[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.

The effect of sodium taurocholate on the hepatic metabolism of sulfobromophthalein sodium (BSP). The role of bile flow.

The influence of bile salts on the hepatic metabolism of sulfobromophthalein sodium (BSP) was studied in the perfused rat liver. During sodium taurocholate infusions, hepatic uptake of BSP from plasma was increased and appeared to be related to an enhanced transit of BSP from liver into bile. BSP-glutathione conjugation was not affected by the bile salt infusions, although bile salts inhibited the enzyme system in vitro. The major effect of bile salts was to increase the BSP transport maximum (Tm). When sodium taurocholate was infused in saline at a rate of 30 mumoles/hr, both bile flow and the BSP Tm increased, and remained at peak levels of 1.5 +/-0.12 mul/min per g liver and 21 +/-3.0 mug/min per g liver, respectively. In contrast, during saline infusion alone both levels were significantly lower (1.06 +/-0.17 mul/min per g liver and 15.8 +/-4.16 mug/min per g liver, respectively), and both fell progressively after the 2nd hr of perfusion. This decline in bile flow and BSP Tm was associated with a decrease in biliary bile salt excretion and was reversed by adding bile salts to the perfusate. Since the biliary concentration of BSP remained within a narrow range in all experiments, the BSP Tm was primarily determined by the rate of bile flow. Dependence of BSP Tm on the rate of bile production was further confirmed by changing the temperature of the perfusate during a constant infusion of taurocholate. BSP Tm paralleled temperature-induced changes in bile flow irrespective of changes in the level of bile salt excretion. Since the biliary concentration of BSP remained within a narrow range in all experiments, the concentrating capacity for BSP in bile may be the major limiting factor in BSP transport. Thus two independent factors appear to determine the BSP Tm: the bile BSP concentration, and the rate of bile production. Because taurocholate enhanced BSP transport only when it increased bile production, its effect on the BSP Tm appears to be attributable to its choleretic properties.

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.

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.

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.

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.

[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.

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.

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.

[Effect of protein-free diet on sulfobromophthalein hepatic transport (author's transl)]. / Effecto de la dieta aproteica sobre el transporte hepatico de bromosulfaleina

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.

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.

[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.

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.