 4. The results indicate that bile acids can influence to some extent biliary excretion of PSP in the rat, although this component is of minor importance at low bile acid secretory rates.     

Effect of gomisin A (TJN-101), a lignan compound isolated from Schisandra fruits, on liver function in rats

TJN-101 [+)-(6S, 7S, R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy- 6,7-dimethyl-10,11-methylenedioxy-6-dibenzo [a, c] cyclooctenol) is one of the lignan compounds isolated from Schisandra fruits. When TJN-101 was administered orally at the doses of 3-100 mg/kg/day for 4 days, bile secretion, hepatic excretion of dye or hepatic hemodynamics 24 hr after the last dose was investigated in comparison with the phenobarbital (100 mg/kg/day)-treated group. Bile flow was dose-dependently increased; in contrast, biliary concentration of bile acids was decreased in TJN-101 (30 and 100 mg/kg/day)-treated groups. Similar changes were also observed in the phenobarbital-treated group. These results suggested that the enhancement of bile secretion caused by TJN-101 or phenobarbital was due to an increase of a bile acid-independent fraction. In the bromosulfophthalein (BSP) clearance test for liver function, both TJN-101 (30 and 100 mg/kg/day) and phenobarbital accelerated the disappearance from the blood and biliary excretion of BSP. Hepatic hemodynamics was examined by the hydrogen clearance method and measurement of liver wet and dry weight. Liver blood flow tended to increase in the TJN-101 (10-100 mg/kg/day) or phenobarbital-treated group. On the other hand, TJN-101 (3-100 mg/kg/day) or phenobarbital hardly altered the water content of the liver. These results suggested that the liver enlargement caused by both compounds was not accompanied with hepatic edema and that the enhancement of bile secretion or hepatic excretion of BSP might be related to an increase of liver blood flow.     

Taurocholate excretion and bile formation in the isolated perfused rat liver

Summary The excretory transport maximum (Tm) for taurocholate was determined in the perfused rat liver and compared to that obtained in the intact rat. An in situ liver perfusion system employing a semisynthetic perfusion medium containing Krebs-Ringer-bicarbonate solution, bovine erythrocytes and bovine albumin was used.In contrast to the bromosulfophthalein (BSP)-Tm reported previously, the taurocholate-Tm was 45% smaller in vitro (196±17 nmol/min per g liver) than in vivo (357±10 nmol/min per g liver), indicating that bile salt transport is more susceptible to alterations induced by the conditions of the perfusion than BSP transport. These findings add to the differences observed previously between the hepatic handling of anionic dyes and bile salts.At a low taurocholate infusion rat (57 nmol/min per g liver) the normal relationship between bile salt excretion and bile flow observed in vivo was maintained in the perfused liver. At higher taurocholate infusion rates, however, bile flow, for a given bile salt excretion rate, was smaller than in vivo.These findings should be taken into account when the isolated perfused rat liver is employed for studies of bile formation.     

THE INFLUENCE OF DIETHYL MALEATE ON THE BILIARY EXCRETION OF INFUSED SULPHOBROMOPHTHALEIN SODIUM AND ITS GLUTATHIONE CONJUGATE IN GUINEA-PIGS

1. Hepatic sulphobromophthalein (BSP) transport was studied in guinea-pigs pretreated intraperitoneally with 0 7 ml of diethyl maleate to depress hepatic glutathione levels. 2. Diethyl maleate depressed the hepatic transport of infused conjugated BSP from hepatocytes into bile without influencing hepatic uptake. 3. Unconjugated BSP transport was also depressed markedly as a result of (a) a reduction in the intrahepatic conjugation of BSP with glutathione and (b) suppression of conjugated BSP excretion.     

