E. Fischer

Biliary excretion of bromsulphthalein and glutathione conjugate of bromsulphthalein in rats pretreated with diethyl maleate

Abstract In rats diethyl maleate (DEM, 0.7 ml/kg i.p.) decreased the hepatic glutathione level to one tenth of the control value. Owing to the low glutathione level the conjugation of bromsulphthalein (BSP) with glutathione was markedly depressed. DEM-treated rats were given BSP and a glutathione conjugate of BSP (BSP-GSH) intravenously at various dose levels, and their biliary excretion and tissue concentrations were determined. No significant difference between the hepatic transport maxima for BSP (673 μg/min/kg) and for BSP-GSH (689 μg/min/kg) was found. BSP-GSH increased the biliary flow, BSP diminished it. Depending on the dose, 52–83 per cent of the BSP administered was taken up by the liver in 45 min, whereas the BSP-GSH predominantly appeared in extra-hepatic tissues. The half saturation doses for transport maxima were 75 mg/kg for BSP and 31 mg/ kg for BSP-GSH. After administration of these doses the hepatic concentration of BSP was approximately ten times as high as the hepatic concentration of BSP-GSH.

Bile Flow and Biliary Excretion Rate of Some Organic Anions in Phenobarbital-Pretreated Rats

Bile flow and the biliary excretion of indocyanine green, bromcresol green, eosine, bromsulphthalein-glutathione conjugate (BSP-GSH), amaranth and iodoxamic acid were investigated in control and phenobarbital-pretreated rats (75 mg/kg i.p. daily for 5 days). The bile flow was increased by phenobarbital from an average of 50.6 to 77.7 microliter/kg/min. Depending on the dose, the biliary excretion rate of bromcresol green was increased by 48-496% and that of eosine by 30-149%. After phenobarbital pretreatment the excretion of BSP-GSH was also enhanced by 34-52%, that of amaranth by 37-53% and that of iodoxamic acid by 40-56%. However, the biliary excretion of indocyanine green remained unchanged. There was no parallelism between the increase in bile flow and biliary excretion of the drugs.

Age dependence of hepatic transport in control and phenobarbital-pretreated rats

Abstract Eosine is excreted in rat bile unchanged, which makes it suitable for the study of age dependent changes in hepatic uptake and excretion. Bile flow was approximately 40 μl/kg/min in 20-day-old rats and twice as high in 30-day-old animals. In 60- and 120-day-old rats the bile volume was decreased, moreover in 220-day-old ones it fell to the level of 20-day-old rats. The biliary excretion of eosine (150 μmol/kg i.v.) was highest in 60-day-old rats, however, the biliary flow reached its peak in 30-day-old rats. After phenobarbital (PB) pretreatment (75 mg/kg i.p. daily for five days) each age group showed enhancement in liver weight and bile volume. On the other hand, the hepatic concentration of eosine did not change after PB pretreatment caused an increase in the biliary excretion of eosine in 30-, 60-, 120- and 220-day-old rats but no significant change in 20-day-old animals. Our results indicate that the hepatic transport in young rats was immature and was not induced by PB. However, PB can increase the low excretion rate in old rats.

A validated HPLC-FLD method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat

A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse(®) XDB-C8 column using isocratic elution with a mobile phase consisting 0.05M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50ng/mL and the limit of quantification (LOQ) was 100ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30μg/mL standardized Capsicum extract - containing capsaicin and dihydrocapsaicin - was luminally perfused for a 90min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.

HPLC Quantification of 4-Nitrophenol and its Conjugated Metabolites from Bile

An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for simultaneous analysis of 4-nitrophenol (PNP), 4-nitrophenyl β-glucuronide (PNP-G), and 4-nitrophenyl sulfate (PNP-S) in rat bile samples using 4-ethylphenol (ETP) as internal standard. Chromatographic separation was achieved on a C18 column by isocratic elution with a mobile phase consisted of methanol-0.01 M citrate buffer pH 6.2 (47:53 v/v) containing 0.03 M TBAB. The flow rate was 1.0 ml min−1, the detection was affected at 290 nm. Calibration plots were generated over the concentration range 1–100 μM PNP, PNP-G, PNP-S with a common lower limit of quantification of 2.5 μM. Intra- and inter-day precision and repeatability were determined at six different concentrations. Results obtained by application of the method for determination of PNP, PNP-G and PNP-S in bile fractions collected during intestinal perfusion of PNP in hyperglycemic rats are presented.

