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Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane.

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.

Animals

Mirex-induced impairment of hepatobiliary function. Suppressed biliary excretion of imipramine and sulfobromophthalein.

Effect of pre-exposure to the pesticide mirex on hepatic uptake, metabolism, and biliary excretion of the tricyclic antidepressant, imipramine (IMP), was studied by using isolated perfused rat liver preparations obtained from control and treated animals. Pretreatment of male rats with mirex (50 mg/kg/day, po, for 3 days) resulted in a marked suppression (91%) of biliary excretion of endogenously formed polar metabolites of IMP. This impairment of biliary excretory function is related neither to the bile flow nor to the rate of metabolism of IMP. These conclusions are supported by the following observations: 1) pre-exposure to mirex results in a significant increase in bile secretion; 2) endogenously formed metabolites of IMP accumulate in increasing concentrations in the perfusate of liver preparations from mirex-treated rats; 3) over 98% of the IMP was metabolized by livers of both control and mirex-treated rats. Furthermore, biliary excretion of exogenously provided polar metabolites of IMP was also suppressed by 88% of that of control livers. Addition of mirex (5 X 10(-5) M) to the perfusate of control liver preparations was without effect on biliary excretion of IMP metabolites. Biliary excretion of sulfobromophthalein was also markedly suppressed (90%) by preexposure to mirex. These results suggest that mirex-induced impairment of hepatobiliary function is not specific to substrates. The mechanism of mirex-induced impairment is located at the site of transfer of otherwise readily excretable substances such as metabolites of IMP and sulfobromophthalein.

Animals

Influence of taurocholate, taurochenodeoxycholate, and taurodehydrocholate on sulfobromophthalein transport into bile.

To test the hypothesis that incorporation of sulfobromophthalein (BSP) into mixed micelles could account for the increase in its biliary transport maximum (Tmax) by bile salts, we have compared in hamsters the influence on BSP Tmax of taurocholate and taurochenodeoxycholate (two micelle-forming physiological bile salts) to that of taurodehydrocholate, a bile salt which, in vitro, does not form micelles. In a first series of experiments, it was observed that taurocholate and taurochenodeoxycholate increased the secretion of phospholipid (40 and 53%, respectively), and cholesterol (50 and 110%, respectively), whereas taurodehydrocholate decreased the secretion of phospholipid (-31%) and cholesterol (-43%). This result suggests that, in vivo, taurodehydrocholate or its metabolites do not form mixed micelles. In a second series of experiments, it was seen that the three bile salts induced a similar increase in BSP Tmax (63% with taurocholate, 52% with taurochenodeoxycholate, and 51% with taurodehydrocholate). These results provide circumstantial evidence for the hypothesis that mixed micelle formation is not an important determinant of maximal BSP secretion into bile.

Animals

Extrasplanchnic effect of sulfobromophthalein (BSP) on plasma estrogen levels in the dog.

The effect of the drug sulfobromophthalein (BSP) on plasma estrogens in the dog were studied during intravenous infusions of 3H-labeled estrogens. During [3H]estrone infusion, BSP administration caused a marked increase in arterial plasma levels of the radioactive conjugated estrogens, estrone glucosiduronate, estradiol-17 beta glucosiduronate(s), and estrone sulfate. Levels of the unconjugated estrogens, estrone and estradiol-17 beta, were substantially unaltered. Possible mechanisms were investigated. Splanchnic extraction of the conjugates did not change significantly during BSP administration, and renal excretion rose promptly in proportion to the plasma levels, thus virtually excluding decreased biliary or renal excretion. There was no net discharge of estrogen glucosiduronate radioactivity from adipose tissue or muscle following BSP. During [3H]estrone glucosiduronate infusion, BSP again caused an increase in plasma estrone glucosiduronate, thus excluding increased formation (of this conjugate, at least). BSP caused decreased extraction of estrone glucosiduronate by the hindlimb, indicating that decreased metabolism was the probable cause of the elevated plasma levels. BSP also caused decreased formation of unconjugated estrogens by the lungs, indicating that the decreased metabolism includes decreased hydrolysis of estrogen glucosiduronates.

Animals

Effect of pregnenolone-16 alpha-carbonitrile on bilirubin-and sulfobromophthalein-binding to hepatic Y and Z proteins in the rat.

Pregnenolone-16 alpha-carbonitrile (PCN), administered twice daily p.o. for 3 days at a dose level of 20 mumol/100 g body weight, significantly enhances in vivo binding of 14C-bilirubin and sulfobromophthalein (BSP) to hepatic Y and Z proteins in female Charles River CD rats. 14C-bilirubin-binding to Y protein showed a 61% increase, while binding of the same moiety to the Z protein fraction was augmented by 59%. BSP-binding in vivo demonstrated rises of 114 and 71% in relation to Y and Z proteins, respectively. These data correlate well with previous investigations in which PCN was found to have a beneficial influence on experimentally induced hyperbilirubinemias and, furthermore, there is an indication that phenobarbital, another potent microsomal enzyme inducer, acts via a similar mechanism.

Animals

Difference in the effect of bucolome on the hepatic transport maximum of sulfobromophthalein and indocyanine green.

