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Quantitation
Kinds of Quantitation Terms modified by Quantitation Selected AbstractsQuantitation of valproic acid in pharmaceutical preparations using dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection without prior derivatizationDRUG TESTING AND ANALYSIS, Issue 7 2010Hamid Reza Sobhi Abstract Dispersive liquid-liquid microextraction (DLLME), coupled with gas chromatography-flame ionization detection (GC-FID), has been successfully used for the extraction and determination of valproic acid (VPA) in pharmaceutical preparations. In the developed method, an appropriate mixture of extracting and disperser solvents was rapidly injected into an aqueous sample. Having formed a cloudy solution, the mixture was centrifuged and then the extracting solvent was sedimented at the bottom of a conical test tube. The extract was then injected into a GC system directly, without any further pretreatment. Initially, microextraction efficiency factors were optimized and the optimum experimental conditions found were as follows: tetrachloroethylene (9.0 µL) as extracting solvent; acetone (1.0 mL) as disperser solvent; 5 mL acidic aqueous sample (pH 1) without salt addition. Under the selected conditions, the calibration curve showed linearity in the range of 0.1,5.0 mg/L with regression coefficient corresponding to 0.9998. The limit of detection was found to be 0.05 mg/L. Finally, the method was applied for the determination of VPA in two different pharmaceutical preparations. A reasonable intra-assay (3.9,10.8%, n = 3) and inter-assay (5.6,11.4%, n = 3) precision illustrated the good performance of the analytical procedure. The protocol proved to be rapid and cost-effective for screening purposes. Copyright © 2010 John Wiley & Sons, Ltd. [source] Quantitation of Ventricular Size and Function:ECHOCARDIOGRAPHY, Issue 8 2000Accuracy of Transthoracic Rotational Scanning, Principles Two-dimensional echocardiography is a readily applicable method for the quantification of ventricular volumes. However, it is limited by assumptions regarding ventricular shape. Three-dimensional echocardiography has emerged as a more accurate and reproducible approach to ventricular volume and functional assessment compared with two-dimensional echocardiography. We review the principles of transthoracic rotational scanning and its clinical application for quantitative assessment of ventricular volume and function. [source] Electrochemically Induced Iron Release of Adsorbed Horse Spleen Ferritin: Quantitation of Iron Using Long Optical Path Length Thin-Layer SpectroelectrochemistryELECTROANALYSIS, Issue 23 2007Abstract In this work, long optical path length thin-layer electrochemical cell was constructed using indium-tin oxide on glass as the electrode material. Iron release from ferritin adsorbed on the electrode was induced by applying a negative potential sweep in the presence of 1,10-phenanthroline. The usefulness of spectroelectrochemistry as a means of determining the quantity of iron released from an adsorbed layer of ferritin is demonstrated. [source] Field-amplified sample injection-micellar electrokinetic capillary chromatography for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers and derivatives in canned soft drinksELECTROPHORESIS, Issue 9 2010Héctor Gallart-Ayala Abstract Conditions were established for the separation and analysis of bisphenol A, bisphenol F, and their diglycidyl ethers by micellar electrokinetic capillary chromatography (MECC). Good resolution was obtained for all compounds, although in order to achieve the separation of ortho,ortho, ortho,para, and para,para isomers of bisphenol F diglycidyl ether (BFDGE), BFDGE·2H2O and BFDGE·2HCl, it was necessary to use a 25,,m id fused silica capillary. To increase sensitivity, a field-amplified sample injection (FASI)-MECC method was developed using 10,mM SDS solution as injection matrix and a 75,,m id fused silica capillary. Instrumental quality parameters such as LODs (<55,,g/L with standards), linearity (r2>0.999), and run-to-run and day-to-day precisions (RSD values lower than 12.5%) were determined. Finally, the suitability of the FASI-MECC method for the analysis of bisphenol A, bisphenol F, and their diglycidyl ethers in canned soft drinks was evaluated. Quantitation was performed by matrix-matched calibration using a plastic-bottled isotonic drink as matrix. The results showed that FASI-MECC is an economic method for the screening and quantitation of these kinds of compounds in soft drink beverages, with no loss of reproducibility, and effective at concentrations lower than the specific migration level values established by the European Union. [source] Chiral separation of the plant lignan matairesinol by capillary electrophoresis,ELECTROPHORESIS, Issue 