The influence of taurocholate and dehydrocholate choleresis on plasma disappearance and biliary excretion of indocyanine green in the rat

Summary The pharmacokinetics of indocyanine green (ICG; 3.9 moles/kg and 12.9 moles/kg) were investigated in rats given infusion of either saline, taurocholate (106 moles/h) or dehydrocholate (106 or 268 moles/h). During the infusion of saline and taurocholate the plasma concentration of ICG decreased in a mono-exponential manner. However, with dehydrocholate the clearance of ICG from plasma showed two phases with different half lives. The half life of the rapid component (2.2 min) was about the same as the one found in the control experiments.After injection of 12.9 moles/kg ICG the biliary excretion of the dye increased by 138% during taurocholate administration, while an equimolar dehydrocholate infusion resulted in a mean increament of 55%. Under these circumstances the bile flow was stimulated by 195% and 297% resp.With the lower dose of ICG (3.9 moles/kg) however, there was no stimulation of the biliary ICG excretion with taurocholate. At this dose level an infusion of dehydrocholate (106 mol/h) enchanced the biliary output of ICG by approximately 54%, while administration of 268 mol/h resulted in a slight but significant decrease of 31%.These observations can be explained by assuming interaction of the bile acids with the hepatic transport of ICG at different sites. The appearance of the second component of the plasma curve during dehydrocholate infusion is possibly related to a diminished hepatic storage capacity for ICG and is not due to an effect on the primary hepatic uptake or biliary output of the dye.     

Multidrug resistance proteins 2 and 3 provide alternative routes for hepatic excretion of morphine-glucuronides

Glucuronidation is a major hepatic detoxification pathway for endogenous and exogenous compounds, resulting in the intracellular formation of polar metabolites that require specialized transporters for elimination. Multidrug resistance proteins (MRPs) are expressed in the liver and can transport glucuronosyl-conjugates. Using morphine as a model aglycone, we demonstrate that morphine-3-glucuronide (M3G), the predominant metabolite, is transported in vitro by human MRP2 (ABCC2), a protein present in the apical membrane of hepatocytes. Loss of biliary M3G secretion in Mrp2(-/-) mice results in its increased sinusoidal transport that can be attributed to Mrp3. Combined loss of Mrp2 and Mrp3 leads to a substantial accumulation of M3G in the liver, from which it is transported across the sinusoidal membrane at a low rate, resulting in the prolonged presence of M3G in plasma. Our results show that murine Mrp2 and Mrp3 provide alternative routes for the excretion of a glucuronidated substrate from the liver in vivo.     

Inhibition of biliary excretion of indocyanine green by the thiol-oxidizing agent, diazenedicarboxylic acid bis[N,N'-dimethylamide].

Biliary excretion of Indocyanine green (ICG) in Sprague-Dawley rats during constant intravenous infusion of the dye in vivo was inhibited by intraperitoneally administered diazenedicarboxylic acid bis[N,N'-dimethylamide] (diamide, 0.5 mmol/kg body wt), a glutathione-specific thiol-oxidizing agent. Significant inhibition of ICG excretion was observed also when ICG was injected rapidly 90 min after diamide administration. Disappearance of ICG from the plasma was not affected by diamide. Oxidized glutathione in bile increased transiently following diamide administration but returned to the basal level within 30 min. Hepatic concentrations of reduced and oxidized glutathione were not different from those of controls when determined 90 min after diamide administration. The inhibition of ICG excretion was completely prevented by subsequent administration of dithiothreitol (0.5 mmol/kg) 30 min after that of diamide. The results, therefore, suggest that the biliary excretion of ICG was inhibited by secondary changes in the redox status of thiols in hepatocytes caused by a transient increase in oxidized glutathione.     

