Attila Almási | Pécsi Tudományegyetem (original) (raw)

Papers by Attila Almási

Research paper thumbnail of HPLC and HPLC-MS Analysis of Intestinal Elimination and Phase 2 Metabolism of 4'-hydroxy-4-Methoxychalcone and its Bis-Mannich Analog In The Rat

The Open Medicinal Chemistry Journal

Aims: The aim was to study the elimination and Phase 2 biotransformation of 4'-hydroxy-4-meth... more Aims: The aim was to study the elimination and Phase 2 biotransformation of 4'-hydroxy-4-methoxychalcone (1) and its bis-Mannich analog (2) in the small intestine of the rat. Background: Earlier studies indicated that chalcones are promising starting points for drug design. Aminomethylation of drugs is considered to improve their delivery into the human body. Objectives: To set up validated HPLC-UV methods to quantitate the investigated chalcones in the rat intestinal perfusates. Comparison of intestinal disappearance and Phase 2 metabolic profile of the 4’-hydroxychalcone (1) and a bis-Mannich analog (2). Methods: Chalcones 1 and 2 were luminally perfused in the small intestine of rats at a concentration of 240 μM and 280 μM, respectively. Analysis of the collected intestinal perfusate samples was performed by a validated HPLC-UV method. Using HPLC-MS, the samples were analyzed for Phase 2 metabolites as well. Results: Elimination kinetics of the two 4’-hydroxychalcones display...

Research paper thumbnail of Effect of experimental hyperglycemia on intestinal elimination and biliary excretion of ibuprofen enantiomers in hyperglycemic rats

Journal of Pharmaceutical and Biopharmaceutical Research

Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associate... more Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics. An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion of ibuprofen enantiomers in the rat. Experimental diabetes was induced by intravenous (i.v.) administration of streptozotocin. The intestinal perfusion medium contained 250 µM racemic ibuprofen. A validated isocratic chiral HPLC method with UV detection was developed to determine the amount of the two enantiomers in the intestinal perfusate and the bile. The results indicated that experimental diabetes doesn’t cause a statistically significant difference in the disappearance of ibuprofen enantiomers from the small intestine. Analysis of the bile samples d...

Research paper thumbnail of The Intestinal and Biliary Metabolites of Ibuprofen in the Rat with Experimental Hyperglycemia

Molecules

Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the... more Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics; furthermore, it can result in the formation of non-enzyme catalyzed oxidative metabolites. The present work aimed to investigate how experimental hyperglycemia affects the intestinal and biliary appearance of the oxidative and Phase II metabolites of ibuprofen in rats. In vivo studies were performed by luminal perfusion of 250 μM racemic ibuprofen solution in control and streptozotocin-treated (hyperglycemic) rats. Analysis of the collected intestinal perfusate and bile samples was performed by HPLC-UV and HPLC-MS. No oxidative metabolites could be detected in the perfusate samples. The biliary appearance of ibuprofen, 2-hydroxyibuprofen, ibuprofen glucuronide, hydroxylated ibuprofen glucuronide, and ibuprofen taurate was depressed in the hyperglycemic ...

Research paper thumbnail of The Relative Importance of the Small Intestine and the Liver in Phase II Metabolic Transformations and Elimination of p-Nitrophenol Administered in Different Doses in the Rat

Scientia Pharmaceutica, 2020

Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimin... more Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimination of p-nitrophenol (PNP) in the rat. A jejunal loop was cannulated and recirculated with isotonic solutions containing PNP in different concentrations (0, 20, 100, 500, 1000 µM). Samples were obtained from the perfusate at given intervals. To investigate the metabolic and excretory functions of the liver, the bile duct was cannulated, and the bile was collected. Metabolites of PNP were determined by validated HPLC (high pressure liquid chromatography) methods. The results demonstrated the relative importance of the small intestine and the liver in phase II metabolic transformations and elimination of PNP. There were significant differences between the luminal and biliary appearances of p-nitrophenol-glucuronide (PNP-G) and p-nitrophenol–sulfate (PNP-S). The PNP-G appeared in the intestinal lumen at the lower PNP concentrations (20 µM and 100 µM) at higher rate than in the bile. No si...

