Biomedical subjects

Yazen Alnouti

Publications and source records attributed to Yazen Alnouti.

9 recordsLinked to original sources

PubMed2006

Tissue distribution and ontogeny of sulfotransferase enzymes in mice.

Sulfotransferases (Sults) are phase-II conjugation enzymes that catalyze the transfer of a sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to target endo and xenobiotics. PAPS is formed from inorganic sulfate by the action of the enzyme PAPS synthase (PAPSs). In the present study, the tissue distribution and developmental changes in the mRNA expression of 11 Sult isozymes and 2 PAPSs isoforms in mice were quantified. Sult1a1, 1b1, 1c1, 1c2, 1d1, 1e1, 2a1/2, 2b1, 3a1, 4a1, 5a1, PAPSs1, and PAPSs2 mRNA expression was quantified in 14 tissues from male and female mice using the branched DNA signal amplification assay. Sult2a1/2 and 3a1 expression were highest in liver; Sult1b1, 2b1, and PAPSs2 in small intestine; Sult1a1 in large intestine; Sult1c2 in stomach; Sult1d1 in kidney; Sult1e1 in placenta; and Sult4a1 in brain. Sult1c1, 5a1, and PAPSs1 were ubiquitously expressed in most tissues. These enzymes demonstrated three different ontogenic expression patterns in liver. Sult1a1, 1c2, 1d1, 2a1/2, and PAPSs2 hepatic expression gradually increased from birth until about 3 weeks of age and then declined somewhat thereafter, Sult1c1 expression was highest before birth and declined after that, and Sult3a1 mRNA expression was very low in fetal livers and remained low until 30 days of age, when expression in females dramatically increased, whereas it never increased in males. The organ-specific distribution of Sults as well as the different expression of the Sults in young animals may affect the pharmacokinetic behavior and organ-specific toxicity of xenobiotics.

Age Factors↗

PubMed2006

Method for internal standard introduction for quantitative analysis using on-line solid-phase extraction LC-MS/MS.

A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation.

Animals↗

PubMed2005

Tissue distribution and ontogeny of organic cation transporters in mice.

Organic cation transporters (Octs) play an important role in transporting cationic xeno- and endobiotics across biological membranes. Little is known about Octs in mice; therefore, the tissue distribution and developmental changes in the mRNA expression of Octs in mice were quantified. Oct1, Oct2, Oct3, Octn1, Octn2, and Octn3 mRNA expression was quantified in 14 tissues from male and female mice using the branched DNA signal amplification assay. Oct1 mRNA expression was highest in kidney, followed by liver. Oct2 mRNA was almost exclusively expressed in kidney, with male mice having twice that in female mice. The higher expression of Oct2 in male mice is due to testosterone. Oct3 mRNA was most highly expressed in placenta, ovary, and uterus, but was expressed at low levels in most tissues. Octn1 and Octn2 mRNA expression was similar, with the highest levels in kidney followed by small intestine. Octn3 mRNA was almost exclusively expressed in testes. The developmental expression of Oct1, Oct2, Octn1, and Octn2 mRNA in kidneys as well as Oct1 in liver was determined in young mice. Ontogenic expression data indicate that each of the Octs approached adult expression levels by about 3 weeks of age. The gender difference in Oct mRNA expression did not become apparent until day 30 after birth. The differences in tissue distribution of the Octs may play an important role in drug disposition to various tissues. Furthermore, low expression of the Octs in young animals may affect the pharmacokinetic behavior of drugs compared with that in adults.

Aging↗

PubMed2005

Development and application of a new on-line SPE system combined with LC-MS/MS detection for high throughput direct analysis of pharmaceutical compounds in plasma.

A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)-mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C18 and monolithic columns. Online SPE LC-MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC-MS/MS with conventional C18 and monolithic columns were in the range of 88-111% and 0.5-14%, respectively. Total analysis cycle time of 4 min per sample was demonstrated using the C18 column. Monolithic column allowed for 2 min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41-108% and 3-81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.

Chemical Fractionation↗

PubMed2005

Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection.

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.

