Bromosulphthalein Research Articles

Efficient biliary excretion of susalimod, probably via the bromosulphthalein carrier, studied in a chronic bile fistula model in dogs.

Susalimod is a structural analogue of sulphasalazine, known to be extensively excreted in the bile in various animal species and for inducing bile duct hyperplasia after long-term treatment of the dog with doses exceeding 25 mg kg(-1). In this study local concentrations of susalimod in the bile duct were determined after oral administration in dogs. A chronic bile fistula experimental model was designed to affect the bile duct as little as possible. The dogs received repeated oral doses of 25-150 mg kg(-1) day(-1) for 5 days; these doses had been used in previous toxicology studies. Extremely high biliary concentrations of unchanged susalimod (20,000-43,000 microM) were measured. Biliary excretion approached saturation at the higher doses, resulting in super-proportional increases in peripheral plasma concentrations as the dose was increased. The maximal bile/plasma concentration ratio was 4300. The high biliary clearance was indicative of almost complete first-pass elimination at doses below saturation of the elimination process. Interaction studies with the biliary excretion marker bromosulphthalein (BSP) demonstrated that susalimod and BSP probably share the same carrier transport system in biliary excretion. The elimination of BSP from plasma was prolonged 20 times and the biliary excretion rate was markedly reduced when susalimod was co-administered with BSP. These results show that susalimod is highly enriched in the bile, in a saturable manner, after oral administration. The compound interacts with the biliary excretion of BSP, suggesting that it shares the same carrier-mediated transport system.

Binding of bilirubin and bromosulphthalein to albumin: Implications for understanding the pathophysiology of liver failure and its management

The binding/transporting functions of albumin provide the rationale for using albumin dialysis (e.g., molecular adsorbents recirculating system [MARS]) in liver failure. This study investigates these properties in vitro, validating the findings in vivo. In vitro bromosulphthalein (BSP) and bilirubin-spiked plasma were dialyzed against albumin and sampled. In vivo serum biochemistry was analyzed in: 7 MARS-treated liver failure patients; 98 MARS-treated patients from the MARS Registry; and 8 patients receiving albumin infusion. In vitro BSP concentrations did not equilibrate, but the molar ratio of BSP to albumin (C(BSP)/C(alb)) did, with no subsequent transmembrane transport, suggesting that the C(BSP)/C(alb) gradient (rather than simple diffusion) drives BSP transport. Bilirubin was transported similarly. In vivo serum bilirubin reduction during MARS sessions (n = 26) correlated with pre-treatment bilirubin (r = 0.42), but better (r = 0.85) with pre-treatment molar ratio of bilirubin to albumin (C(bilirubin)/C(alb)). The strongest correlation was between C(bilirubin)/C(alb) reduction and pre-treatment C(bilirubin)/C(alb) (r = 0.9). A similar pattern was observed in the MARS Registry patients. After albumin infusion (n = 8), both serum albumin and bilirubin increased, while C(bilirubin)/C(alb) remained unchanged. C(bilirubin)/C(alb) appears to be important in albumin dialysis, and generally in liver disease patients, reinforcing the importance of the toxin-binding functions of albumin in liver disease.

Hepatic arterial perfusion decreases intrahepatic shunting and maintains glucose uptake in the rat liver.