Research paper thumbnail of Sci Pharm www.scipharm.at Research article Open Access HPLC Quantification of 4-Nitrophenol and its

This is an Open Access article distributed under the terms of the Creative Commons Attribution Li... more This is an Open Access article distributed under the terms of the Creative Commons Attribution License

Research paper thumbnail of Investigation of drug metabolism in various segments of small intestine in the rat

Acta Physiologica Hungarica, 2013

In the extrahepatic drug metabolism the intestinal tract can play an important role. These experi... more 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.

Research paper thumbnail of Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat

Acta Physiologica Hungarica, 2013

The aim of these experiments was the investigation of the correlation between the metabolic enzym... more 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 PNPsulfate: 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. Signifi cant differences were calculated by the Student's 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 β-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No signifi cant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these fi ndings, the biliary excretion of PNP-S was signifi cantly 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.

Research paper thumbnail of Investigation of Intestinal Absorption and Excretion of Paracetamol in Streptozotocin-Induced Hyperglycemia

International Journal of Molecular Sciences

The phenolic drug molecules can be metabolized, among others, by the small intestine’s enterocyte... more The phenolic drug molecules can be metabolized, among others, by the small intestine’s enterocytes. The conjugation reactions (glucuronidation and sulfation) show great importance in these transformations, although the oxidation reactions can be significant. These processes are dependent on the substituents of the phenolic compounds or the reacting functional groups (hydroxyl or carboxyl). Pathologic conditions, e.g., permanent hyperglycemia and diabetes, can alter the activities of the conjugative and possibly the oxidative enzymes, thus forming a change in the metabolic pattern and eventually provoking oxidative stress. A rat intestinal perfusion model was used to investigate the way in which experimental hyperglycemia affects the paracetamol’s intestinal elimination and metabolism. Hyperglycemia was induced by the administration of streptozotocin. Two hundred and fifty µM paracetamol was used in the intestinal perfusion solution. For the quantitation of the paracetamol and its ma...

Research paper thumbnail of Changes in hepatic metabolic enzyme activities and biliary excretion of 4-nitrophenol in streptozotocin induced diabetic rats

Brazilian Journal of Pharmaceutical Sciences

Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and... more Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and biliary excretion of its glucuronide (PNP-G) and sulfate (PNP-S) conjugates have been investigated in control and streptozotocin (STZ)-induced diabetic rats. 500 μM PNP solution was luminally perfused in a cannulated jejunal loop for 90 minutes. It was found that biliary excretion of PNP-G was significantly decreased in the diabetic rats. This effect of STZ could be completely reversed by administration of rapidacting insulin. Activity of hepatic UDP-glucuronyltransferase and β-glucuronidase was also depressed by the STZ pretreatment. Administration of insulin antagonized the inhibitory action of STZ on UDPglucuronyltransferase, but the reduced activity of β-glucuronidase was not reversed. Biliary excretion of PNP-S was also depressed in the diabetic rats. Whereas, different effects of insulin administration were observed. Namely, the lower biliary excretion rate of PNP-S was not changed after administration of insulin. Activity of the sulfotransferase and the arylsulfatase enzymes was not altered either by STZ pretreatment or by insulin administration. Biliary excretion of PNP was also significantly depressed by STZ and this depression was not changed after insulin administration. The results call attention to hepatobiliary circulation of low molecular weight xenobiotics and their glucuronide and sulfate conjugates.

Research paper thumbnail of Toxicokinetic Study of a Gastroprotective Dose of Capsaicin by HPLC-FLD Method

Molecules

Background: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have sho... more Background: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have shown to prevent development of gastric mucosal damage of alcohol and non-steroid anti-inflammatory drugs. Based on this experimental observation, a drug development program has been initiated to develop per os applicable capsaicin containing drugs to eliminate gastrointestinal damage caused by non-steroid anti-inflammatory drugs. Methods: As a part of this program, a sensitive and selective reverse-phase high-performance liquid chromatography-based method with fluorescence detection has been developed for quantification of capsaicin and dihydrocapsaicin in experimental dog’s plasma. Results: The method was evaluated for a number of validation characteristics (selectivity, repeatability, and intermediate precision, LOD, LOQ, and calibration range). The limit of detection (LOD) was 2 ng/mL and the limit of quantification (LOQ) was 10 ng/mL for both capsaicin and dihydrocapsaicin. The method ...

