[Hepatoprotective properties of cytochrome C in traumatic toxicosis].

Experiments on rats showed that traumatic toxicosis (crush syndrome) was accompanied by disorders of both excretion and detoxication functions of the liver and a decrease in the energy potential of the liver. Systemic administration of cytochrome C (10 mg/kg) immediately after trauma and decompression increased the level of endogenous cytochrome C, recovered the pool of adenine nucleotides, normalized bromsulfaleine excretion from the blood, and decreased the content of toxic metabolites in the blood. The obtained experimental data show that cytochrome C possesses high hepatoprotective properties with respect to the development of traumatic toxicosis.

Nrf2 activation enhances biliary excretion of sulfobromophthalein by inducing glutathione-S-transferase activity.

Sulfobromophthalein (BSP) is used to study hepatobiliary excretory function. BSP is conjugated with glutathione (GSH), whereas its dibrominated analog disulfobromophthalein (DBSP) is not conjugated with GSH prior to biliary excretion. In addition, both BSP and DBSP are transported into hepatocytes via organic anion-transporting polypeptides and excreted into bile via multidrug resistance-associated protein 2 (Mrp2). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that under basal conditions is targeted for proteasomal degradation in the cytosol by kelch-like ECH-associated protein 1 (Keap1). Electrophilic and oxidative stress facilitate Nrf2 nuclear translocation and subsequent induction of cytoprotective genes, including GSH synthetic enzymes, GSH-S-transferases (Gsts), and Mrp transporters. The current study determined whether varying the amount of Nrf2 activation would effect the elimination of BSP and DBSP. Male wild-type (WT), Nrf2-null, and Keap1-knockdown (Keap1-kd) mice were administered BSP or DBSP. Within 30 min, Nrf2-null mice excreted 25%, WT mice 52%, and Keap1-kd mice 80% of the injected BSP. Liver GSH content was not altered by BSP. The biliary excretion of GSH and messenger RNA (mRNA) expression of major Gsts were directly proportional to the amount of Nrf2. Moreover, BSP-GSH conjugation activity in the liver of Nrf2-null and Keap1-kd mice was 42% and 237% of WT mice, respectively. In contrast to BSP, there were no differences in biliary excretion or plasma disappearance of DBSP among the three genotypes, suggesting that the modest differences in Mrp2 mRNA expression among genotypes do not affect BSP or DBSP biliary excretion. Collectively, these results indicate that increased biliary excretion of BSP, and possibly other compounds, is due to Nrf2-induced Gst mRNA expression and enzyme activity.

Rotor-type hyperbilirubinaemia has no defect in the canalicular bilirubin export pump.

BACKGROUND: The cause of Rotor syndrome (RS), a rare-familial conjugated hyperbilirubinaemia with normal liver histology, is unclear. We hypothesized that RS can be an allelic variant of Dubin-Johnson syndrome, caused by mutation in ABCC2, and investigated ABCC2 (gene) and ABCC2 (protein) in two patients with RS. METHODS: A 57-year-old male presented with a 5-year history of predominantly conjugated hyperbilirubinaemia (170 micromol/l). Urinary porphyrin excretion was increased; cholescintigraphy revealed no chromoexcretion. A 68-year-old male presented with lifelong conjugated hyperbilirubinaemia (85 micromol/l). Bromosulfophthalein elimination was typical for RS. Both patients had histologically normal liver, without pigment. ABCC2 expression was investigated by confocal fluorescence microscopy. ABCC2 was sequenced from genomic DNA and cDNA, and exon deletions/duplications were sought by comparative genomic hybridization on a custom micro-array. RESULTS: In both patients, ABCC2 was expressed unremarkably at the apical membrane of hepatocytes and no sequence alterations were found in 32 exons, adjacent intronic regions and the promoter region of ABCC2. CONCLUSIONS: Rotor-type hyperbilirubinaemia is not an allelic variant of ABCC2 deficiency.

Does paracellular permeability play a role in cholephilic dye-induced cholestasis?

Changes in hepatic paracellular permeability were investigated during the development of cholephilic dye-induced cholestasis in rats. For this purpose, four dyes with different cholestatic potency (phenol red, sulfobromophthalein, bromcresol green and rose bengal) were infused at a high, potentially damaging dose (280 nmol/min per 100 g body wt., i.v.), and changes in paracellular permeability were continuously monitored by measuring the access into bile of the permeability probe -14C-sucrose. The cholestatic potency of the different dyes was: rose bengal > bromcresol green > sulfobromophthalein > phenol red. All dyes increased [14C]sucrose bile-to-plasma ratio, producing a displacement towards curves of higher permeability. The capability of the dyes to increase biliary permeability followed the same order as their respective cholestatic potencies. The possible implications of the present results for cholephilic dye-induced cholestasis are discussed.

