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At least 19 recordsLinked to original sources
PubMed1976

Comparison of sulfobromophthalein (BSP) and sulfobromophthalein glutathione (BSP-GSH) disposition under conditions of altered liver function and in the isolated perfused rat liver.

Disposition of sulfobromophthalein (BSP) and sulfobromophthalein glutathione (BSP-GSH) was compared in control, phenobarbital, or alpha-naphthylisothiocyanate-(ANIT)-treated rats, and in the isolated perfused rat liver preparation. After dye administration, BSP-GSH was found to have a more rapid early plasma disappearance rate, a more rapid appearance in the liver, and a greater rate of biliary excretion both in vivo and in the isolated perfused liver, than that of BSP. In considering these observations, it is concluded that hepatic uptake as well as biliary excretion of BSP-GSH is faster than that of BSP. Comparing BSP and phenobarbital, augmentation or reduction in plasma dye concentration, mean plasma half-life (2 to 30 minutes), hepatic dye content, and bile dye concentration, were of the same order of magnitude for both dyes. However, with dye infusion of 3.6 mumoles per kilogram per minute, phenobarbital significantly enhanced the rate of biliary excretion of BSP but not BSP-GSH and ANIT treatment had a greater inhibitory effect on biliary excretion of BSP-GSH than BSP.

1-Naphthylisothiocyanate↗
PubMedDate not supplied

Biliary excretion of sulfobromophthalein compounds in normal and mutant Corriedale sheep. Evidence for a disproportionate transport defect for conjugated sulfobromophthalein.

Biliary excretion of dye was evaluated in three normal and one mutant Corriedale sheep (characterized by depressed biliary transport of many organic anions) during infusion of unconjugated sulfobromophthalein (BSP) and its preformed glutathione conjugate (BSP-GSH). Maximal dye excretion rates in bile in normal sheep were higher when BSP-GSH rather than BSP was administered. In confirmation of earlier studies, dye excretion in bile was markedly depressed in the mutant animal. However, movement of unconjugated BSP from liver cells into bile remained relatively intact whereas transport of conjugated BSP compounds was virtually absent. Preservation of unconjugated dye entry into bile suggests the presence of a transport mechanism for unconjugated BSP which is preserved in mutant sheep and is distinct from that involved in BSP-GSH excretory transport. Renal clearance of conjugated BSP compounds was greater than of unconjugated BSP and clearance values were comparable in the normal and mutant sheep. Thus, no excretory defect comparable to that present in liver was identifiable in the kidney.

Animals↗
PubMed1976

The hepatic handling of 131I-labeled sulfobromophthalein in the dog. Comparison with sulfobromophthalein.

131I-labeled BSP is obtained by the incorporation of one molecule of radioactive iodine in BSP. The purpose of this work was to study the influence of the added iodine on the hepatic extraction and biliary excretion of BSP. Two types of experiments were performed. In the first, a compartmental analysis of BSP and 131I-labeled BSP disappearance curves was performed after the simultaneous injection of 5 mg per kg of b. wt. of BSP and 10 muCi of 131I-labeled BSP: both, the plasma to liver and liver to bile transfer rates were significantly lower for 131I-labeled BSP than for BSP; the liver to plasma transfer rates were not significantly different. In the second series of experiments, the hepatic uptake of BSP and 131I-labeled BSP was estimated by the multiple indicator dilution technique: the extraction of BSP (59% +/- SD 5) was significantly higher than that of 131I-labeled BSP (35% +/- SD 5) (P less than 0.001). In conclusion the labeling of BSP with 131I significantly modified the hepatic handling of the dye; 131I-labeled BSP cannot therefore be used as a tracer of BSP.

Animals↗
PubMed1996

Hepatocellular sulfobromophthalein uptake at physiologic albumin concentrations: kinetic evidence for a high affinity/low capacity sinusoidal membrane system.