Research paper thumbnail of Investigation of intestinal elimination and biliary excretion of ibuprofen in control and hyperglycemic rats

Canadian Journal of Physiology and Pharmacology

An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affe... more An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion in the rat. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg, i.v.). The intestinal perfusion medium contained 250 μM (±)-ibuprofen. An isocratic high-performance liquid chromatography method with UV–visible detection was developed to quantitate ibuprofen in the intestinal perfusate, while a gradient method was applied to quantitate ibuprofen and ibuprofen-β-d-glucuronide in the bile. The limit of quantitation of ibuprofen was found to be 0.51 μM in the perfusate of the small intestine. In the bile, the limit of quantitation of ibuprofen and ibuprofen-β-d-glucuronide was 4.42 and 10.3 μM, respectively. Unconjugated ibuprofen and ibuprofen-β-d-glucuronide were detected in the bile; however, no β-d-glucuronide of ibuprofen could be detected in the intestinal perfusate. The results indicate that experimental dia...

Research paper thumbnail of HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Research paper thumbnail of Effects of Mesalazine on Morphological and Functional Changes in the Indomethacin-Induced Inflammatory Bowel Disease (Rat Model of Crohn’s Disease)

Pathology & Oncology Research, 2016

Morphological and functional changes have been investigated in the rat model of Crohn&amp... more Morphological and functional changes have been investigated in the rat model of Crohn's disease. The inflammatory bowel disease was induced by indomethacin (1 × 10 mg/kg s.c. for 3 days). Morphological alterations were evaluated by macroscopic scoring system and on the base of histological changes in the small intestine. Functional activities were studied by determination of the intestinal and hepatic elimination of p-Nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) during the luminal perfusion of PNP. It was found that the indomethacin induced severe macroscopic changes (hyperaemia, petechia, bleeding, erosions, ulcerations) and significant histological alterations in the small intestine of rats which were definitely inhibited by mesalazine (1000 mg/kg by gastric tube for 3 days). Disappearance of PNP from the luminal perfusion solution was diminished by indomethacin which was corrected by administration of mesalazine. Significant depression was found in the luminal appearance of PNP metabolites by giving of indomethacin and these alterations could not be compensated by mesalazine.Hepatic elimination of PNP (biliary excretion of PNP and its metabolites) was decreased definitely by indomethacin which was - at least partly - compensated by mesalazine.The findings of the present study suggest that the indomethacin-induced inflammation in the small intestine represents a useful rat model of Crohn's disease. Morphological and functional alterations caused by indomethacin can be compensated by mesalazine.

Research paper thumbnail of Effect of experimental diabetes and insulin replacement on the intestinal metabolism and excretion of 4–nitrophenol in the rat

Canadian Journal of Physiology and Pharmacology, 2015

Luminal appearance of 4-nitrophenol (PNP) metabolites (4-nitrophenol-β-glucuronide (PNP-G) and 4-... more 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. ...

Research paper thumbnail of A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Journal of Biochemical and Biophysical Methods, 2006

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a... more Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-β-D-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol-water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6-1200 μM (PNP) and 2.5-100 μM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 μM and 2.1 μM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.

Research paper thumbnail of HPLC quantification of 4-nitrophenol and its conjugated metabolites from bile

Scientia Pharmaceutica, 2011

An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for s... more 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 C 18 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.

Research paper thumbnail of A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Journal of Biochemical and Biophysical Methods, 2006

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a... more Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-β-D-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol-water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6-1200 μM (PNP) and 2.5-100 μM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 μM and 2.1 μM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.

Research paper thumbnail of HPLC and HPLC-MS Analysis of Intestinal Elimination and Phase 2 Metabolism of 4'-hydroxy-4-Methoxychalcone and its Bis-Mannich Analog In The Rat

The Open Medicinal Chemistry Journal

Aims: The aim was to study the elimination and Phase 2 biotransformation of 4'-hydroxy-4-meth... more Aims: The aim was to study the elimination and Phase 2 biotransformation of 4'-hydroxy-4-methoxychalcone (1) and its bis-Mannich analog (2) in the small intestine of the rat. Background: Earlier studies indicated that chalcones are promising starting points for drug design. Aminomethylation of drugs is considered to improve their delivery into the human body. Objectives: To set up validated HPLC-UV methods to quantitate the investigated chalcones in the rat intestinal perfusates. Comparison of intestinal disappearance and Phase 2 metabolic profile of the 4’-hydroxychalcone (1) and a bis-Mannich analog (2). Methods: Chalcones 1 and 2 were luminally perfused in the small intestine of rats at a concentration of 240 μM and 280 μM, respectively. Analysis of the collected intestinal perfusate samples was performed by a validated HPLC-UV method. Using HPLC-MS, the samples were analyzed for Phase 2 metabolites as well. Results: Elimination kinetics of the two 4’-hydroxychalcones display...