17 2008Ulrike Müller Abstract Lignans are dimeric phenylpropanoid compounds in plants that enjoy increasing medicinal interest because of their phytoestrogen activity. Lignans are chiral compounds and for most natural occurring lignans, chirality is not known. Separation of racemic matairesinol by CE in a non-coated silica capillary with carboxymethyl-,-cyclodextrin as chiral selector in phosphate buffer was successful. Electrolyte and selector concentrations and pH were systematically optimized in order to obtain baseline separation and short analysis times. Matairesinol from safflower fruit was determined as (,)-enantiomer. Quantitation results for matairesinol with the optimized method after calibration with authentic lignan were very similar to those by HPLC. The limit of detection is 2,,g/mL sample by DAD detection. [source] Quantitation of reduced glutathione and cysteine in human immunodeficiency virus-infected patientsELECTROPHORESIS, Issue 10-11 2004Elena Sbrana Abstract Plasma viral load (VL) values and CD4+ cell count are employed clinically for initiation of therapy in the treatment of patients infected with human immunodeficiency virus (HIV), as previous clinical studies have shown a marked prevalence of acquired immunodeficiency sydrome (AIDS) development in seropositive individuals with VL values over 30,000 copies/mL. Many studies have shown that reduced glutathione (GSH) and cysteine (Cys) deficiency play an important role in the infection. We have developed capillary zone electrophoresis (CZE)-based assays and have used them to investigate the relationship between plasma and intracellular thiol levels and HIV-1 viremia in plasma. Blood samples from healthy volunteers and seropositive patients undergoing different antiretroviral regimes were analyzed in the study. The VL assay was based on CZE-UV detection of viral RNA at 260 nm. Determination of endogenous reduced Cys and GSH was achieved by CZE-UV detection of their mercurial complexes at 200 nm. We found that a decrease in GSH and Cys levels may be associated with disease progress. In fact, reduced GSH and Cys levels appear progressively reduced with increasing VL. [source] Quantitation of talinolol and other ,-blockers by capillary electrophoresis for in vitro drug absorption studiesELECTROPHORESIS, Issue 15 2003Bilal Awadallah Abstract A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-,-cyclodextrin (HDAS-,-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-,-CD and at 20°C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other ,-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determing talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments. [source] Quantitation of non-motor symptoms in Parkinson's diseaseEUROPEAN JOURNAL OF NEUROLOGY, Issue 2008K. Ray Chaudhuri Background:, Disabling non-motor symptoms (NMS) associated with Parkinson's disease (PD), such as dementia and loss of balance, do not respond well to levodopa therapy and can lead to eventual death in patients with the disease. In 2006, a multidisciplinary group of experts and patient representatives developed an NMS screening questionnaire (NMSQuest) and a unified Non-Motor Symptoms Scale (NMSS) to address the need for simple identification and comprehensive assessment of NMS in patients with PD. Methods and Results:, An international pilot study of 96 healthy controls and 123 patients with various stages of treated and untreated PD was conducted to demonstrate that the NMSQuest is a feasible, valid, and accepted tool. Conclusion:, The majority of patients and caregivers felt that the questionnaire was clear and relevant to their daily lives. Data from 242 PD patients with no dementia were analysed in a pilot study on the clinimetric validation of NMSS. Similar to the NMSQuest study, the NMSS study revealed a significant correlation between progression of PD and increasing NMS burden. These studies suggest that the NMSQuest accurately detects the NMS, and that the NMSS closely correlates with quality of life for PD patients. [source] Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probeFEBS JOURNAL, Issue 7 2007Jing Jing Gao Quantitation of superoxide radical (O,2,·) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O2,· using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O2,· in living cells. Better selectivity for O2,· over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 × 10,9,3.33 × 10,6 m. The detection limit was 1.68 × 10,9 m. Fluorescence images of probe-stained macrophages stimulated with 4,-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope. [source] Regulation of human and mouse procathepsin E gene expressionFEBS JOURNAL, Issue 9 2001Matthew Cook Cathepsin E is an intracellular aspartic proteinase that is considered to have a number of