Intrahepatic shunts have an important function in the regulation of portal venous pressure in the normal rat liver. The present study determined their location, the region of confluence between the hepatic arterial and portal venous vasculatures, regions within the liver that are bypassed and the effects of hepatic arterial perfusion upon the intrahepatic redistribution of portal venous flow. Livers of male Sprague-Dawley rats were excised and perfused in vitro. Hepatic bromosulphthalein (BSP) and glucose uptake were measured in hepatic venous samples. Diversion of the hepatic arterial supply into the portal venous vasculature opened the portal venous intrahepatic shunts and resulted in a 50% reduction in portal venous glucose uptake from 29.6+/-1.6% to 14.9+/-0.9% ( P<0.0001 Student's paired t-test). Portal venous injection of 15-microm-diameter microspheres also opened the intrahepatic shunts and reduced portal venous glucose uptake to 10% from 29.0+/-1.6% to 7.8+/-0.9% ( P<0.0001 Student's paired t-test). No significant reductions in portal venous BSP uptake (43.0+/-6.9 to 48.0+/-4.8%) or hepatic arterial glucose uptake, from an average value of 94.0%, occurred. Therefore, cessation of hepatic arterial perfusion or portal venous injection of microspheres reduced portal venous glucose uptake without affecting BSP uptake. It is concluded that intrahepatic shunts divert perfusate away from the perivenous, sinusoidal (zone III) regions and into the hepatic venous vasculature, distal to zone III. The microsphere data indicate that confluence between the hepatic arterial and portal venous vasculatures occurs mainly in sinusoidal zone II.

Hepatic function during prolonged isolated rat liver perfusion using a new miniaturized perfusion circuit

Previous designs of isolated rat liver perfusion circuits used for toxicological investigations are often expensive, cumbersome, or traumatic in relation to hepatic biocompatibility following extended perfusion times. A new, miniaturized circuit that incorporates a novel design of organ bath, to maintain a buoyant preparation, and a high-efficiency miniaturized membrane tubing oxygenator is described. Livers from male Sprague-Dawley rats were perfused continuously for 6 hr in vitro using rat blood diluted to a perfusate hematocrit of 9.75 ± 0.35% with Krebs-Henseleit buffer (KHB). Hepatic function was evaluated by measurement of perfusion pressure, flow rate, bile volume production, bile bilirubin content, hepatic oxygen uptake (HOU), bromosulphthalein (BSP) removal, hepatic enzyme activities, and electrolyte concentrations, and finally by histological examination. Perfusion pressure and flow rate remained stable at 8.7 ± 1.7 mmHg and 1.92 ± 0.06 mL min −1 g −1 liver, respectively. Bile volume production and HOU were maximal at 784 ± 84 μL h −1 and 0.99 μmol/L min −1 g −1 respectively. Erythrocyte damage in the perfusate was evaluated by measurement of reduction in perfusate hematocrit from 9.75 ± 0.35% to 9.27 ± 0.24%, and increases in plasma free hemoglobin, which rose from 85.8 ± 12.3 mg% to 650.1 ± 53.3 mg% over the 6-hr perfusion period studied. Using bile volume production, hepatic oxygen uptake, and the liberation of plasma free hemoglobin as the most sensitive indices of adverse conditions, the new circuit was capable of supporting an isolated perfused rat liver for periods of up to 6 hr under close-to-physiological conditions.