The biliary elimination of amaranth, indocyanine green and nitrazepam in germ-free rats

In anaesthetised bile duct-cannulated rats the overall rate of bile flow was 15-50% lower in both male and female germ-free (GF) rats. Except in the case of amaranth in female GF rats, this was reflected in a lower rate of biliary excretion of the three test xenobiotics, of which two (amaranth and indocyanine green) are excreted unchanged and one (nitrazepam) is excreted solely as metabolites. It was also noted that compared with conventional (CV) rats the relative liver weights (g/kg body weight) were about 20% lower in GF animals. After the intravenous injection of 14C-nitrazepam, thin-layer chromatographic separation of biliary nitrazepam-derived radioactivity revealed three loci (A, B and C in decreasing order of polarity). The relative proportions of A, B and C were similar in GF and CV rats, with C and B being the major and minor "metabolites" respectively. When 14C-nitrazepam was given intragastrically to non-anaesthetised rats, by 9 days about 20% and 70% of the dose had been recovered in the urine and faeces respectively of both GF and CV rats. The rate of elimination of urinary radioactivity was similar in GF and CV rats. However, faecal elimination was much slower in GF animals, for example after 24 hr the respective amounts of radioactivity excreted in GF and CV rats corresponded to 13% and 52% of the dose. These findings indicate that the indigenous bacterial population of an animal may indirectly affect the disposition of a xenobiotic whether or not it is metabolised by the bacteria.     

Involvement of breast cancer resistance protein (ABCG2) in the biliary excretion mechanism of fluoroquinolones.

Fluoroquinolones are effective antibiotics for the treatment of bile duct infections. It has been shown that the biliary excretion of grepafloxacin is partly accounted for by multidrug resistance-associated protein 2 (MRP2/ABCC2), whereas neither MRP2 nor P-glycoprotein is involved in the biliary excretion of ulifloxacin. In the present study, we examined the involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion of fluoroquinolones (grepafloxacin, ulifloxacin, ciprofloxacin, and ofloxacin). In Madin-Darby canine kidney II cells expressing human BCRP or mouse Bcrp, the basal-to-apical transport of grepafloxacin and ulifloxacin was greater than that of the mock control, which was inhibited by a BCRP inhibitor, 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143). Plasma and bile concentrations of fluoroquinolones were determined in wild-type and Bcrp(-/-) mice after i.v. bolus injection. The cumulative biliary excretion of fluoroquinolones was significantly reduced in Bcrp(-/-) mice, resulting in a reduction of the biliary excretion clearances to 86, 50, 40, and 16 of the control values, for ciprofloxacin, grepafloxacin, ofloxacin, and ulifloxacin, respectively. Preinfusion of sulfobromophthalein significantly inhibited the biliary excretion of grepafloxacin in Bcrp(-/-) mice. There was no change in the tissue/plasma concentration ratios of fluoroquinolones in the liver or brain, whereas those in the kidney were increased 3.6- and 1.5-fold for ciprofloxacin and grepafloxacin, respectively, in Bcrp(-/-) mice but were unchanged for ofloxacin and ulifloxacin. The present study shows that BCRP mediates the biliary excretion of fluoroquinolones and suggests that it is also involved in the tubular secretion of ciprofloxacin and grepafloxacin.     

Increase in bile flow and biliary excretion of glutathione-derived sulfhydryls in rats by drug-metabolizing enzyme inducers is mediated by multidrug resistance protein 2.

Glutathione (GSH) is an important cellular constituent for normal liver homeostasis. Certain drug-metabolizing enzyme inducers (i.e., phenobarbital [PB] and pregnenolone-16alpha-carbonitrile [PCN]) increase biliary excretion of GSH-derived sulfhydryls (SH) as well as bile flow, whereas other drug-metabolizing enzyme inducers (i.e., 3-methylcholanthrene [3MC] and benzo(a)pyrene [BaP]), do not. The purpose of the study was to determine whether rat multidrug resistance protein 2 (Mrp2) is the inducible transporter responsible for increasing biliary SH excretion and bile flow. Sprague-Dawley (SD) rats were injected ip daily for 4 days with PB, PCN, 3MC, BaP, or vehicle; Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were injected ip daily for 4 days with PCN or vehicle. Although no drug-metabolizing enzyme inducer altered hepatic GSH in SD rats, PB and PCN significantly increased the rate of biliary SH excretion and bile flow. Neither 3MC nor BaP affected the biliary SH excretion rate or bile flow. In control EHBR rats, despite elevated hepatic GSH, the rate of biliary SH excretion was almost completely eliminated and bile flow was dramatically reduced compared with SD rats. Furthermore, PCN treatment did not affect bile flow or the biliary SH excretion rate in EHBR rats. PB and PCN also increased Mrp2 protein levels, but 3MC and BaP did not. None of the drug-metabolizing enzyme inducers tested significantly increased Mrp2 mRNA levels. PCN increased Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner. In conclusion, Mrp2 is the inducible efflux transporter responsible for increased biliary SH excretion and bile flow after administration of some drug-metabolizing enzyme inducers.     