physiological roles including antigen processing. Quantitation of procathepsin E mRNA by LightCyclerÔ technology indicated that the gene was transcribed in lung but not in kidney of both human and mouse origin. In contrast, the transcript was present in mouse spleen and alveolar macrophages but not in the counterpart tissue/cells from humans. Regulation of human and mouse procathepsin E gene expression was shown not to be influenced by the extent of CpG methylation but depended on the recognition of potential binding motifs in each promoter region by transcription factors such as GATA1, PU1 and YY1, as revealed by functional analysis using a series of promoter/luciferase reporter gene fusion constructs. Thus the extent to which the procathepsin E gene is expressed in a particular cell type may depend on the balance between the effects produced by positive-acting, cell-specific transcription factors such as GATA1 and PU1 and the negative influence of the ubiquitous YY1 factor. In this way, the relative abundance and influence of general and cell-specific transcription factors can govern the production of cathepsin E and thereby account for the sporadic cell and tissue distribution of this enzyme in different species. [source] Impact of estragole and other odorants on the flavour of anise and tarragonFLAVOUR AND FRAGRANCE JOURNAL, Issue 2 2007Annette Zeller Abstract Since estragole has been described as potent carcinogen and did not show any flavour impact on the odour of fennel, the flavour of anise and tarragon was examined by combinations of instrumental and sensory analyses. In anise fruits, trans- anethole, 2-isopropyl-3-methoxypyrazine, and anisaldehyde showed high flavour dilution (FD) factors, followed by those of eugenol, cis- anethole, estragole and linalool. Quantitation of the odorants showing higher FD factors in anise tea, and calculation of odour activity values (OAVs) by dividing the concentrations of the respective compound by its recognition threshold in water revealed the highest OAV for trans- anethole, followed by those of anisaldehyde and estragole. Still, an aroma impact of estragole next to trans- anethole could be neglected, due to former sensory studies of aqueous models of fennel tea. For tarragon, AEDA revealed the highest FD factors for eugenol, 7-methoxycoumarin, , -ionone, 2-isopropyl-3-methoxypyrazine, estragole and linalool. Of these, estragole and 7-methoxycoumarine were most abundant in tarragon leaves, showing contents of 2900 and 220 mg/kg, respectively. To calculate OAVs, the contents of potent odorants were divided by their odour threshold in starch or cellulose, as these were the main constituents of the leaves. In this way, , -ionone and eugenol showed the highest OAVs of 5862 and 2653, repectively, followed by estragole (OAV = 460), cis- 1,5-octadiene-3-one (OAV = 140), vanilline (OAV = 139) and 2-isopropyl-3-methoxypyrazine (OAV = 130). Therefore, it was concluded that , -ionone and eugenol made the greatest contribution to the overall flavour of tarragon, whereas estragole and the other odorants were of minor importance. Copyright © 2006 John Wiley & Sons, Ltd. [source] Quantitation of suspected allergens in fragrances (Part I): evaluation of comprehensive two-dimensional gas chromatography for quality controlFLAVOUR AND FRAGRANCE JOURNAL, Issue 2 2004Robert Shellie Abstract An evaluation of comprehensive two-dimensional (2D) gas chromatography (GC×GC) was performed to assess its suitability for the analysis of volatile fragrance components, recognized by the European Commission's Scienti,c Committee on Cosmetics and other Non-food Products (SCCNFP) as possible skin sensitizers. The 24 volatile components listed by the SCCNFP were baseline-resolved or better within one 30 min analysis. High-quality calibration data for standard mixtures were obtained, with R2 > 0.998 over the concentration range 2,1000 mg/l. However, the analysis of small spiked amounts of target compounds in truly complex fragrances was problematic, due to uncertainty in component assignment. The bene,ts and limitations of GC×GC are reported, and a discussion of the proposed directions for the solution of this analysis is provided. Copyright © 2004 John Wiley & Sons, Ltd. [source] Preoperative hCG, and CA 72-4 are prognostic factors in gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 6 2004Johanna Louhimo Abstract In gastric cancer, the role of tumour markers in assessment of prognosis is unconfirmed. In our study, we evaluated the prognostic significance of serum tumour markers carcinoembryonic antigen (CEA), CA 19-9, CA 72-4, CA 242 and free , subunit of human chorionic gonadotropin (hCG,) in gastric cancer. Preoperative serum samples were obtained from 146 patients with gastric cancer, including 29 with stage I, 11 with stage II, 