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

1. In confluent primary cultures of rat hepatocytes, membrane effects of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) were recorded with conventional microelectrodes. In addition, cell pH and cell Ca2+ were monitored by use of the fluorescent dyes BCECF and fluo-3, respectively. Uptake of DIDS was determined by measuring intracellular DIDS fluorescence between 470 and 520 nm (excitation wavelength 390 nm). 2. In the presence of 0.2 mM DIDS, membrane voltages hyperpolarized from -44.0 +/- 1.8 to -73.1 +/- 1.9 mV (n = 16). This change was monophasic and occurred with a time constant of 170 +/- 25 s. The effect was only partly reversible. 3. Cable analysis revealed a concomitant decrease in the specific cell membrane resistance from 3.2 to 1.5 k omega cm2. 4. In ion substitution experiments, a 10-fold elevation of external K+ (from 2.5 to 25 mM) depolarized cell membranes by 6.2 +/- 1.5 mV (n = 5). In the presence of 0.2 mM DIDS, this membrane response was increased 5-fold to 32.2 +/- 4.1 mV. 5. Replacement of Cl- by 99% with gluconate depolarized the cells by 9.3 +/- 1.9 mV. In contrast, with 0.2 mM DIDS present, Cl- removal led to a membrane hyperpolarization of 5.9 +/- 0.9 mV (n = 4). 6. DIDS had no effect on cytosolic pH or Ca2+. 7. To determine the sidedness of the DIDS effect, i.e. to analyse if the increase in K+ conductance is mediated by uptake of the compound, DIDS was added in the presence of different substrates of hepatocellular anion transport. Taurocholate (50 microM) and frusemide (0.5 mM), which are both taken up via the sinusoidal multi-specific bile acid transporter, did not change DIDS-induced membrane hyperpolarization. 8. In contrast, 0.5 mM bromosulphthalein (BSP), a substrate of the bilirubin transporter, competitively inhibited the membrane hyperpolarization elicited by various concentrations of DIDS (0.1-1.0 mM). 9. Hepatocellular uptake of BSP is known to be, in part, Cl- dependent and to be competitively inhibited by Indocyanine Green. When 0.2 mM DIDS was added to a superfusate, in which 99% of Cl- had been exchanged by gluconate, the velocity of membrane hyperpolarization was decreased by 45%. In the presence of Indocyanine Green (0.1 mM) DIDS-induced membrane hyperpolarization was reduced to approximately 20%. 10. Addition of 0.2 mM DIDS to hepatocyte monolayers led to a time-dependent increase in cell fluorescence which was absent at 4 degrees C and which was completely blocked by 0.5 mM BSP.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical chemical diagnosis of diseases assisted by logistic regression illustrated by diagnosis of canine primary and secondary hepatobiliary diseases.

The purpose of the present study was to demonstrate the use of logistic regression models in the prediction of diseases using the prediction of canine primary and secondary hepatobiliary diseases as an example. Briefly, in a logistic regression model independent variables (i.e. the analytical results) are combined in a linear equation that is used to estimate the logarithm of the odds (logit) of an event (i.e. having primary or secondary hepatobiliary disease). From the estimated logit given by the logistic regression model, a conditional probability of the event (i.e. having primary or secondary hepatobiliary disease) can be calculated. Twenty-six dogs with verified primary and secondary hepatobiliary diseases and 19 dogs, initially suspected to have hepatobiliary diseases, but with apparently other diseases, were included in the study. The following clinical chemical parameters were measured: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (AP), bilirubin(Total) (TB), urea, glucose, retention of bromosulphthalein (BSP), fasting and postprandial total serum bile acid concentration (FSBA and PSBA). Logistic regression analysis, using the CATMOD procedure in SAS, was used to select which of the measured parameters should be included in the model, and to derive a logistic regression model using the selected parameters. To observe more closely the potential of the logistic regression model, the model was also used to classify a test group consisting of 13 dogs (6 dogs with hepatobiliary diseases and 7 dogs with other diseases). By logistic regression analysis, ASAT and PSBA were selected to be included in the final model, and the final logistic regression model was Y = -3.194 + 0.044.PSBA + 3.251.ASAT. The logistic regression model classified correctly 38 (84%) of 45 dogs in the present study. Specifically, 21 (81%) of 26 dogs with verified primary or secondary hepatobiliary diseases and 17 (90%) of 19 dogs with various other diseases were correctly classified by the logistic regression model. When the model was used on the test group, 5 (83%) of 6 dogs with hepatobiliary diseases and 7 dogs (100%) of 7 dogs with other diseases were correctly classified. Even though the logistic regression model derived in the present study only serves as an example, thus reducing the practical usefulness of the derived logistic regression model, the present study indicates a great potential of logistic models for the diagnosis of diseases.

