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Pierina Sueli Bonato - One of the best experts on this subject based on the ideXlab platform.
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combination of hollow fiber liquid phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver Microsomal Fraction
Electrophoresis, 2013Co-Authors: Leandro Augusto Calixto, Pierina Sueli BonatoAbstract:Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver Microsomal Fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 μm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver Microsomal Fraction.
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stereoselective liquid chromatographic determination of 1 oxobufuralol and 1 hydroxybufuralol in rat liver Microsomal Fraction using hollow fiber liquid phase microextraction for sample preparation
Journal of Separation Science, 2011Co-Authors: Thiago Barth, Rodrigo Almeida Simoes, Monica Tallarico Pupo, Laura Tiemi Okano, Pierina Sueli BonatoAbstract:A three-phase hollow-fiber liquid-phase microextraction (HF-LPME) method for the stereoselective determination of bufuralol metabolites 1′-oxobufuralol (1′-Oxo-BF) and 1′-hydroxybufuralol (1′-OH-BF) in Microsomal preparations is described for the first time. The HPLC analysis was carried out using a Chiralcel OD-H column with hexane/2-propanol/methanol (97.5:2.0:0.5, v/v/v) plus 0.5% diethylamine as the mobile phase, and UV detection at 248 and 273 nm. The HF-LPME optimized conditions involved: n-octanol as the organic solvent, 0.2 mol/L acetic acid as the acceptor phase, donor phase pH adjusted to 13, sample agitation at 1500 rpm and extraction for 30 min. By using this extraction procedure, the recovery rates were in the range of 63–69%. The method was linear over the concentration range of 100–5000 ng/mL for each enantiomer of 1′-Oxo-BF (r>0.9978) and of 100–2500 ng/mL for each stereoisomer of 1′-OH-BF (r>0.9957). The quantification limits were 100 ng/mL for all analytes. The validated method was used to assess the in vitro biotransformation of bufuralol using rat liver Microsomal Fraction that demonstrated predominant formation of (S)-1′-Oxo-BF and (R,R)-1′-OH-BF.
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In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver Microsomal Fraction
European Journal of Drug Metabolism and Pharmacokinetics, 2011Co-Authors: Leandro Augusto Calixto, Anderson Rodrigo Moraes Oliveira, Valquíria Aparecida Polisel Jabor, Pierina Sueli BonatoAbstract:Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N -desmethyl rosiglitazone ( N -Dm-R) and ρ-hydroxy rosiglitazone (ρ-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver Microsomal Fraction is described for the first time. Maximum velocity ( V _max) values of 87.29 and 51.09 nmol/min/mg protein were observed for N -Dm-R and ρ-OH-R, respectively. Michaelis–Menten constant ( K _m) values were of 58.12 and 78.52 μM for N -Dm-R and ρ-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N -Dm-R than of ρ-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (ο-OH-R) and two isomers of N -desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.
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simultaneous determination of rosiglitazone and its metabolites in rat liver Microsomal Fraction using hollow fiber liquid phase microextraction for sample preparation
Journal of Separation Science, 2010Co-Authors: Leandro Augusto Calixto, Pierina Sueli BonatoAbstract:A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in Microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of Microsomal preparation, were 47–70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50–6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver Microsomal Fraction.
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capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the Microsomal Fraction of liver homogenates
Electrophoresis, 2006Co-Authors: Valquiria A P Jabor, Carmem Dickow Cardoso, Pierina Sueli BonatoAbstract:A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the Microsomal Fraction of liver homogenates. After liquid-liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50 microm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated-beta-CD and 30 mg/mL hydroxypropyl-beta-CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (
Leandro Augusto Calixto - One of the best experts on this subject based on the ideXlab platform.