Toxic effects of indocyanine green on rat liver mitochondria.

Indocyanine green (ICG) (68 to 171 nmol/mg of protein) inhibited the oxygen consumption of rat liver mitochondria in vitro. In state IV respiration, an acceleration of oxygen consumption occurred prior to this inhibition. ICG induced mitochondrial swelling in an isotonic KCl and K⁺ acetate medium but not in a 0.27 m sucrose medium. The increase in the ⁴²K diffusion space of isolated mitochondria was significantly larger than that of the [¹⁴C]sucrose diffusion space, indicating that ICG renders the mitochondrial membrane permeable to K⁺ ions. At the same time, an efflux of H⁺ ions from mitochondria was observed. It is concluded that the inhibition of mitochondrial oxygen consumption could be the result of a decrease in substrate penetration, as suggested by the diminution in NAD reduction by glutamate or isocitrate in the presence of ICG, without change in the activity of the corresponding dehydrogenases. A nigericin-like-mediated K⁺ for H⁺ antiport is proposed to account for the effects of ICG upon mitochondrial membrane permeability.     

Increased biliary excretion of glutathione is generated by the glutathione-dependent hepatobiliary transport of antimony and bismuth.

We have recently demonstrated that the hepatobiliary transport of arsenic is glutathione-dependent and is associated with a profound increase in biliary excretion of glutathione (GSH), hepatic GSH depletion and diminished GSH conjugation (Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 41: 937-944 and Gyurasics A, Varga F and Gregus Z, Biochem Pharmacol 42: 465-468, 1991). The present studies in rats aimed to determine whether antimony and bismuth, other metalloids in group Va of the periodic table, also possess similar properties. Antimony potassium tartrate (25-100 mumol/kg, i.v.) and bismuth ammonium citrate (50-200 mumol/kg, i.v.) increased up to 50- and 4-fold, respectively, the biliary excretion of non-protein thiols (NPSH). This resulted mainly from increased hepatobiliary transport of GSH as suggested by a close parallelism in the biliary excretion of NPSH and GSH after antimony or bismuth administration. Within 2 hr, rats excreted into bile 55 and 3% of the dose of antimony (50 mumol/kg, i.v.) and bismuth (150 mumol/kg, i.v.), respectively. The time courses of the biliary excretion of these metalloids and NPSH or GSH were strikingly similar suggesting co-ordinate hepatobiliary transport of the metalloids and GSH. However, at the peak of their excretion, each molecule of antimony or bismuth resulted in a co-transport of approximately three molecules of GSH. Diethyl maleate, indocyanine green and sulfobromophthalein (BSP), which decreased biliary excretion of GSH, significantly diminished excretion of antimony and bismuth into bile indicating that hepatobiliary transport of these metalloids is GSH-dependent. Administration of antimony, but not bismuth, decreased hepatic GSH level by 30% and reduced the GSH conjugation and biliary excretion of BSP. These studies demonstrate that the hepatobiliary transport of trivalent antimony and bismuth is GSH-dependent similarly to the hepatobiliary transport of trivalent arsenic. Proportionally to their biliary excretion rates, these metalloids generate increased biliary excretion of GSH probably because they are transported from liver to bile as unstable GSH complexes. The significant loss of hepatic GSH into bile as induced by arsenic or antimony may compromise conjugation of xenobiotics with GSH.     

Multidrug resistance in Serratia marcescens and cloning of genes responsible for the resistance

Six clinically isolated strains of Serratia marcescens were tested for their drug resistance. All showed fairly high resistance to many antimicrobial agents tested including norfloxacin, streptomycin, ampicillin, erythromycin, tetracycline, chloramphenicol, and antimicrobial dyes. Using the drug-hypersensitive strain of Escherichia coli KAM32 as the host, we cloned the genes responsible for multidrug resistance from chromosomal DNA of one of the strains of S. marcescens, NUSM8906. We obtained 28 hybrid plasmids that made host cells resistant to several antimicrobial agents. Many of the transformants harboring each of the plasmids showed multidrug resistance, and some showed resistance to specific drugs. The hybrid plasmids were classified into several groups based on their drug specificity. It appears that each class of plasmid carries different types of drug resistance genes. Analysis of such genes will reveal the multiple mechanisms involved in multidrug resistance in S. marcescens.     