Antiarrhythmic drugs impair hepatic uptake and secretory function by different mechanisms in the isolated perfused rat liver

In the present study the effect of various antiarrhythmic drugs on hepatic perfusi organic anions and biliary secretion using the isolated perfused rat liver was examined. Infusion of verapamil (VP), diltiazem, N-propyl-ajmaline (NPAB), and quinidine at pharmacological doses induced consistently a 1.4–1.6-fold increase in portal pressure accompanied by a ∼60% decrease in bile flow and a ∼65% inhibition of biliary taurocholate (TC) excretion. Furthermore, hepatic uptake of oxygen, bromosulphthalein (BSP), and TC was significantly reduced. All these effects were dose-dependent and reversible upon withdrawal of the drugs. Studies of the hepatic circulation using a Trypan blue staining technique demonstrated a patchy perfusion pattern during infusion of the antiarrhythmic drugs as compared to the homogenously stained control organ. The hemodynamic alterations and the impairment of the hepatic initial uptake function could be entirely prevented by concomitant administration of the vasodilator papaverine. Bile flow and biliary TC excretion, however, were still inhibited under these conditions. The present results indicate that antiarrhythmic drugs produce cholestasis in the isolated perfused rat liver independently of their adverse effect on hepatic hemodynamics.

The clearance of bromosulphthalein from plasma as a measure of the changes in hepatic function during pregnancy and lactation in ewes

Single intravenous injections of bromosulphthalein (BSP) were given to sheep and the change in plasma BSP concentration with time was analysed by computer to obtain the proportionality transfer constants ‘ a’, ‘ h’ and ‘ b’ and plasma volume. The BSP half-time, fractional clearance and retention values were also calculated. Alterations in BSP clearance were compared with changes in bilirubin and liver specific enzyme activities in ewes in late pregnancy and early lactation. The measurement of BSP clearance, particularly the proportionality transfer constants, yielded more information about the alterations in hepatocellular function during pregnancy and lactation than the changes in clinical chemistry. It is suggested that these alterations in liver function are physiological rather than pathological at these times.

Altered hepatic function in vitamin D-deprived rats

Rats were rendered vitamin D-deficient by housing them in a room free of ultraviolet light and maintaining them for 20 weeks on a diet devoid of only vitamin D. The vitamin D-deficiency state was confirmed by the undetectable levels of circulating vitamin D metabolites, severe hypocalcaemia and significantly reduced intestinal calcium transport. Liver function and protein metabolism in these rats were assessed by bromosulphthalein (BSP) clearance, liver histology, plasma transaminases and alkaline phosphatase, and 14C-labelled amino acid incorporation into liver and plasma proteins. Subtle alterations in hepatic function, as manifested by delayed BSP clearance, elevated levels of plasma transaminases and alkaline phosphatase, were noticed. Liver histology revealed changes consistent with periportal necrosis. Synthesis of liver and plasma proteins were reduced by 26–34% ( P < 0.01), without affecting the circulating levels of plasma proteins, suggesting reduced protein turnover in vitamin D-deprived rats. The results strongly suggest the direct/indirect involvement of vitamin D in mediating the altered liver function.

Clearance of bromosulphthalein by the liver of sheep

The application of a serial sampling technique for assessing bromosulphthalein (BSP) clearance by the liver of sheep is described. After a single intravenous injection BSP was almost completely cleared by the liver into the bile. Clearance by the liver was in two phases which allowed the calculation of the proportionality transfer constants a (from plasma to liver), h (from liver to bile) and b (from liver back to plasma). The ratio of a:h was approximately 4:1 in sheep. The proportionality transfer constants had the advantage over fractional clearance and retention tests of relating the test more precisely to the condition of the liver at the cellular level. The single injection method was simple, non-invasive and repeatable and suitable for conscious animals.