Induction of multidrug resistance-associated protein 2 in liver, intestine and kidney of streptozotocin-induced diabetic rats

Many studies have demonstrated that Mrp2 is highly regulated in some physiopathological situations. The aim of this study was to investigate effects of diabetes mellitus on function and expression of multidrug resistance-associated protein 2 (Mrp2) in rat liver, kidney and intestine. Diabetic rats were induced by an intraperitoneal administration of streptozotocin (65?mg/kg) and randomly divided into diabetic (DM) rats and insulin-treated diabetic rats. Sulfobromophthalein (BSP), a substrate of Mrp2, was used to evaluate Mrp2 function in vivo. Data from excretion experiments demonstrated that compared with normal rats, diabetes markedly enhanced BSP excretion via bile, urine and intestinal perfusate, which contributed to the elevated plasma clearance of BSP after intravenous administration of 45 μmol/kg BSP. Western blot results showed higher levels of hepatic, renal and intestinal Mrp2 protein in DM rats, although no difference was observed in renal Mrp2. Insulin treatment partly reversed these alterations. Induction of Mrp2 by diabetes was in parallel with the increase in bile flow, levels of biliary and plasma total bile acid (TBA), and plasma conjugated bilirubin in DM rats. Diabetes may enhance Mrp2 function and expression in liver, kidney and intestine, which might be due to insulin deficiency, increased TBA and conjugated bilirubin.     

Hepatic transport and biliary excretion of sulfobromophthalein in aflatoxin B1-treated rats

The effects of a single dose of aflatoxin B1 (AFB1) on sulfobromophthalein (BSP) plasma disappearance, hepatic transport maximum (Tm), and relative storage capacity (S), were examined in rats 48 hr after AFB1 injection. BSP plasma concentration decay was delayed, and the BSP biliary excretion was diminished in treated animals. S and Tm values were unaltered. However, the Tm was reached in treated rats at a higher infusion rate. A Lineweaver-Burk plot of BSP biliary excretion rate vs BSP serum concentration curve showed a higher apparent Km in the AFB1-treated rats.     

Comparison of biliary excretion of organic anions in mice and rats

Biliary excretion of cholephilic organic acids in anesthetized, male Swiss-Webster mice was compared to that in male Sprague-Dawley rats. The mouse excreted six of the eight compounds examined at a faster or equal rate than the rat. Indocyanine green, rose bengal, phenol-3,6-dibromsulphthalein disulfonate, and eosine were excreted in mice at a rate 120 to 460% higher than in rats. The excretion rates of bromcresol green and sulfobromophthalein glutathione conjugate were similar in the two species, whereas amaranth was excreted at a slightly lower rate in mice than in rats. Biliary excretion of sulfobromophthalein (BSP), especially its glutathione conjugate, was significantly lower in the mouse which corresponded to a difference in BSP-glutathione transferase activities between the two species (mouse, 0.97; rat, 1.35 μmol/min/g liver). The depression of bile production by cholestatic organic anions was stronger, and the stimulation of bile flow by choleretic acids was weaker in mice than in rats. Differences in biliary bile acid excretion (mouse, 3.62; rat, 1.42 μmol/kg/min), bile flow (mouse, 102; rat, 69 μl/kg/min), and liver weight (mouse, 57; rat, 38 g/kg) but not hepatic ligandin concentration (mouse, 132; rat, 214 nmol BSP/g liver) may explain the variations in the biliary organic anion excretion between mice and rats.     

Pharmacokinetics and biliary excretion of bromosulphophthalein, [3H]-ouabain and [3H]-taurocholic acid in rats with glycerol-induced acute renal failure

The pharmacokinetics and biliary excretion of bromosulphophthalein (BSP), ouabain and taurocholic acid (TChA) have been studied in rats with glycerol-induced acute renal failure (ARF). In rats with ARF, the hepatic uptake and initial biliary excretion of BSP were decreased. In addition, the rate of BSP conjugation with glutathione by rat liver homogenates was also decreased. This latter change may contribute to the initial decrease in the biliary excretion of BSP. No change was found in the hepatic uptake and biliary excretion of ouabain, but the area under the concentration-time curve was increased and the plasma clearance (Clp) decreased in rats with ARF. This decrease in Clp was not due to reduced renal excretion. The decreased Clp of ouabain in rats with ARF may come from reduced tissue binding and a concomitant decrease in its volume of distribution (Vd). The hepatic handling of TChA appeared unaltered in ARF, but the rate constant for the terminal part of the concentration-time curve (beta) was decreased. This change probably resulted from a large increase in Vd in rats with ARF. It is concluded that the decreased uptake of BSP was not due to a non-specific disturbance of hepatocyte function in ARF because the hepatic handling of ouabain and TChA were unaltered.