BACKGROUND/AIMS: Controversy exists regarding the nature of the hepatocyte membrane transport system for sulfobromophthalein and other organic anions and its driving forces. Most studies have been performed in the absence of albumin, the plasma sulfobromophthalein binding protein, or using very low albumin concentrations. We have shown that in the latter case uptake kinetics reflect dissociation/diffusion events and not membrane transport. In contrast, without albumin very high sulfobromophthalein concentrations reach the cell surface and may overwhelm a high affinity/low capacity system operating in vivo. The aim of this study was to test the latter hypothesis. METHODS: Sulfobromophthalein uptake was measured by rapid filtration in isolated hepatocytes without albumin (up to 13 microM sulfobromophthalein) and with 600 microM albumin (sulfobromophthalein:albumin from 0.03:1 to 1:1), a physiologic setting which greatly reduces the unbound BSP concentration. Unbound sulfobromophthalein concentration was estimated according to a three binding site model. RESULTS: In the absence of albumin, kinetic parameters for sulfobromophthalein uptake were similar to those reported in the literature (K(m):7.1 +/- 1.2 microM; V(max): 452 +/- 37 pmol/min/5 x 10(4) cells). In the presence of albumin, sulfobromophthalein uptake displayed much greater affinity and much lower capacity (K(m): 80 +/- 11 nM; V(max): 60 +/- 9 pmol/min/5 x 10(4) cells). CONCLUSIONS: These findings suggest that in the absence of albumin, resulting high sulfobromophthalein concentrations overload (and make undetectable) a high affinity/low capacity system operating at physiologic albumin concentrations (i.e. at low unbound sulfobromophthalein concentrations). Previously characterized transport systems may be operating only under defined conditions. These findings could explain the apparent controversy regarding the nature of the sulfobromophthalein transport system and its driving forces.

Animals↗
PubMed1981

Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro.

As part of a study of the mechanism whereby organic anionic dyes such as sulfobromophthalein and bilirubin enter hepatocytes, the binding of [35S]sulfobromophthalein and of its glutathione conjugate to two rat liver plasma membrane fractions were studied in vitro. Both fractions reversibly and saturably bound conjugated and unconjugated sulfobromophthalein. Three classes of binding site were necessary to account for the observed sulfobromophthalein binding, their maximal binding capacities being 3.5 . 10(-11), 1.6 . 10(-7) and 5.4 . 10(-7) mol/mg membrane protein. The corresponding association constants were 5.5 . 10(7), 1.5 . 10(5) and 1.3 . 10(3) M-1. Binding of the glutathione conjugate could be accounted for by two classes of binding site only, their association constants being 2.0 . 10(8) and 1.9 . 10(3) M-1 and their maximal binding capacities 5.0 . 10(-11) and 2.2 . 10(-7) mol/mg protein, respectively. Conjugated and unconjugated sulfobromophthalein mutually competed for binding, KI being 7.8 . 10(-7) and 5.5 . 10(-5) M for conjugated and unconjugated sulfobromophthalein, respectively. Similarly, bilirubin and indocyanine green, but not taurocholate, competitively inhibited sulfobromophthalein binding. Treatment with trypsin and phospholipases reduced, while treatment with neuraminidase did not affect binding. Neither changes in pH nor substitution of other cations for Na+ in the incubation mixture significantly affected sulfobromophthalein binding. Heating the membranes increased binding. This was due to an increase in maximal binding capacity of the low-affinity, high-capacity sites. The description of saturable binding sites on hepatocellular surface membranes, the affinity of one of the sites exceeding the reported affinities for albumin and ligandin, supports the hypothesis that a membrane-located membrane carrier is responsible for hepatic uptake and biliary excretion of organic anionic dyes. Based on the studies with enzyme treatments, it is speculated that this carrier is a phospholipid-dependent, integral membrane protein.

Animals↗
PubMedDate not supplied

Induction of cystine transport activity in isolated rat hepatocytes by sulfobromophthalein and other electrophilic agents.