The effect of preoperative oral fluid and ranitidine on gastric fluid volume and pH

One hundred unpremedicated daycare patients were randomly assigned to one of four groups. Between two and three hours preoperatively all patients received either oral ranitidine 150 mg, or placebo, with bromosulphthalein (BSP) 50 mg in 10 ml water, immediately followed by either 150 ml water or no further fluid. The residual gastric volume (RGV) in the two placebo groups was significantly lower in patients given 150 ml water (20.6 +/- 14.1 ml) than in those who continued fasting (29.9 +/- 18.2 ml) (p less than 0.05). The RGV was further significantly decreased in the two ranitidine groups (10.0 +/- 8.8, 9.7 +/- 10.5 ml) compared with the two placebo groups (20.6 +/- 14.1, 29.9 +/- 18.9 ml) (p less than 0.01). Mean pH values were significantly higher in the two ranitidine groups (6.71 +/- 0.99; 6.31 +/- 1.81) than in the two placebo groups (2.05 +/- 1.41; 1.72 +/- 0.33) but were not significantly different in the fluid versus non-fluid groups. In patients who ingested 150 ml water there was no correlation between the premedication interval and either RGV or pH values. The extremely low percentage of the original BSP (less than 0.9 per cent) in the gastric fluid of these patients demonstrated that gastric emptying of the ingested water was virtually complete prior to surgery. The combination of RGV of 25 ml or more with pH less than 2.5 was present in 56 per cent of patients who had only a sip of water with placebo, in 28 per cent of those who received 150 ml water with placebo, and in only two per cent of those patients who received ranitidine.

THE USE OF A MODIFIED BROMOSULPHTHALEIN EXCRETION TEST FOR THE MEASUREMENT OF HEPATIC BLOOD FLOW IN CALVES

Single intravenous injections of bromosulphthalein (BSP) were given to calves before and at intervals after the oral administration of 1000 metacercariae of Fasciola hepatica. The change in BSP concentration with time was analysed by computer to obtain the proportion of BSP extracted from plasma by hepatic cells (E) and the rate of hepatic blood flow (F). The increasing age of the normal calves during the 34 weeks of experiment had no significant effect on E or F. There was no significant effect of the duration of the fasciola lesion on E or F. When all tests on infected calves were compared as a group with tests on uninfected calves, there was a significant reduction in E(P less than 0.05) and in F(P less than 0.001) in the calves infected with F. hepatica. A survey of previously reported results suggests that sheep and cattle have a wide range of F values and that F is probably lower in 3-month-old calves than in adult cattle.

Plasma Clearance of Bromosulphthalein in Surgically-Prepared Sheep

SUMMARY Plasma clearance of bromosulphthalein (BSP) was investigated as a method to assess the normality of liver function in conscious sheep after major surgery of the liver. In sheep with gall bladder fistulation (GBF, three ewes), bile duct catheterization (BDC, five ewes) or hepatic and portal vein catheterization (HPVC, two ewes), 25 experiments gave a BSP fractional clearance of 0·35 ± 0·014/min (mean ± SEM) after single i. v. injection of 1·8 to 7·9 mg BSP/kg bodyweight. The notional volumes of plasma cleared of BSP in unit time (ml/kg/min) were 10·3 for control observations (5), 11·65 for GBF (10), 11·26 for BDC (6) and 11·43 for HPVC (4) which were not significantly different between animals or treatments.

The effect of bromosulphthalein (BSP) on biliary lipid secretion

The effect of bromosulphthalein (BSP) on biliary lipid secretion

Conjugation of bromosulphthalein (BSP) with reduced glutathione in the gastrointestinal tract in vertebrate species

Present study has revealed the existence of bromosulphthalein-glutathione conjugating enzyme activity in the gastrointestinal tract of all the vertebrate species tested. The enzyme activity demonstrated in the intestine is comparable to that in kidneys. It is inferred that like the liver and kidney, the gastrointestinal tract may have detoxicating/conjugating function.

Further studies of binding of bromosulphthalein sodium by human serum albumin: effects of albumin concentration and buffer composition.