Research paper thumbnail of Effect of experimental hyperglycemia on intestinal elimination and biliary excretion of ibuprofen enantiomers in hyperglycemic rats

Journal of Pharmaceutical and Biopharmaceutical Research

Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associate... more Diabetic complications are mostly due to hyperglycemia. Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics. An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion of ibuprofen enantiomers in the rat. Experimental diabetes was induced by intravenous (i.v.) administration of streptozotocin. The intestinal perfusion medium contained 250 µM racemic ibuprofen. A validated isocratic chiral HPLC method with UV detection was developed to determine the amount of the two enantiomers in the intestinal perfusate and the bile. The results indicated that experimental diabetes doesn’t cause a statistically significant difference in the disappearance of ibuprofen enantiomers from the small intestine. Analysis of the bile samples d...

Research paper thumbnail of The Intestinal and Biliary Metabolites of Ibuprofen in the Rat with Experimental Hyperglycemia

Molecules

Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the... more Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics; furthermore, it can result in the formation of non-enzyme catalyzed oxidative metabolites. The present work aimed to investigate how experimental hyperglycemia affects the intestinal and biliary appearance of the oxidative and Phase II metabolites of ibuprofen in rats. In vivo studies were performed by luminal perfusion of 250 μM racemic ibuprofen solution in control and streptozotocin-treated (hyperglycemic) rats. Analysis of the collected intestinal perfusate and bile samples was performed by HPLC-UV and HPLC-MS. No oxidative metabolites could be detected in the perfusate samples. The biliary appearance of ibuprofen, 2-hydroxyibuprofen, ibuprofen glucuronide, hydroxylated ibuprofen glucuronide, and ibuprofen taurate was depressed in the hyperglycemic ...

Research paper thumbnail of The Relative Importance of the Small Intestine and the Liver in Phase II Metabolic Transformations and Elimination of p-Nitrophenol Administered in Different Doses in the Rat

Scientia Pharmaceutica, 2020

Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimin... more Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimination of p-nitrophenol (PNP) in the rat. A jejunal loop was cannulated and recirculated with isotonic solutions containing PNP in different concentrations (0, 20, 100, 500, 1000 µM). Samples were obtained from the perfusate at given intervals. To investigate the metabolic and excretory functions of the liver, the bile duct was cannulated, and the bile was collected. Metabolites of PNP were determined by validated HPLC (high pressure liquid chromatography) methods. The results demonstrated the relative importance of the small intestine and the liver in phase II metabolic transformations and elimination of PNP. There were significant differences between the luminal and biliary appearances of p-nitrophenol-glucuronide (PNP-G) and p-nitrophenol–sulfate (PNP-S). The PNP-G appeared in the intestinal lumen at the lower PNP concentrations (20 µM and 100 µM) at higher rate than in the bile. No si...

Research paper thumbnail of Sci Pharm www.scipharm.at Research article Open Access HPLC Quantification of 4-Nitrophenol and its

This is an Open Access article distributed under the terms of the Creative Commons Attribution Li... more This is an Open Access article distributed under the terms of the Creative Commons Attribution License

Research paper thumbnail of Investigation of drug metabolism in various segments of small intestine in the rat

Acta Physiologica Hungarica, 2013

In the extrahepatic drug metabolism the intestinal tract can play an important role. These experi... more 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.

Research paper thumbnail of Metabolic enzyme activities and drug excretion in the small intestine and in the liver in the rat

Acta Physiologica Hungarica, 2013

The aim of these experiments was the investigation of the correlation between the metabolic enzym... more 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 PNPsulfate: 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. Signifi cant differences were calculated by the Student's 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 β-glucuronidase was about six times higher, the activity of the arylsulfatase was approximately seven times greater in the liver than in the jejunum. No signifi cant difference was found between the luminal appearance and the biliary excretion of PNP-G. Contrary to these fi ndings, the biliary excretion of PNP-S was signifi cantly 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.