42 with stage III and 64 patients with stage IV cancer. Quantitation of CEA, CA 19-9, CA 72-4 and CA 242 in serum was performed with commercial assays. HCG, was measured with an in-house immunofluorometric assay based on monoclonal antibodies specific for the free ,-subunit of hCG. Survival analysis was performed with Kaplan-Meier life-tables and log-rank test, and with multivariate Cox regression analysis. Disease-specific cumulative 2-year survival rate was 40%. Serum levels of CEA, CA 72-4, CA 242 and hCG, showed significant correlation with stage (p<0.027); for CA 19-9 the association was of borderline significance (p=0.056). Of the studied markers, CA 19-9, CA 72-4, CA 242 and hCG, were found to be prognostic factors in univariate analysis (p< 0.022). In multivariate analysis, stage had the statistically most significant association with prognosis followed by hCG,, tumour histology according to the Laurén classification and by CA 72-4. In gastric cancer, tumour markers hCG, and CA 72-4 are independent prognostic factors in addition to stage and histological type of the tumour. © 2004 Wiley-Liss, Inc. [source] Quantitation of cytological parameters of malignant lymphocytes using computerized image analysisINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2008S. A. HAMID JAHANMEHR Summary Computerized image analysis may add to morphological evaluation by turning qualitative data into quantitative values. In this study, image analysis program was used to quantitate cytological parameters of lymphocytes in B-cell lymphoproliferative disorders. Chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and B-cell prolymphocytic leukemia (B-PLL) were selected to represent typically small, medium, and large-sized lymphocytes, respectively. Image analysis was performed to determine the morphological parameters. A set of measurements was generated for quantitation of total cell area, cell diameter, cytoplasm area, nuclear area, nuclear/cell ratio, and nuclear density. The quantitated parameters substantiated morphological characteristics of the tumor cells. Comparative assessments demonstrated that CLL, MCL, and PLL can be differentiated by the quantitative descriptors. The results from image analysis may assist in defining morphological criteria and in developing quantitative cell morphology. [source] Determination of peripheral blood stem cells by the Sysmex SE-9500INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 4 2001Liming Peng The Sysmex SE-9500 automated haematology analyser provides an estimate of immature cells, referred to as ,haematopoietic progenitor cells' (HPC). The aim of this study was to evaluate the reliability and usefulness of the SE-9500 HPC parameter as compared with the CD34 + cell count and to determine whether the HPC count was of value in predicting the optimal harvesting time for peripheral blood stem cells (PBSC). Studies were performed on 112 samples from 21 patients with haematological malignancies and 13 healthy donors undergoing progenitor cell mobilisation. Coefficients of variation for the HPC count were 30%, 23.8%, 12.4% and 8.3% respectively for samples with low (4 × 106/l), medium (13 × 106/l), high (250 × 106/l) and very high (2413 × 106/l) counts. There was good linearity for HPC measurement in both peripheral blood (PB) and purified CD34 + cell suspensions (r > 0.995), and no detectable carryover was observed. There was an acceptable correlation between HPC and CD34 + cell counts for PB samples (r=0.669) and for CD34 + cell suspensions (r=0.859). Analysis of purified CD34 + cells using the SE-9500 HPC mode revealed that they appear both in the blast cell area and the immature granulocyte area of the analyser cell display. Quantitation of CD34 + cells and HPC during PBSC mobilisation showed good agreement between these parameters with regard to the optimal time for PBSC harvesting. These findings suggest that HPC counting with the Sysmex SE-9500 may be clinically useful for optimising the timing of PBSC collection. [source] Clinicians Treating Hypertension Need to Mind Their "P's" (Precision) and "Q's"(Quantitation): A Lesson From Left Ventricular Hypertrophy Regression and Atrial FibrillationJOURNAL OF CLINICAL HYPERTENSION, Issue 5 2007Thomas D. Giles MD No abstract is available for this article. [source] Tumor R2* is a prognostic indicator of acute radiotherapeutic response in rodent tumorsJOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 4 2004Loreta M. Rodrigues MSc Abstract Purpose To test the prognostic potential of tumor R2* with respect to radiotherapeutic outcome. Blood oxygenation level dependent (BOLD) MRI images are sensitive to changes in deoxyhemoglobin concentration through the transverse MRI relaxation rate R2* of tissue water, hence the quantitative measurement of tumor R2* may