1. Binding isotherms of equilibrium solution concentration of bromosulphthalein (BSP) determined on the number of moles of BSP bound per mole of human serum albumin (HSA) in 310 ideal milliosmolar pH 7.4, Krebs-Henseleit and Krebs improved mammalian Ringer number 1 buffers at 37 degrees C were determined using continuous diafiltration. The albumin concentration range was from about 10 to 30 g/litre.2. The results indicate a competition between HSA polymerization and HSA binding BSP, confirming in more physiological conditions, the findings of Crawford, Jones, Thompson & Wells (1972) with pH 7.4 phosphate buffer.3. The results in Krebs-Henseleit buffer were markedly different from those in Krebs mammalian Ringer buffer and it is suggested that the differences in ionic composition influence the HSA conformation and so affect the competition between HSA polymerization and HSA binding BSP.

Binding of bromosulphthalein sodium by human serum albumin using a continuous diafiltration technique.

1. The recently introduced ultrafiltration-cum-dialysis technique termed continuous diafiltration has been used to obtain detailed binding isotherms of equilibrium solution concentration of bromosulphthalein (BSP) versus the number of molecules of BSP bound per molecule of human serum albumin for pure human serum albumin solutions (albumin concentrations: 1.59-3.39 g/100 ml) and for three dilutions of pooled human serum (albumin concentrations: 1.45-2.85 g/100 ml) in 310 ideal milliosmolar phosphate buffer at pH 7.4 and 22 degrees C.2. Qualitative analysis of the isotherms seems to indicate that there is a competition between the polymerization of the albumin to dimers and high oligomers and the binding of BSP by albumin.3. The binding capacity of pure human serum albumin at total BSP (bound and free) concentrations below 2.3 mM is greater than that of the pooled serum at an equivalent albumin concentration, possibly indicating the blockage of binding sites by more strongly binding ligands.4. The binding capacity of pure albumin at total BSP concentrations greater than 2.3 mM is less than that of pooled serum at an equivalent albumin concentration, suggesting the presence of other non-ultrafilterable materials in the serum capable of binding BSP.

Liver Function in Dairy Cows at Parturition

SUMMARY The possibility that subclinical liver damage may occur normally during late pregnancy and at parturition has been investigated in healthy dairy cows by measuring the plasma concentrations of the liver-specific enzyme G.D. and of G.O.T. and G.P.T., and also by measuring the rate of clearance of bromosulphthalein (B.S.P.) from the plasma approximately one week before, at, and one week after calving. Dimidium bromide, a known liver poison, was used to produce liver damage in one cow, and its effect on the same parameters have been studied. There were no significant changes in the concentration of G.D. during the 2 to 3 weeks spanning parturition, even though 3 cows showed evidence of milk fever, but there was a significant decrease in B.S.P. clearance one week after calving compared with the pre-calving levels. The mean values of k, the fractional clearance constant between 2 and 15 minutes after dosing, and kV, the plasma clearance before and after calving were −0·151 min.−1, and 6·74 l./min., and −0·121 min.−1 and 4·95 l./min. respectively. In contrast, a dose of dimidium bromide to one cow sufficient to cause a large rapid increase in the plasma concentration of G.D. 40 days after dosing did not produce any change in the liver’s capacity to clear B.S.P. from plasma at that time. It seems likely, therefore, that changes in liver function at calving in the cow are not degenerative in nature, but purely functional and related to the metabolic demands of lactation.

Studies on the influence of sex hormones on the avian liver. I. Sexual differences in avian liver clearance curves.

SUMMARY 1. A modified method of determining the avian liver clearance of intravenously administered sodium bromosulphthalein (BSP) is described. 2. Using this test, a sexual difference has been observed in the clearance gradients over the periods 5–10 and 10–15 min after injection of BSP. This appears to be determined by sex hormones, in that males caponized by stilboestrol possess a female type of clearance. 3. Surgically caponized birds appear to occupy an anomalous position which can be variously interpreted as showing female or male trends, according to the particular clearance gradients selected. Androgen, however, causes a clear reversion to the male type of excretion. 4. High initial BSP concentrations in the plasma of certain females have been correlated with a relatively smaller blood volume in proportion to body weight increase.