Animals↗

PubMed2005

Simultaneous determination of zidovudine and lamivudine from rat tissues by liquid chromatography/tandem mass spectrometry.

A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid, placental, and fetal tissues. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C18 column (5 microm, 150 x 3.9 mm i.d). The mobile phase consisted of 30% methanol and 7.5 mM ammonium acetate (pH 6.5). The method was validated in the range of 0.05-25 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport following co-administration of these compounds at a dose of 25 mg/kg each in a pregnant rat.

Amniotic Fluid↗

PubMed2004

Determination of lamivudine in plasma, amniotic fluid, and rat tissues by liquid chromatography.

An HPLC method for the quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal tissues has been developed, validated and applied to the study of the placental transport of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-liquid extraction enhanced by salting out the sample using a saturated solution of ammonium sulfate. Plasma and amniotic fluid samples were processed by protein precipitation using 2 M perchloric acid. Reverse phase chromatography was performed using a phenyl column (5 microm, 150 mm x 2 mm i.d.) under a flow rate of 0.2 ml/min. The mobile phase consisted of 5% methanol in 20 mM dibasic phosphate buffer (pH 6). The method was validated over the range from 0.1 to 50 microg/ml for plasma and amniotic fluid and 0.2-50 microg/ml for the placental and fetal tissues.

Amniotic Fluid↗

PubMed2004

Simultaneous quantitation of zidovudine and zidovudine monophosphate from plasma, amniotic fluid and tissues by micellar capillary electrophoresis.

Zidovudine (AZT) therapy given during pregnancy has been shown to reduce the vertical transmission of the human immunodeficiency virus (HIV) from mother to fetus. In order to investigate the efficacy of AZT, it is important to know the concentration of its active phosphorylated metabolites. We have developed the first CE method for the simultaneous quantitation of AZT and zidovudine monophosphate (AZT-MP) from rat plasma, amniotic fluid and fetal tissues. Sample extractions were performed by protein precipitation using acetonitrile for the plasma and amniotic fluids, while in fetal tissues solid phase extraction using Waters Oasis HLB extraction cartridges was used. Recoveries ranged from 78 to 92% for AZT, AZT-MP and 3'-azidouridine (internal standard, AZDU), in the three matrices. The optimum separation conditions were achieved using a 40 mm sodium dodecylsulfate (SDS) in 50 mm phosphate buffer (pH 7) with a run voltage of 15 kV. The CE system consists of a 75 microm i.d., 50 cm effective length uncoated fused silica capillary. The method was validated over the range 0.5-100 microg/ml ( micro g/g for tissues). Intra-day precision (RSD) and accuracy (%error) for AZT ranged from 0.13 to 11 and 0.68 to 11.1%, respectively, while for AZT-MP it ranged from 2.05 to 11.1 and 4.22 to 11.7%. Inter-day precision and accuracy for AZT ranged from 3.82 to 11.2 and 3.14 to 9.01%, while for AZT-MP it ranged from 3.9 to 9.32 and 3.44 to 9.37%, respectively. We also report the enzymatic dephosphorylation of AZT-MP in the placental tissue of rats. This new enzymatic pathway provides increased understanding of the mechanism of anti-viral transport in the rat during pregnancy.

Amniotic Fluid↗

PubMed2004

Simultaneous determination of zidovudine and lamivudine from rat plasma, amniotic fluid and tissues by HPLC.

A sensitive HPLC method has been developed and validated for the simultaneous quantification of zidovudine (AZT) and lamivudine (3TC) in rat plasma, amniotic fluid and placental and fetal tissues. Samples were processed by solid-phase extraction using C2 cartridges. Chromatography was performed using a phenyl column (5 microm, 150 x 2 mm i.d.) under a flow rate of 0.2 mL/min. The mobile phase consisted of 8% acetonitrile in 5 mM 1-heptane sulfonic acid dissolved in 30 mM ammonium formate buffer (pH 3.3). The method was validated in the range 0.25-50 microg/mL for both 3TC and AZT in the four biological matrices. Finally, the method was applied to a study involving fetal transport of co-administration of these compounds in a pregnant rat.

Amniotic Fluid↗