The uptake of cystine in isolated rat hepatocytes was enhanced after incubation of the cells with sulfobromophthalein. The enhancement was time- and dose-dependent, with a lag of about 6 hr, and an approximately 4-fold increase in the activity occurred after 24 hr with 0.2 mM sulfobromophthalein. Actinomycin D or cycloheximide completely blocked the time-related increase in the uptake. The enhanced activity of cystine uptake was almost entirely Na+-independent and inhibited by some anionic amino acids, such as glutamate. The uptake of glutamate was also enhanced by sulfobromophthalein. It is concluded that sulfobromophthalein induces the activity of the Na+-independent, anionic amino acid transport system (System x-c) which mediates the transport of cystine. Some other electrophilic agents also enhanced the cystine uptake. The glutathione level of hepatocytes was rapidly reduced by sulfobromophthalein to about 10% of the control level at 6 hr, but at 12 hr it began to rise. It seems likely that this restoration resulted from the induced activity of the cystine uptake because inhibitors of the cystine uptake blocked the restoration and because the uptake of other sulfur amino acids was not enhanced significantly by sulfobromophthalein. System x-c may thus participate in the detoxication of electrophilic agents in hepatocytes.

Animals↗
PubMedDate not supplied

Plasma sulfobromophthalein disappearance in Gilbert's syndrome.

BACKGROUND/AIMS: We studied the metabolism of sulfobromophthalein and its relationship with serum bilirubin levels in 40 patients with Gilbert's syndrome (type I 30; type II 6; type III 4). MATERIAL AND METHODS: Plasma sulfobromophthalein disappearance studies were carried out and 72 hours later, serum bilirubin concentrations (total and unconjugated fraction) were determined at baseline and after 24 and 48 hours of dietary restriction to 400 calories/day. RESULTS: The fractional transfer rate of sulfobromophthalein from plasma to liver was significantly higher in types I (14.7 +/- 3.4 ml/min) and II (14.9 +/- 2.7 ml/min) than in type III (8.7 +/- 1.5 ml/min). The fraction of the plasma sulfobromophthalein pool irreversibly cleared per min was significantly higher in type I (12.2 +/- 2.6 ml/min) than in types II (9.5 +/- 1.5 ml/min) and III (9.3 +/- 3.8). In all patients, serum bilirubin concentrations were significantly higher after fasting as compared with baseline. There was a significant correlation between the increments of serum unconjugated bilirubin levels after the fasting test and the transfer rate of sulfobromophthalein from plasma to liver (F = 9.8411, r = -0.4535, p = 0.003). CONCLUSION: These findings indicate the presence of an active uptake system shared by bilirubin and sulfobromophthalein.

Adult↗
PubMed1994

Induction of a dose-related increase in sulfobromophthalein uptake velocity in freshly isolated rat hepatocytes by phenobarbital.

To determine whether phenobarbital affects hepatocellular bilirubin/sulfobromophthalein uptake mechanism, we administered it to male Sprague-Dawley rats, body weight 175 +/- 25 gm, at doses of 1 to 75 mg/kg body wt/day for 7 days. Control rats were given an equivalent volume of physiological saline solution. On day 8, hepatocytes were isolated by means of collagenase perfusion, suspended in Hanks' solution without albumin and incubated with high specific activity (3 Ci/mmol) [35S]sulfobromophthalein, which was synthesized in our laboratory and purified by means of a new reverse-phase high-pressure liquid chromatography procedure. The initial uptake rate of sulfobromophthalein was determined at sulfobromophthalein concentrations of 1 to 50 mumol/L with a rapid filtration technique. The maximum uptake velocity and Michaelis constant for sulfobromophthalein uptake at each phenobarbital dose were determined by means of a computer analysis. In control studies, maximum uptake and Michaelis constant were 48.0 +/- 16.7 (mean +/- S.D.) pmol/50,000 cells/min and 22 +/- 4 mumol/L, respectively. Maximum uptake velocity increased linearly with the log of the phenobarbital dose (r = 0.98, p < 0.01), the increase achieving statistical significance at a dose of 3 mg/kg/day. Michaelis constant, however, was essentially unchanged at phenobarbital doses of 50 mg/kg/day or less. The maximal observed increase in maximum uptake velocity of sulfobromophthalein (to 619% of control values) was appreciably greater than the maximal increase in UDP-glucuronyltransferase activity (200% of control) or immunoreactive ligandin concentrations (260% of control) seen in earlier studies, suggesting a direct effect on the plasma membrane transport mechanism.