be related to tissue oxygenation. Methods and Materials Tumor growth inhibition in response to radiation was established for both GH3 prolactinomas and RIF-1 fibrosarcomas with animals breathing either air or carbogen during radiation. In a separate cohort, the baseline R2* and carbogen (95% O2, 5% CO2)-induced ,R2* of rat GH3 prolactinomas and murine RIF-1 fibrosarcomas were quantified using multigradient echo (MGRE) MRI prior to radiotherapy, and correlated with subsequent tumor growth inhibition in response to ionizing radiation, while the animals breathed air. Results A radiation dose of 15 Gy caused pronounced growth delay in both tumor models and transient regression of the GH3 prolactinomas. When the animals breathed carbogen during radiation, the growth delay/regression was enhanced only in the GH3 prolactinomas. The GH3 prolactinomas, which exhibit a relatively fast baseline R2* and large ,R2* in response to carbogen breathing prior to radiotherapy, showed a substantial reduction in normalized tumor volume to 66 ± 3% with air breathing and 36 ± 5% with carbogen seven days after 15 Gy irradiation. In contrast, the effect of 15 Gy on the RIF-1 fibrosarcomas, which give a relatively slow baseline R2* and negligible ,R2* response to carbogen prior to treatment, showed a much smaller growth inhibition (143 ± 3% with air, 133 ± 12% with carbogen). Conclusion Quantitation of tumor R2* and carbogen-induced ,R2* by MGRE MRI provides completely noninvasive prognostic indicators of a potential acute radiotherapeutic response. J. Magn. Reson. Imaging 2004;19:482,488. © 2004 Wiley-Liss, Inc. [source] Quantitation of sleep and spinal curvature in an unusually longevous owl monkey (Aotus azarae)JOURNAL OF MEDICAL PRIMATOLOGY, Issue 6 2006Juri Suzuki Abstract Background, A table summarizing the primary literature on 19 species of longevous non-human primates, other than owl monkey, is presented. Methods, We prospectively quantitated the sleep of a longevous female owl monkey (Aotus azarae), aged >30 years, longitudinally for 2 years and also evaluated the senility-induced change in spinal curvature. Results, The mean daily total sleep time (TST) of this monkey ranged between 790 and 1106 minutes, and was markedly higher in comparison with its female progeny (aged 16 years and used as a control) whose daily TST during the same experimental period ranged between 612 and 822 minutes. Conclusions, The calculated kyphotic index (KI) of 2.27 for this monkey, compared with the KIs 4.83 and 5.42, for its progeny and female grandprogeny (aged 1 year) respectively, confirmed the prominent spinal curvature. [source] Measurement of HIV RNA in patients infected by subtype C by assays optimized for subtype B results in an underestimation of the viral loadJOURNAL OF MEDICAL VIROLOGY, Issue 2 2004Bat-Sheva Gottesman Abstract Quantitation assays of HIV-1 RNA used currently were designed and optimized for subtype B viruses. However, infection with non-B HIV viruses has become more common worldwide. Unfortunately, little information is available regarding the suitability of these assays for measurement of viral load in specific non-B subtypes. The performance of two commercial HIV-1 RNA quantitation assays was evaluated in 82 HIV subtype C-infected patients and in 43 HIV-1 subtype B-infected patients. Blood samples were tested by the Amplicor HIV-1 Monitor Assay, Version 1.5, and by the nucleic acid sequence-based amplification HIV-1 assay (NucliSens). The results were compared by using a paired, two-tailed Student's t -test; the difference between the assays was found to be significant only for subtype C. Discordant results (>0.5 log difference) between the two assays were detected in 39% of subtype C samples, compared to 23.2% of subtype B samples. In all cases in which a discordant result was detected, the lower results were obtained by the NucliSens assay. Discordant results between CD4 and viral load (CD4,<,200 cells/ml with a viral load <5,000 copies/ml) were observed in eight of the subtype C-infected patients when a viral load was measured by NucliSens (9.7%), compared to three patients (3.6%) when measured by the Amplicor assay. In conclusion, in patients with HIV subtype C infection, measurement of HIV RNA by the NucliSens assay resulted in a significant underestimation of the viral load as compared to the Amplicor assay. As a consequence, such an underestimation may result in sub-optimal care of patients infected with HIV subtype C. J. Med. Virol. 73:167,171, 2004. © 2004 Wiley-Liss, Inc. [source] Quantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapyJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Karine Gourlain Abstract In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan® PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan® PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan® PCR >10/150,000 cells (RR: 27.6, IC95: 7.1,107.2), the CD4 cell count <75 cells/mm3 and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan® PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml. J. Med. Virol. 