The effects of bucolome (BC, 1-cyclohexyl-5-n-butyl-2,4,6-trioxoperhydropyrimidine, a non-steroid antiinflammatory agent), a potent choleretic, on the maximal biliary excretion rates (Tm) of sulfobromophthalein (BSP) and indocyanine green (ICG) were investigated in rats. With continuous infusion of BSP or ICG, Tm and bile flow rate in control rats and rats given BC (40--100 min after 20 mg/100 g body weight i.p. injection) were compared. The BSP Tm was not significantly different in control and BC administered rats (BC rats), while the bile flow rate and endogenous bile salts excretion rate were significantly higher in BC rats. On the other hand, ICG Tm was significantly higher in BC rats, although ICG concentration in bile was significantly lower in BC rats. The rates of the rise in plasma concentration of the dyes were significantly lower in BC rats. However, the plasma concentration of ICG was significantly higher in BC rats at corresponding time intervals than in control rats throughout the experimental period. The difference in the effect of BC on the transport of these two dyes indicates variability in the regulatory mechanisms for hepatic anion transport.

Animals

Sulfobromophthalein metabolism in isolated perfused rat liver. Factors determining the applicability of the model.

Sulfobromophthalein (BSP) was used to test the applicability of the isolated perfused rat liver in the study of drug metabolism. Flow of perfusate through the liver averaged 35 ml per min and the bile flow rate was 0.75 microliters per min per g of liver. BSP was delivered into the system as a single dose of 4.7 to 9.5 mumoles. Samples of perfusate from the pre and posthepatic circuits and bile samples were obtained at different time intervals. The following parameters were elimination half life, mean hepatic clearance, mean intrinsic clearance, time course of hepatic and bile concentrations and percent dose excreted into bile. The results demonstrated that bile flow and BSP biliary excretion depended on hepatic blood flow. Time course of hepatic concentration decay and mean intrinsic clearance were a reflection of the inherent ability of the liver for BSP elimination into bile. The parameters tested and their correlations gave overall information on isolated liver function that may be applied to the study of other exogenous or endogenous compounds.

Animals

The handling of bilirubin and sulfobromophthalein by the isolated perfused rat liver.

The isolated perfused rat liver was used for the study of the characteristics of bilirubin transfer from perfusate to bile in comparison to those of sulfobromophthalein (BSP). The parameters estimated included mean extraction ratio, removal half life, hepatic clearance, intrinsic or metabolic clearance rate of biliary excretion, hepatic concentration and biliary concentration for both compounds. The results obtained favoured the conclusion that bilirubin was less efficiently handled than BSP by the isolated rat liver.

Animals

Competition for binding to multiple sites of human serum albumin for cholecystographic agents and sulfobromophthalein.

The binding of two cholecystographic agents, iophenoxate and iopanoate, to human serum albumin was studied with 11 putative competitors; the results were qualitatively consistent with competitive binding to common sites. A more precise analysis of competition was achieved with four pairs of compounds for which the free and bound concentration of each was determined. The results were analyzed by a computer program and the dissociation constants calculated for both binder and competitor at specified sites on albumin. With numbering based on the rank order of dissociation constants for iophenoxate, the highest binding of the four compounds occurs at different sites: iophenoxate at site I; iopanoate at site II, sulfobromophthalein at site III; and bromphenol blue at site II. For a given compound, there is close agreement in the calculated affinities at different sites regardless of the competitor.

Binding Sites

Inhibition by sulfobromophthalein of mitochondrial translocation of anions and adenine nucleotides: effects upon liver adenosine triphosphate and possible correlation with inhibition of bile flow in the rat.

Unconjugated sulfobromophthalein (BSP) inhibits state III respiration of rat liver mitochondria. It competitively inhibits the translocation into mitochondria of citrate, malate, phosphate and adenosine diphosphate, as studied by the inhibitor stop method. A double-beam spectrophotometric study strongly suggests that glutamate translocation is similarly inhibited. After perfusion of 65 mumol/hr/100 g for 90 minutes, bile flow is inhibited by 82% and liver adenosine triphosphate (ATP) falls by 60%. The amount of mitochondrial BSP can be computed form the amount of [35S] BSP still bound to mitochondria that are prepared at the end of such experiments; the amount of BSP lost during the isolation procedure is estimated from parallel experiments following binding of BSP in vitro. Comparison of the kinetic constants of mitochondrial transport and of their inhibition by BSP on the one hand and of liver concentration of substrates and BSP on the other gives rise to the conclusion that a strong inhibition of transports, mainly of phosphate, occurs in vivo and is responsible for the concomitant decrease in bile flow.

Adenine Nucleotides

[Sulfobromophthalein kinetics in normal subjects and patients with alcoholic cirrhosis (author's transl)].

The AA. studied sulfobromophthalein kinetics in normal subjects and patients with alcoholic cirrhosis by compartmental analysis and by mathematical resolution of disappearance plasmatic curve of BSF. The comparison between the two methods gave some interesting clues for understanding BSF metabolism in alcoholic cirrhosis. From obtained data it seems that BSF clearance in these patients, besides on hepatic uptake, like normal subjects, largerly depends on intact secretory function of the liver.

Female

Sulfobromophthalein and indocyanine green binding by the hepatic cytoplasmic proteins.

The elution patterns of human and rat liver supernate mixed with BSP or ICG have been demonstrated using G-100 Sephadex gel filtration. The elution pattern in the rat liver remained the same as that shown by Levi et al. In the human liver BSP localized to Y protein could hardly be demonstrated, whereas ICG was detected. This pattern was quite similar in Rotor's hyperbilirubinemia and in Crigler-Najjar syndrome Type II. These observations suggest that the impaired hepatic transportation of organic anions in congenital hyperbilirubinemias may not be related to cytoplasmic organic anion-binding proteins.

Animals