Animals↗
PubMed1982

Reconstitution in vitro of sulfobromophthalein transport by bilitranslocase.

Liposomes containing 150 mM KCl and 0.48 mM sulfobromophthalein have been prepared. The internal pH was set at 6.5, a value at which sulfobromopthalein is colorless. When brought to alkaline pH a certain amount of the dye is deprotonated and can be read spectrophotometrically as external sulfobromophthalein. Upon addition of Triton X-100 the membrane is dissolved and all sulfobromophthalein present in the preparation may be measured. Addition of bilitranslocase to such a preparation of liposomes causes the internal sulfobromophthalein to leave the internal compartment. The rate of this phenomenon may be followed directly and shown to be greatly accelerated by the addition of valinomycin. The latter finding indicates that sulfobromophthalein transport occurs in response to a membrane diffusion potential created by permeabilisation to K+ of liposomes brought about by valinomycin (uniport). The permeability change induced by bilitranslocase is specific and does not reflect an alteration of the normal impermeability of liposomes to small ions such as protons or Ca2+.

Animals↗
PubMed1989

The hepatocellular transport of sulfobromophthalein-glutathione by clofibrate treated, perfused rat liver.

The hypolipidemic drug clofibrate is known to affect the hepatic transport of various organic anions including bilirubin, fatty acids and sulfobromophthalein. Changes in the rate of metabolism and/or intracellular transport have been claimed responsible for the effect. To evaluate these possibilities, the transport of sulfobromophthalein-glutathione, a model compound that does not require metabolism for biliary excretion, was studied in perfused livers isolated from clofibrate-treated and control rats. Cytosolic fatty acid binding protein and glutathione S-transferase activity were also measured. Clofibrate treatment significantly increased liver weight; as a result glutathione S-transferase activity (toward 1-chloro-2,4-dinitrobenzene) fell if expressed per gram of liver (4560 +/- 420 (SE) vs 7010 +/- 260 nmoles/min for clofibrate treated and controls respectively, p less than 0.002), but was unchanged when expressed per total liver (60.8 +/- 6.5 vs 64.6 +/- 3.5 mumoles/min for clofibrate and controls p greater than 0.5). Irrespective of how it was expressed fatty acid binding protein was significantly increased by the drug treatment. Steady state sulfobromophthalein-glutathione removal velocity was saturable with increasing concentrations of sulfobromophthalein-glutathione in both control and clofibrate-treated livers. Steady state extraction ratio, as well as Vmax and Km for removal, did not differ between the two groups. In keeping with other observations, these data collectively indicate that the hepatic steady state removal of nonmetabolized compounds is not affected by clofibrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Preparation of [35S]sulfobromophthalein of high specific activity.

Study of the hepatocyte transport mechanism of organic anions such as bilirubin and sulfobromophthalein has been limited by the relatively low specific activities of these ligands. [3H]Bilirubin and [35S]sulfobromophthalein have been available with specific activities of only approximately 100 mCi/mmol. We now report a relatively simple procedure to prepare [35S]sulfobromophthalein at a specific activity of approximately 3000 mCi/mmol. This compound is radiochemically pure and serves as a tracer for authentic sulfobromophthalein as judged by chromatography, hepatocyte uptake, metabolism, and biliary excretion. Use of this material as a photoaffinity probe and as a transported ligand may permit dissection and understanding of its transport mechanism.

Animals↗
PubMed1987

Phenobarbital specifically increases the hepatocellular uptake of sulfobromophthalein-glutathione.