Loperamide effects on hepatobiliary function, intestinal transit and analgesia in mice.

Loperamide effects on hepatobiliary function, analgesia and gut transit were studied in mice. Varying doses of the antidiarrheal drug, loperamide, were administered to mice by intracerebroventricular, intravenous, subcutaneous and intragastric routes. Gut motility was determined by intestinal transit of India ink, analgesia by warm water tail flick latency, and hepatobiliary function by retention of the anionic dye, sulfobromophthalein in plasma and liver. When given by all routes at modest doses, loperamide slowed intestinal transit. Analgesia, a centrally mediated opiate effect, was only detected after intracerebroventricular or subcutaneous loperamide at high, near-toxic doses. Elevations of plasma and liver sulfobromophthalein were noted at routes and doses which slowed gut transit, well below those needed for analgesia. Intragastric loperamide at one fortieth its LD50 caused marked elevation of sulfobromophthalein levels and gut slowing, but no analgesia. Sulfobromophthalein elevation and gut slowing by intragastric loperamide were not affected by spinal cord transection but were reversed by naltrexone, an opiate antagonist. Non-toxic doses of loperamide slow gut transit and modify hepatobiliary function in mice by opiate actions at peripheral sites.

[Biliary scintigraphy. Application to the study of chronic cholestasis]. / Scintigraphie biliaire. Application à l'étude des cholestases chroniques.

In a sample population of 49 subjects (7 normal, 42 with various liver diseases), the parameters of the activity/time curve of trimethylbromo-iminodicetic acid (TBIDA) biliary scintigraphy were compared with the clearances of bromosulfophthalein (BSP) and indocyanine green (ICG). Correlation between T1/2 and P2 BSP slope was r = 0.50 (n = 33; P < 0.01). Correlation between Tmax TBIDA and fractional ICG clearance (P ICG) was r = 0.65 (n = 44; P < 0.001). In 23 cases of chronic cholestasis correlations remained significant (T1/2-P2 BSP: r = 0.53; n = 17; P = 0.02; Tmax-P ICG: r = 0.59; n = 17; P < 0.01). A prospective study of 11 cases of chronic intrahepatic cholestasis (primary biliary cirrhosis 8, primary sclerosing cirrhosis 3) showed that these two types of tests varied concordantly. Biliary scintigraphy, therefore, seems to be an accurate method to explore hepatocellular mass (degree of hepatic insufficiency) and cholestasis. The validation of biliary TBIDA scintigraphy as hepatobiliary functional exploration method and the possibility to study intrahepatic "regions of interest" defined a priori would make it possible to obtain a functional estimate of hepatic segments or lobes, for example before wide liver excision.

Methods for the quantitation of bromosulfophthalein and its glutathione conjugate in biological fluids.

This paper describes a solvent-gradient HPLC method which was developed for the quantitation of bromosulfophthalein (BSP) in erythrocyte and albumin containing (blood) perfusate samples, and of BSP and its glutathione conjugate (BSP-GSH) in bile samples obtained from rat liver perfusion experiments. Phenolphthalein was used as an internal standard. The cysteinyl-, N-acetylcysteinyl-, and cysteinylglycinyl-BSP conjugates did not interfere with the HPLC assay and cysteine, N-acetylcysteine, [3H]GSH, and [3H]GSSG eluted within the first 6 min postinjection onto the HPLC system. A spectrophotometric method was also developed for the rapid quantitation of BSP in blood perfusate; tracer [14C]urea was utilized as the internal standard. Although the spectrophotometric method was less sensitive than the HPLC method, good correlation was found to exist between the methods.

Influence of hepatic and renal failure on pharmacokinetic properties of the novel recombinant plasminogen activator BM 06.022 in rats.

BM 06.022 is a novel recombinant, unglycosylated plasminogen activator comprising only the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA), and it has a longer half-life than t-PA. Because t-PA is mainly cleared by the liver, rat models of hepatic and renal insufficiency were used to identify the main catabolic organ of BM 06.022, compared with alteplase (recombinant t-PA). Hepatic insufficiency in rats was chemically induced by pretreatment with carbon tetrachloride for 1 day; renal insufficiency was achieved by acute bilateral, surgical nephrectomy. Plasma concentration of functionally active BM 06.022 or alteplase was measured by an indirect spectrophotometric assay. Intravenous administration of 200 kU/kg of BM 06.022 or alteplase over 15 sec to rats with hepatic failure or olive oil pretreatment as control did not significantly alter the total plasma clearance (CL) of BM 06.022 vs. control (4.9 +/- 0.5 vs. 5.7 +/- 0.5 ml.min-1 x kg-1, NS) in contrast to alteplase (32.1 +/- 6.5 vs. 82.3 +/- 12.9 ml.min-1 x kg-1, p < 0.05). Renal insufficiency increased the CL of BM 06.022 vs. sham surgery (3.1 +/- 0.4 vs. 6.3 +/- 0.5 ml.min-1 x kg-1, p < 0.05) in contrast to alteplase (33.2 +/- 5.2 vs. 37.2 +/- 7.2 ml.min-1 x kg-1, NS). In vitro incubation of 2000 U/ml BM 06.022 or alteplase in citrate blood and plasma demonstrated a decrease of the plasma concentration with a shorter (p < 0.001) half-life for BM 06.022 than for alteplase in blood (71.9 +/- 3.1 vs. 130 +/- 3.3 min) and in plasma (62.9 +/- 1.2 vs. 129.9 +/- 5.3 min).(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic determination of sulphobromophthalein and its conjugates.