Research paper thumbnail of Investigation of Intestinal Absorption and Excretion of Paracetamol in Streptozotocin-Induced Hyperglycemia

International Journal of Molecular Sciences

The phenolic drug molecules can be metabolized, among others, by the small intestine’s enterocyte... more The phenolic drug molecules can be metabolized, among others, by the small intestine’s enterocytes. The conjugation reactions (glucuronidation and sulfation) show great importance in these transformations, although the oxidation reactions can be significant. These processes are dependent on the substituents of the phenolic compounds or the reacting functional groups (hydroxyl or carboxyl). Pathologic conditions, e.g., permanent hyperglycemia and diabetes, can alter the activities of the conjugative and possibly the oxidative enzymes, thus forming a change in the metabolic pattern and eventually provoking oxidative stress. A rat intestinal perfusion model was used to investigate the way in which experimental hyperglycemia affects the paracetamol’s intestinal elimination and metabolism. Hyperglycemia was induced by the administration of streptozotocin. Two hundred and fifty µM paracetamol was used in the intestinal perfusion solution. For the quantitation of the paracetamol and its ma...

Research paper thumbnail of Changes in hepatic metabolic enzyme activities and biliary excretion of 4-nitrophenol in streptozotocin induced diabetic rats

Brazilian Journal of Pharmaceutical Sciences

Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and... more Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and biliary excretion of its glucuronide (PNP-G) and sulfate (PNP-S) conjugates have been investigated in control and streptozotocin (STZ)-induced diabetic rats. 500 μM PNP solution was luminally perfused in a cannulated jejunal loop for 90 minutes. It was found that biliary excretion of PNP-G was significantly decreased in the diabetic rats. This effect of STZ could be completely reversed by administration of rapidacting insulin. Activity of hepatic UDP-glucuronyltransferase and β-glucuronidase was also depressed by the STZ pretreatment. Administration of insulin antagonized the inhibitory action of STZ on UDPglucuronyltransferase, but the reduced activity of β-glucuronidase was not reversed. Biliary excretion of PNP-S was also depressed in the diabetic rats. Whereas, different effects of insulin administration were observed. Namely, the lower biliary excretion rate of PNP-S was not changed after administration of insulin. Activity of the sulfotransferase and the arylsulfatase enzymes was not altered either by STZ pretreatment or by insulin administration. Biliary excretion of PNP was also significantly depressed by STZ and this depression was not changed after insulin administration. The results call attention to hepatobiliary circulation of low molecular weight xenobiotics and their glucuronide and sulfate conjugates.

Research paper thumbnail of Toxicokinetic Study of a Gastroprotective Dose of Capsaicin by HPLC-FLD Method

Molecules

Background: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have sho... more Background: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have shown to prevent development of gastric mucosal damage of alcohol and non-steroid anti-inflammatory drugs. Based on this experimental observation, a drug development program has been initiated to develop per os applicable capsaicin containing drugs to eliminate gastrointestinal damage caused by non-steroid anti-inflammatory drugs. Methods: As a part of this program, a sensitive and selective reverse-phase high-performance liquid chromatography-based method with fluorescence detection has been developed for quantification of capsaicin and dihydrocapsaicin in experimental dog’s plasma. Results: The method was evaluated for a number of validation characteristics (selectivity, repeatability, and intermediate precision, LOD, LOQ, and calibration range). The limit of detection (LOD) was 2 ng/mL and the limit of quantification (LOQ) was 10 ng/mL for both capsaicin and dihydrocapsaicin. The method ...

Research paper thumbnail of Investigation of intestinal elimination and biliary excretion of ibuprofen in control and hyperglycemic rats

Canadian Journal of Physiology and Pharmacology

An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affe... more An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion in the rat. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg, i.v.). The intestinal perfusion medium contained 250 μM (±)-ibuprofen. An isocratic high-performance liquid chromatography method with UV–visible detection was developed to quantitate ibuprofen in the intestinal perfusate, while a gradient method was applied to quantitate ibuprofen and ibuprofen-β-d-glucuronide in the bile. The limit of quantitation of ibuprofen was found to be 0.51 μM in the perfusate of the small intestine. In the bile, the limit of quantitation of ibuprofen and ibuprofen-β-d-glucuronide was 4.42 and 10.3 μM, respectively. Unconjugated ibuprofen and ibuprofen-β-d-glucuronide were detected in the bile; however, no β-d-glucuronide of ibuprofen could be detected in the intestinal perfusate. The results indicate that experimental dia...