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combination of hollow fiber liquid phase microextraction and capillary electrophoresis for pioglitazone and its main metabolites determination in rat liver Microsomal Fraction
Electrophoresis, 2013Co-Authors: Leandro Augusto Calixto, Pierina Sueli BonatoAbstract:Pioglitazone (PGZ), a thiazolidinedione antidiabetic agent, is reported as a potent and selective activator of peroxisome proliferator-activated receptor γ (PPAR γ). This drug has been widely prescribed for the treatment of Type 2 diabetes mellitus. In this regard, this manuscript presents, for the first time, an alternative electrophoretic method for PGZ and its main metabolites determination in rat liver Microsomal Fraction. The electrophoretic analyses were performed using an uncoated fused-silica capillary of 50 μm id, 48 cm in total length and 40 cm in effective length, and 50 mmol/L sodium phosphate buffer solution (pH 2.5). All experiments were carried out under the normal mode. The capillary temperature was set at 35°C and a constant voltage of +30 kV was applied during the analyses. Samples were introduced into the capillary by hydrodynamic injection (50 mbar, 15 s) and detection was performed at 190 nm. The sample preparation procedure, based on hollow-fiber liquid-phase microextraction, was optimized using multifactorial experiments. Next, the following optimal condition was established: sample agitation at 1500 rpm, extraction for 15 min, 0.01 mol/L hydrochloric acid as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 6.0. The method demonstrated LOQs of 200 ng/mL. Additionally, it was linear over the concentration range of 200-25,000 ng/mL for PGZ and 200-2000 ng/mL for the metabolites. Finally, the validated method was employed to study the in vitro metabolism of PGZ using rat liver Microsomal Fraction.
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In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver Microsomal Fraction
European Journal of Drug Metabolism and Pharmacokinetics, 2011Co-Authors: Leandro Augusto Calixto, Anderson Rodrigo Moraes Oliveira, Valquíria Aparecida Polisel Jabor, Pierina Sueli BonatoAbstract:Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N -desmethyl rosiglitazone ( N -Dm-R) and ρ-hydroxy rosiglitazone (ρ-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver Microsomal Fraction is described for the first time. Maximum velocity ( V _max) values of 87.29 and 51.09 nmol/min/mg protein were observed for N -Dm-R and ρ-OH-R, respectively. Michaelis–Menten constant ( K _m) values were of 58.12 and 78.52 μM for N -Dm-R and ρ-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N -Dm-R than of ρ-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (ο-OH-R) and two isomers of N -desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.
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simultaneous determination of rosiglitazone and its metabolites in rat liver Microsomal Fraction using hollow fiber liquid phase microextraction for sample preparation
Journal of Separation Science, 2010Co-Authors: Leandro Augusto Calixto, Pierina Sueli BonatoAbstract:A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in Microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of Microsomal preparation, were 47–70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50–6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver Microsomal Fraction.
A Pla - One of the best experts on this subject based on the ideXlab platform.
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divergent effects of classical inducers on rat plasma and Microsomal Fraction paraoxonase and arylesterase
Environmental Toxicology and Pharmacology, 1997Co-Authors: Antonio F Hernandez, Ma Carmen Gonzalvo, Fernando Gil, E Villanueva, A PlaAbstract:The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver Microsomal Fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in Microsomal Fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.
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differences in the kinetic properties effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and Microsomal Fraction from rat liver
Biochemical Pharmacology, 1994Co-Authors: Fernando Gil, Antonio F Hernandez, E Villanueva, M C Gonzalvo, A PlaAbstract:The properties of a rat hepatic Microsomal enzyme that hydrolyses O,O-diethyl-p-nitrophenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the Microsomal Fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver Microsomal Fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and Microsomal Fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the Microsomal Fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and Microsomal Fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver Microsomal Fraction. In general we found more differences than analogies between the rat plasma and liver enzyme which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant Fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the Microsomal Fraction of rat liver.
A M Rubtsov - One of the best experts on this subject based on the ideXlab platform.
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seasonal changes in Microsomal Fraction enriched with na k atpase from kidneys of the ground squirrel spermophilus undulatus
Biochemistry, 2010Co-Authors: E V Basevich, O D Lopina, A M RubtsovAbstract:The Na,K-ATPase activity in Microsomal Fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.
Fernando Gil - One of the best experts on this subject based on the ideXlab platform.
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divergent effects of classical inducers on rat plasma and Microsomal Fraction paraoxonase and arylesterase
Environmental Toxicology and Pharmacology, 1997Co-Authors: Antonio F Hernandez, Ma Carmen Gonzalvo, Fernando Gil, E Villanueva, A PlaAbstract:The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver Microsomal Fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in Microsomal Fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.
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differences in the kinetic properties effect of calcium and sensitivity to inhibitors of paraoxon hydrolase activity in rat plasma and Microsomal Fraction from rat liver
Biochemical Pharmacology, 1994Co-Authors: Fernando Gil, Antonio F Hernandez, E Villanueva, M C Gonzalvo, A PlaAbstract:The properties of a rat hepatic Microsomal enzyme that hydrolyses O,O-diethyl-p-nitrophenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the Microsomal Fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver Microsomal Fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and Microsomal Fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the Microsomal Fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and Microsomal Fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver Microsomal Fraction. In general we found more differences than analogies between the rat plasma and liver enzyme which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant Fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the Microsomal Fraction of rat liver.