The transport of sulfobromophthalein glutathione was studied in perfused livers isolated from phenobarbital treated and control rats. Phenobarbital increased the cell size and the uptake of sulfobromophthalein glutathione. The effect on uptake is specific since in phenobarbital treated livers each unit of hepatocyte surface area takes up more sulfobromophthalein glutathione than controls. The cellular hypertrophy does not involve all cell functions; total and specific content of cytosolic fatty acid binding protein for example, were unchanged by phenobarbital. The increase in Vmax and influx rate constant for sulfobromophthalein glutathione uptake suggest that phenobarbital increases the amount of membrane carriers or their rate of cycling.

Animals↗
PubMed1988

Binding of sulfobromophthalein to rat and human ligandins: characterization of a binding-site peptide.

Photoaffinity techniques were employed to affect the covalent binding of [35S]sulfobromophthalein to proteins of rat and human liver cytosol. In rat liver cytosol at low concentrations, sulfobromophthalein bound to the 22 kDa subunit of ligandin. In human liver cytosol, binding to a 23.5 kDa subunit was observed. At higher concentrations, sulfobromophthalein also bound to 12, 23.5, 37, and 42 kDa peptides. When the peptides resulting from CNBr cleavage of [35S]sulfobromophthalein-ligandin complex were resolved by high-performance liquid chromatography, radioactivity was associated with two peptides. The peptide containing 80% of the radioactivity was isolated and characterized. Its molecular weight is 3.4 kDa, it contains the single tryptophan residue of ligandin and has a glutamate (glutamine) as the N-terminal amino acid.

Amino Acids↗
PubMed1987

Measurement of sulfobromophthalein uptake in isolated rat hepatocytes by a direct spectrophotometric method.

A spectrophotometric technique is described for the continuous recording of sulfobromophthalein uptake by isolated hepatocytes. The technique is based on the principle that sulfobromophthalein behaves as a pH-indicator and may be followed photometrically when moving from the medium at pH 7.8 into the interior of the cell. Data show that upon addition of cells to a sulfobromophthalein solution, an absorbance change can be recorded. The kinetics of the process is biphasic and the initial rate is linearly related to the amount of cells added. By this technique it was confirmed that the substrate dependence of the initial velocity of transport is a compound function including a saturable portion with an apparent Km in the mu molar region. Experiments carried out either in the presence of valinomycin or of high concentrations of potassium chloride indicate that the presence of a membrane potential opposes the entry of sulfobromophthalein into isolated hepatocytes. This finding is in agreement with previous observations in isolated plasma membrane vesicles and in liposomes reconstituted with purified bilitranslocase which indicate a rheogenic type of transport for the dye. Low concentrations of nicotinate (1.6 microM) efficiently inhibit the saturable transport. It is suggested, in addition, that the sensitivity of the transport to valinomycin could be used as an early indication of the functional integrity of cell preparations.

Animals↗
PubMed1982

Conjugation does not influence initial rates of uptake of sulfobromophthalein into isolated hepatocytes.

The organic dye sulfobromophthalein is rapidly taken up and conjugated with glutathione by isolated hepatocytes. To examine whether intracellular conjugation influences the preceding uptake process of the organic dye, conjugation was blocked by depleting cellular glutathione with styrene oxide. Complete inhibition of the conjugation step did not affect initial rates of uptake of 5 and 10 microM sulfobromophthalein. Moreover the maximal cellular accumulation of the dye in glutathione-depleted hepatocytes representing almost exclusively free sulfobromophthalein is similar to the transient maximum concentration of the organic anion found with untreated cells where conjugation takes place. In the case of the untreated cells maximal accumulation is attained at about 20 min, thereafter intracellular levels decrease due to the release of sulfobromophthalein-glutathione conjugate into the medium.

Animals↗
PubMed1975

[Effect of protein-free diet on sulfobromophthalein hepatic transport (author's transl)].

Initial plasma disappearance rate and biliary excretion of intravenously injected sulfobromophthalein were studied in Wistar female rats fed a normal or a protein-free diet. Proteins and sulfobromophthalein distribution in the liver were studied by a gel filtration method. The results suggest that protein deficiency may produce an impaired sulfobromophthalein transference from plasma to liver cell due to a reduction of total hepatic proteins and of sulfobromophthalein-binding protein fraction responsible for liver uptake.

Animals↗