Research paper thumbnail of HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Research paper thumbnail of Effects of Mesalazine on Morphological and Functional Changes in the Indomethacin-Induced Inflammatory Bowel Disease (Rat Model of Crohn’s Disease)

Pathology & Oncology Research, 2016

Morphological and functional changes have been investigated in the rat model of Crohn&amp... more Morphological and functional changes have been investigated in the rat model of Crohn's disease. The inflammatory bowel disease was induced by indomethacin (1 × 10 mg/kg s.c. for 3 days). Morphological alterations were evaluated by macroscopic scoring system and on the base of histological changes in the small intestine. Functional activities were studied by determination of the intestinal and hepatic elimination of p-Nitrophenol (PNP) and its metabolites (PNP-glucuronide: PNP-G and PNP-sulfate: PNP-S) during the luminal perfusion of PNP. It was found that the indomethacin induced severe macroscopic changes (hyperaemia, petechia, bleeding, erosions, ulcerations) and significant histological alterations in the small intestine of rats which were definitely inhibited by mesalazine (1000 mg/kg by gastric tube for 3 days). Disappearance of PNP from the luminal perfusion solution was diminished by indomethacin which was corrected by administration of mesalazine. Significant depression was found in the luminal appearance of PNP metabolites by giving of indomethacin and these alterations could not be compensated by mesalazine.Hepatic elimination of PNP (biliary excretion of PNP and its metabolites) was decreased definitely by indomethacin which was - at least partly - compensated by mesalazine.The findings of the present study suggest that the indomethacin-induced inflammation in the small intestine represents a useful rat model of Crohn's disease. Morphological and functional alterations caused by indomethacin can be compensated by mesalazine.

Research paper thumbnail of Effect of experimental diabetes and insulin replacement on the intestinal metabolism and excretion of 4–nitrophenol in the rat

Canadian Journal of Physiology and Pharmacology, 2015

Luminal appearance of 4-nitrophenol (PNP) metabolites (4-nitrophenol-β-glucuronide (PNP-G) and 4-... more 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. ...

Research paper thumbnail of A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Journal of Biochemical and Biophysical Methods, 2006

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a... more Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-β-D-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol-water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6-1200 μM (PNP) and 2.5-100 μM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 μM and 2.1 μM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.

Research paper thumbnail of HPLC quantification of 4-nitrophenol and its conjugated metabolites from bile

Scientia Pharmaceutica, 2011

An isocratic ion pair RP-HPLC method with UV-Vis detection has been developed and validated for s... more 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 C 18 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.

Research paper thumbnail of A simple and rapid ion-pair HPLC method for simultaneous quantitation of 4-nitrophenol and its glucuronide and sulfate conjugates

Journal of Biochemical and Biophysical Methods, 2006

Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a... more Because of its simple and well characterized metabolic profile, 4-nitrophenol is widely used as a model substrate to investigate the influence of drug therapy, disease, nutrient deficiencies and other physiologically altered conditions on conjugative drug metabolism in animal studies. For simultaneous determination of 4-nitrophenol (PNP), 4-nitrophenyl-β-D-glucuronide (PNP-G) and 4-nitrophenyl-sulfate (PNP-S) in samples generated in rat small intestine luminal perfusion experiments, an ion-pair HPLC assay coupled with UV detection was set up. The RP-HPLC separation was achieved with a methanol-water mixture (50:50, v/v) containing 0.01 M tetrabutyl-ammonium-bromide with UV detection of the analytes at 290 nm. The isocratic system was operated at ambient temperature and required less than 7 min of chromatographic time. The method provided good enough within-day precision, between-day precision and linearity in the target concentration ranges of 6-1200 μM (PNP) and 2.5-100 μM (PNP-G and PNP-S). The instrumental limit of quantification for PNP-G and PNP-S was found to be 2.7 μM and 2.1 μM, respectively. The assay was applied for determination of PNP, PNP-G and PNP-S in rat small intestine perfusates.