A simple, sensitive and selective high-performance liquid chromatographic method for the determination of sulphobromophthalein and its mercaptide conjugates in rat bile was developed. These pigments, which have an absorption maximum at 580 nm in alkaline solution, were separated isocratically on an alkali-resistant ODS column by paired-ion chromatography. Analysis of bile samples obtained after intravenous administration of sulphobromophthalein to rats showed the presence of at least twenty peaks of metabolites, of which thirteen were identified and seven quantified.

Bromosulfophthalein disposition in chronically bile duct obstructed rats.

The disposition of bromosulfophthalein was studied in chronically bile duct obstructed rats. In this model a catheter was inserted into the common bile duct and the distal tip was sealed. Resumption of bile flow was achieved with great ease. Obstruction of bile duct for 18 days in rats resulted in elevated bilirubin, ALT, AST, and alkaline phosphatase levels. Portal hypertension developed within this period (11.6 +/- 0.5 in obstructed rats vs. 8.6 +/- 0.6 mm Hg in sham-operated group). After the bile duct obstruction was opened, the half-life time for elimination of bromosulfophthalein (42.30 +/- 6.47 min) was longer than in sham-operated rats (21.23 +/- 3.34 min). Plasma clearance was reduced by 70% in bile duct obstructed rats. In spite of increased bile flow rate, biliary excretion of the dye was reduced by 40% in chronically bile duct obstructed rats. Hepatic glutathione levels were significantly reduced by 20% in this model. The specific activity of glutathione S-transferase with chlorodinitrobenzene and styrene oxide, as substrates, was reduced by 50% and 30%, respectively. However, the percent of conjugated bromosulfophthalein in bile was similar to that of sham-operated rats.

Hepatobiliary effects of morphine are mediated in the brain.

Morphine slows hepatobiliary elimination of sulfobromophthalein in rodents, raising dye levels in plasma and liver. Earlier studies showed these effects to be independent of other opiate effects such as bile duct spasm, hypothermia or blood gas changes resulting from respiratory depression. Because opiate receptors are distributed throughout the body, within the central nervous system and at peripheral sites including the gastrointestinal tract, experiments were performed to ascertain whether central or peripheral sites mediate the hepatobiliary effects of morphine. Sulfobromophthalein was administered intravenously to mice and its levels were measured in plasma and liver. Tail-flick latency indicated centrally mediated analgesia. Inhibited intestinal transit of India ink reflected an opiate effect with a significant peripheral component. When injected into a cerebral ventricle morphine was much more potent in producing analgesia and raising sulfobromophthalein levels than when administered intravenously or intraperitoneally. An intravenous dose of naloxone that reversed morphine analgesia also prevented sulfobromophthalein elevation but did not prevent gut slowing. Naltrexone injected in a cerebral ventricle also reversed analgesia and sulfobromophthalein elevation but not intestinal slowing. The polar opiate agonist N-methylmorphine did not cause analgesia or raise sulfobromophthalein levels at peripheral intraperitoneal doses to 100 mg/kg. When given in a central ventricle at 4 x 10(-3) mg/kg, this agent produced analgesia and raised sulfobromophthalein but did not slow intestinal transit. After spinal cord transection, intravenous morphine did not retard the tail-flick response or affect sulfobromophthalein disposition, but peripherally mediated intestinal transit was slowed as it was in intact mice. These experiments demonstrate parallel opiate effects on analgesia and on BSP disposition but not on intestinal transit.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin and liver toxicity in experimental Lantana camara poisoning in albino rats.