Investigation of drug metabolism in various segments of small intestine in the rat

In the extrahepatic drug metabolism the intestinal tract can play an important role. These experiments were designed to study the biotransformation of p-nitrophenol (PNP) in the small intestine in the rat. Various segments of the small intestine (proximal and distal jejunum, terminal ileum) were perfused with isotonic solution in vivo containing different concentrations of PNP (20-100-500-1000 μM) and the concentrations of metabolites (PNP-G: p-nitrophenol glucuronide, PNP-S: p-nitrophenol sulfate) were determined in the perfusion medium. It was found a decreasing tendency in the glucuronidation from the proximal to distal segment of the small intestine: e.g. 430 nmol, 240 nmol, and 100 nmol PNP-G appeared in the perfusion medium in the proximal, distal jejunum and in the terminal ileum, respectively, when 500 μM PNP was luminally perfused for 90 minutes. Similar ratio was found at the luminal perfusion of other PNP-concentrations, too. Luminal appearance of sulfoconjugate of PNP was considerably lower and no clear gradient tendency in the formation of PNP-S could be detected in the small intestine from the proximal to distal segment. Our results show that there are considerable differences in drug metabolism in various segments of the small intestine. We have found a gradient conjugating activity from proximal to distal segment of small intestine in the glucuronidation of PNP.

Effects of barbiturates on the hepatic cytochrome P-450 dependent enzyme system and biliary excretion of exogenous organic anions in rats

Effect of pretreatment (150 mumol/kg i.p. twice daily for 5 days) wtih six barbiturates (barbital, butobarbital, pentobarbital, amobarbital, phenobarbital and thiopental) on the hepatic microsomal drug methabolizing enzyme system and biliary excretion of bromcresol green and bromsulphthalein-glutathione conjugate has been investigated in rats. All six barburates produced microsomal induction. Phenobarbital, thiopental, barbital and butobarbital increased liver weight, biliary flow and biliary excretion of bromcresol green and bromsulphthalein-glutathione conjugate, however, pentobarbital and amobarbital were ineffective in these parameters. Our results seem to indicate that the enhanced biliary excretion of exogenous anions produced by barbiturates is independent of microsomal enzyme induction.

Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat.

The aim of these experiments was the investigation of the correlation between the metabolic enzyme activities and the intestinal and hepatic excretion of p-nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) in the same group of rats (n = 10). A jejunal loop was perfused with isotonic medium containing PNP in a concentration of 500 μM. The samples were obtained from the luminal perfusion medium and from the bile. For enzyme assays tissue samples were obtained from the liver and jejunum at the end of experiments. Significant differences were calculated by the Students t-test. The activity of UDP-glucuronyltransferase and sulfotransferase was about three times higher in the liver than in the small intestine. The activity of the ß-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No significant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these findings, the biliary excretion of PNP-S was significantly higher than the luminal appearance of PNP-sulfate. It can be concluded that no direct correlation exists between the activity of metabolic enzymes and the excretion rate of PNP-metabolites in the liver and in the jejunal segment of the small intestine.

Effect of experimental diabetes and insulin replacement on intestinal metabolism and excretion of 4-nitrophenol in rats

Luminal appearance of 4-nitrophenol (PNP) metabolites (4-nitrophenol-β-glucuronide (PNP-G) and 4-nitrophenol-sulfate (PNP-S)) and activity of the related metabolic enzymes have been investigated in control and experimental diabetic rats. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg i.v.). PNP (500 μmol/L) was luminally perfused in the small intestine and the metabolites were determined in the perfusion solution. Effect of insulin replacement was also investigated in the diabetic rats. It was found that experimental diabetes increased the luminal appearance of PNP-G, which could be completely compensated by rapid-acting insulin administration (1 U/kg i.v.). Activities of the enzymes involved in PNP-G production (UDP-glucuronyltransferase and β-glucuronidase) were also elevated; however, these changes were only partially compensated by insulin. Luminal appearance of PNP-S was not significantly changed by administration of streptozotocin and insulin. Activities of the enzymes of PNP-S production (sulfotransferases and arylsulfatases) did not change in the diabetic rats. The results indicate that experimental diabetes can provoke changes in intestinal drug metabolism. It increased intestinal glucuronidation of PNP but did not influence sulfate conjugation. No direct correlation was found between the changes of metabolic enzyme activities and the luminal appearance of the metabolites.

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat.

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 μm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC-DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5-50 μg/mL. After liquid-liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC-MS. The method was linear over the concentration range 0.25-5.0 μg/mL. Two hydroxylated metabolites of salicylic acid (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3-DHB, 72.5% for 2,5-DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.