Cerebral near infrared spectroscopy for the measurement of indocyanine green elimination in cirrhosis

BACKGROUND: Indocyanine green (ICG) clearance is a useful indicator of hepatic function but most measurement methods are invasive. AIM: To validate a less invasive technique using cerebral near infrared spectrophotometry (NIRS) to measure ICG elimination, and to compare it with the established methods for the determination of ICG clearance in a group of normal controls and patients with cirrhosis. METHOD: NIRS was used to measure ICG elimination in 41 cirrhotic patients and nine healthy volunteers. The first 13 of the cirrhotic patients also had their ICG clearance measured by the conventional spectrophotometric technique. RESULTS: NIRS ICG elimination rate (ICG-k) and spectrophotometry ICG-k values correlated strongly (r= 0. 828, P < 0.001, n=13). There was a significant reduction in the mean NIRS-k in cirrhotic patients and within Child-Pugh classes A, B, and C (P < 0.001). CONCLUSION: Measurement of ICG elimination by the NIRS method is at least as reliable as the conventional spectrophotometric technique in normals and in patients with cirrhosis. This technique merits further development for use as a bedside, less invasive liver function test.     

Studies on the biliary excretion mechanism of drugs. I. Biliary excretion of azo dyes in the rat

The mechanism of biliary excretion of the azo dyes, Azorubin S (AS), Amaranth (AM) and New Coccine (NC), in rats was investigated. It was observed that these azo dyes have an apparent transport maximum (Tm) for their biliary excretion. Further, the biliary excretion of these dyes was markedly depressed by phenolphthalein glucuronide (PPG) and probenecid, which are considered to be actively excreted into bile. The results, therefore, suggest that the biliary excretion of these dyes in rats involves an active transport process.     

Changes in indocyanine green kinetics after the administration of enalapril to healthy subjects.

Enalapril maleate, an angiotensin converting enzyme inhibitor, is a vasodilator liable to modify regional blood flow. The effects of an oral dose of 40 mg enalapril maleate on indocyanine green (ICG) kinetics were assessed in nine healthy subjects. At 4 h after the administration of the drug, a 35% decrease in ICG clearance was observed (P less than 0.01) associated with a 23% decrease in its volume of distribution (P less than 0.02). The half-life of ICG was not altered significantly by enalapril. These results suggest that the administration of enalapril maleate to healthy subjects may reduce apparent liver plasma flow and plasma volume, as a consequence of the pooling of blood in the hepatosplanchnic area.     

Two pathways for biliary copper excretion in the rat. The role of glutathione

To evaluate the role of glutathione in biliary copper excretion, we studied this process in control Wistar rats and in mutant Wistar rats (GY rats), in which the secretion of glutathione into bile is deficient. For comparison, biliary zinc excretion was determined simultaneously. In spite of the markedly reduced bile flow (-45%) in GY rats, biliary output rates of endogenous copper were virtually identical in GY and control rats. In contrast, zinc output was drastically reduced in GY rats compared to controls (-80%). Biliary excretion patterns after intravenous administration of copper, in doses ranging from 65 to 2265 nmol/100 g/body wt, showed a distinct rapid and slow phase in control rats. In GY rats, on the other hand, the rapid phase in copper excretion was absent but the slow phase appeared to be unaffected. Pretreatment of rats with diethylmaleate to deplete hepatic and biliary glutathione abolished the rapid phase of copper excretion in control rats, while the slow phase remained unaffected. No significant effect of diethylmaleate on the hepatic handling of exogenous copper was observed in GY rats. The maximal capacity of the slow copper excretion pathway was 40-45 nmol/hr/100 g body wt, both in control and GY rats; the capacity of rapid excretion pathway depended on the administered copper load. Intravenous injection of copper induced the biliary excretion of a substantial amount of zinc in control rats, but not in GY rats. These results indicate the existence of at least two distinct biliary excretory pathways for copper in the rat, i.e. a slow and a rapid pathway, with a glutathione dependency of the latter only. The basal excretion of (endogenous) copper, in contrast to that of zinc, can proceed independently of glutathione excretion. However, glutathione appears to be involved in the rapid secretion of excess copper.     