Dried alcoholic extract of fresh Lantana camara leaves (LE), on oral administration to albino rats of both sexes, induced photodermatitis during exposure to clear sunlight for 1 hr. Its severity was related to the dose of LE and was maximal in rats exposed to sunlight from 4 to 14 hr after feeding LE and gradually declined over 40 hr. Wavelengths of light about 540 to 570 mu only were effective. In control study, the alcoholic extract of edible spinach leaves was only 1/3 in potency and its effect lasted for less than 20 hr. LE did not raise serum bilirubin, SGOT, SGPT or cause liver injury as assessed by light microscopy. However, like CCL4 but unlike spinach extract, LE impaired excretion of BSP by liver, proportionate to the dose and also maximal at 5.5 hr declining thereafter over 40 hr.

Rapid method for the determination and quantification of bromosulphophthalein and metabolites in cultured hepatocytes, culture media and bile by high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method for the rapid, selective and sensitive analysis of samples containing bromosulphophthalein (BSP) and its conjugates is presented. The method is useful for analysis in bile, culture media and cultured hepatocytes. Two sample preparation methods are described. Even though BSP recovery from albumin binding is complete, only a small percentage of free BSP can be detected in cells, possibly owing to a conjugation-related pool of BSP in cells. As BSP-glutathione recovery is complete, the method offers a useful tool to investigate impairment of glutathione conjugation.

Determination of inorganic radioiodine in 131/-Rose Bengal and 131/-Bromosulphathalein

Desenvolvem-se um sistema rápido de cromatografia miniaturizada para controle radioquímico de Rosa Bengala-131/ e Bromossulfaleina 131/ utilizando como solvente uma soluçäo aquosa de sulfato de amônio 33%, pH 7,5. Os compostos marcados permanecem na origem enquanto os íons iodeto e iodato migram com Rf 0,5 e 0,9, respectivamente

[Duodenogastric reflux and stress ulcer (author's transl)]. / Duodenogastraler Reflux und Stressulkus

Duodenogastric reflux in 14 patients in an intensive care unit was compared with that in 12 healthy controls. In addition, intragastric bromsulphalein concentration was measured up to 60 minutes after intravenous administration. Reflux was much more frequent in the intensive-care patients and correlated with the incidence of gastric erosions and stress ulcers. Reflux is apparently one of three factors (in addition to ischaemia and HCl) which synergistically lead to stress-induced lesions of the gastric mucosa.

Differences of reabsorption of unconjugated BSP and BSP-glutathione from the rat biliary tree after retrograde intrabiliary injection.

It has been shown that water and electrolytes are reabsorbed from the biliary tract of the rat. Furthermore there are some suggestions for the reabsorption of organic compounds during their passage down the biliary tract. Our results presented in this paper demonstrate a different mode of biliary excretion of unconjugated BSP [BSP-U] and BSP- glutathione [BSP-GSH] after retrograde intrabiliary injection, BSP-GSH is excreted to a much greater extent than BSP-U within the first 5 minutes after retrograde administration. In other terms BSP-GSH is reabsorbed to a lesser extent than BSP-U. Additionally the reabsorption of BSP-U and BSP-G after retrograde injection seems to be dependent on the concentration and the contact time in the biliary tree. It is suggested that the different biliary excretion of BSP-U and BSP-GSH after intravenous injection might be explained partly by a different reabsorption mode.

Effect of age on liver function.

Serum concentrations of bilirubin, alkaline phosphatase, and GOT as well as the retention of BSP and 131I-rose bengal were determined in 43 normal subjects over 50 years old for the purpose of evaluating age-dependent variations. The normalcy of the liver in all subjects was confirmed by biopsy. The values of serum bilirubin, alkaline phosphatase, and GOT were unaltered with increasing age. Age also had no effect on the retention of BSP and 131I-rose bengal, in contrast to some previous studies in which the normalcy of the liver was not satisfactorily established. No variations between the sexes were seen in our study. The median of the 45 minute BSP retention test was 4.2 percent, with a ninth decile of 7.5 percent. The median of the retention of 131I-rose bengal was 32 percent, with a ninth decile of 39.4 percent.

Structural analysis of compartmental models for the hepatic kinetics of drugs.

The structure of some compartmental models for the analysis of the hepatobiliary kinetics of bromosulphalein (BSP) was studied in order to evaluate their adequacy in the estimation of the processes involved in the hepatic metabolism of drugs, namely uptake, conugation, and biliary excretion. Biological measurements were obtained from 4 cholecystectomized patients with a biliary T-tube. Blood and bile specimens were taken at various intervals after the administration of a single intravenous dose of BSP and analyzed for both direct BSP quantitation and chromatographic separation and estimation of BSP metabolic fractions. The structural analysis was carried out by using a mathematical model that described the kinetics of BSP. By means of computer simulations different measurement situations were analyzed, showing for each experimental condition the available information and the degree of accuracy of each estimated parameter. The obtained results show that the use of compartmental models can provide a useful theoretical framework by which the experimental data can be interpreted for the evaluation of the hepatobiliary metabolism and for a discriminant analysis between different physiopathological conditions.