Effects of taurocholate infusion on biliary excretion in isolated perfused rat livers. Decreased biliary excretion of dibromosulfophthalein in pregnancy

A crossover experimental design was used to examine the effects of saline (SAL) vs. taurocholate (TC) infusion on hepatic excretory function in isolated perfused livers from pregnant (19-21 days gestation) and nonpregnant female rats. Bile flow, bile acid concentration, bile acid secretory rate, dibromosulfophthalein (DBSP) concentration in bile, and DBSP secretory rates were determined in livers infused continuously with DBSP and initially with SAL (1 ml/hr, 45 min), followed by TC (60 mumol/hr, 1 ml/hr; 75 min) or initially with TC (45 min) followed by SAL (75 min). The order of infusion (SAL-TC vs. TC-SAL) had no significant effect. TC infusion significantly increased all measures in livers from both nonpregnant and pregnant rats. Two-way analysis of variance followed by the Tukey-Kramer test showed that bile flow (microliter/min/g liver) and DBSP concentration in bile (mumol/ml) were significantly decreased during SAL infusions in pregnancy. These two measures plus bile acid and DBSP secretory rates (nmol/min/g liver) were also significantly decreased during TC infusion in pregnancy. Pregnancy had no effect on bile acid concentration in the presence of SAL or TC infusions. When bile flow, bile acid, and DBSP secretory rates were calculated per whole liver, only the DBSP secretory rate was significantly decreased in pregnancy. These data indicate that bile flow and bile acid secretion do not increase in proportion to the increase in liver weight in pregnancy so that these measures are decreased when expressed per g liver. Pregnancy appears to have a real inhibitory effect on DBSP since its secretion is depressed when activity is expressed per g liver or per whole organ.     

A simple method for the correction of biliary excretion curves distorted by the biliary dead space

Biliary solute concentrations measured at the tip of the cannula suffer a delay with respect to bile flow due to the transit time through the biliary tree volume. This study proposes a simple method, which is valid under variable bile flow conditions, to correct the distortion introduced by the biliary tree volume on the kinetic curves of the biliary excretion rate. The biliary transit time (tt) was calculated as the time needed to excrete a bile volume equal to the biliary tree volume by means of the interpolation of biliary cumulative volume versus time curves. Such tt permits one to estimate the canalicular concentration at time t, interpolating the biliary concentration curves at time t-tt. The product between the estimated canalicular concentration and the bile flow allows the calculation of the corrected biliary excretion rate. This method was evaluated by a comparison between biliary excretion rate curves of [14C]taurocholate [( 14C]TC) injected as a bolus under basal and sodium dehydrocholate (DHC)-induced choleresis conditions. Since the canalicular excretion rate of [14C]TC is considered independent of bile flow, the significant differences observed in its excretion kinetics under both conditions were attributed to distortion due to the biliary tree volume. After the correction, both curves showed a significant overlapping. This result indicates that the method improves the time-course representation of canalicular events in biliary excretion kinetic studies.     

Changes in susceptibility to acetaminophen-induced liver injury by the organic anion indocyanine green.

The non-metabolizable organic anion indocyanine green (ICG) has been shown previously to reduce markedly the biliary secretion of acetaminophen, particularly the glutathione conjugate of APAP (APAP-GSH), suggesting that this APAP metabolite may compete with other xenobiotics for excretion into the bile via a canalicular organic anion transport process. This study was conducted to determine whether changes in the biliary disposition of APAP induced by ICG could lead to alterations in susceptibility to APAP hepatotoxicity. To investigate this, groups of overnight-fasted male CD-1 mice received 30 micromol ICG/kg, intravenously, immediately prior to APAP dosing (500 mg/kg, ip). Controls were given propylene glycol vehicle. Mice were killed at 4 h after APAP challenge for immunochemical analysis of cytosolic protein arylation and determination of non-protein sulfhydryl (NPSH) depletion, or at 12 and 24 h for biochemical and histological assessment of liver injury. Elevated plasma sorbitol dehydrogenase activity and centrilobular hepatocellular necrosis was present in control mice receiving APAP at 12 and 24 h. Treatment with ICG did not alter susceptibility to APAP toxicity when measured at 12 h after challenge. However, the severity of histologic lesions in the ICG-APAP group was significantly lower at 24 h after challenge. Furthermore, treatment with ICG did not alter APAP-induced glutathione depletion or cytosolic protein arylation. These data suggest that the organic anion ICG has a protective effect on APAP toxicity that promotes a faster recovery from liver injury.