69:401,407, 2003. © 2003 Wiley-Liss, Inc. [source] RNA editing and alternative splicing of human serotonin 2C receptor in schizophreniaJOURNAL OF NEUROCHEMISTRY, Issue 6 2003Stella Dracheva Abstract Serotonin 2C receptor (5-HT2CR) heterogeneity in the brain occurs mostly from two different sources: (i) 5-HT2CR mRNA undergoes adenosine-to-inosine editing events at five positions, which leads to amino acid substitutions that produce receptor variants with different pharmacological properties; (ii) 5-HT2CR mRNA is alternatively spliced, resulting in a truncated mRNA isoform (5-HT2CR-tr) which encodes a non-functional serotonin receptor. 5-HT2CR mRNA editing efficiencies and the expression of the full-length and the truncated 5-HT2CR mRNA splice isoforms were analyzed in the prefrontal cortex of elderly subjects with schizophrenia vs. matched controls (ns = 15). No significant differences were found, indicating that there are no alterations in editing or alternative splicing of 5-HT2CRs that are associated with schizophrenia in persons treated with antipsychotic medications. Quantitation of 5-HT2CR and 5-HT2CR-tr mRNA variants revealed that the expression of 5-HT2CR-tr was ,,50% of that observed for the full-length isoform. [source] Quantitation of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and central nervous system and its relationship to the clinicopathologic features of autoimmune encephalomyelitisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2006Hiroshi Sakuma Abstract There is controversy whether the amount of autoantigens expressed in the thymus regulates negative selection of autoreactive T cells and determine susceptibility or resistance to experimental autoimmune encephalomyelitis (EAE). In the present study, we have addressed this issue by quantifying neuroantigens in the thymus of two EAE-susceptible (LEW and LEW.1AV1) and one EAE-resistant (BN) rat strains. We further examined whether amounts of neuroantigens in various parts of the central nervous system (CNS) affect the clinical course and lesion distribution of acute and chronic EAE. Real-time PCR and histologic analyses showed that there was no significant difference in the amount and distribution of myelin oligodendrocyte glycoprotein and myelin basic protein in the thymus and CNS among the three strains and that both acute and chronic EAE lesions in the CNS were preferentially distributed in the area where neuroantigens were abundantly present. These findings suggest that susceptibility or resistance to EAE is not regulated by the amount of the neuroantigens expressed in the thymus. Furthermore, the lesion distribution, but not the clinical course, of EAE is related to the neuroantigen expression in the CNS. © 2006 Wiley-Liss, Inc. [source] Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell-penetrating abilityJOURNAL OF PEPTIDE SCIENCE, Issue 1 2010Yvonne Turner Abstract A 12-mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell-penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell-penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA-peptide conjugates to be not primarily related to the cell-penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA-peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Quantitation of protein particles in parenteral solutions using micro-flow imagingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2009Chi-Ting Huang Abstract The U.S. and European Pharmacopeias require subvisible (,10 and ,25 µm) and visible particulate testing of therapeutics to ensure their safety and suitability for clinical use. The objective of this article is to compare the sizing and counting accuracies of light obscuration, which is the standard technique used to measure subvisible particulate matter, and Micro-Flow Imaging (MFIÔ), a new imaging-based technology. An immunoconjugate was selected as the model protein for this study since it could be induced to form particulate matter in PBS. Light obscuration was performed as described in USP chapter <788> while MFI measurements were conducted per the manufacturer's procedures. The two techniques yielded similar results when polystyrene standards were analyzed. However, the MFI measurements indicated the presence of significantly more particles in the protein-containing solution compared to the light obscuration measurements. The presence of nonspherical protein particles as well as particles that possess a refractive index similar to the solvent that they are in appear to be detected by MFI, but not by light obscuration, leading to the difference in the results. Imaging-based technologies could aid in developing formulations and processes that would minimize the formation of protein particulates. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3058,3071, 2009 [source] Quantitation of crystalline and amorphous forms of anhydrous neotame using 13C CPMAS NMR spectroscopyJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2005Thomas J. Offerdahl Abstract Although most drugs are formulated in the crystalline state, amorphous or other crystalline forms are often generated during the formulation process. The presence of other forms can dramatically affect the physical and chemical stability of the drug. The identification and quantitation of different forms of a drug is a significant analytical challenge, especially in a formulated product. The ability of solid-state 13C NMR spectroscopy with cross polarization (CP) and magic-angle spinning (MAS) to quantify the amounts of three of the multiple crystalline and amorphous forms of the artificial sweetener neotame is described. It was possible to quantify, in a mixture of two anhydrous polymorphic forms of neotame, the amount of each polymorph within 1,2%. In mixtures of amorphous and crystalline forms of neotame, the amorphous content could be determined within 5%. It was found that the crystalline standards that were used to prepare the mixtures were not pure crystalline forms, but rather a mixture of crystalline and amorphous forms. The effect of amorphous content in the crystalline standards on the overall quantitation of the two crystalline polymorphic forms is discussed. The importance of differences in relaxation parameters and CP efficiencies on quantifying mixtures of different forms using solid-state NMR spectroscopy is also addressed. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2591,2605, 2005 [source] Plasma pharmacokinetics of warfarin enantiomers in catsJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2000S.A. SMITH The purpose of this study was to determine the dispositions of S-warfarin and R-warfarin in normal cats following intravenous and oral administrations of racemic warfarin. Citrated blood samples were collected from 10 cats prior to and at times 5, 15, and 30 min, 1, 2, 3, 4, 5, 6, 12, 24, 36, 48, 72, 96, and 120 h following a single intravenous bolus of 0.5 mg/kg of racemic warfarin. After a 21-day washout period, samples were then similarly collected in three groups of four cats for 120 h following oral administration of 0.1, 0.25, and 0.5 mg/kg racemic warfarin. S-warfarin and R-warfarin were detected using a high-performance liquid chromatography assay validated for cat plasma. Drug concentration,time curves were subjected to non-compartmental analysis. Median pharmacokinetic parameters associated with the intravenous administration of 0.5 mg/kg racemic warfarin were as follows: t1/2 (S:28.2, R:18.3 h), area under the plasma concentration,time curve (AUC; S:33.0, R:24.6 h*,g/mL), area under the moment curve (AUMC; S:1889, R:527.8 h*h*,g/mL), and mean residence time (MRT; S:38.7, R:20.9 h). For each parameter, S-warfarin was significantly different from R-warfarin (P<0.05). Warfarin was absorbed rapidly after oral administration, and the dosage did not affect the time to maximum concentration (S:0.87, R:0.75 h). Oral dosage significantly influenced maximum plasma concentration (ng/mL, S:1267, R:1355 at 0.5 mg/kg; S:614.9, R:679.4 at 0.25 mg/kg; S:250.5, R:367.6 at 0.1 mg/kg), AUC (h*,g/mL, S:45.12, R:30.91 at 0.5 mg/kg; S:22.98:, R:18.99 at 0.25 mg/kg; S:3.922, R:3.570 at 0.1 mg/kg) and AUMC (h*h*,g/mL, S:2135, R:1062 at 0.5 mg/kg; S:943.1, R:599.9 at 0.25 mg/kg; S:132.2, R:59.03 at 0.1 mg/kg), but not t1/2 (S:23.5, R:11.6 h) nor MRT (S:26.3, R:13.5 h). Both warfarin enantiomers were highly (>96.5%) protein-bound. Quantitation of the warfarin content in commercially available tablets indicated an unequal distribution of the drug throughout the tablet. [source] Pitfalls and advantages of different strategies for the absolute quantification of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRSNMR IN BIOMEDICINE, Issue 10 2009E. Malucelli Abstract This study extensively investigates different strategies for the absolute quantitation of N -acetyl aspartate, creatine and choline in white and grey matter by 1H-MRS at 1.5,T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water-unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post-processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T2, of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole-metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T2s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T2 estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time-consuming alternative to IP in the quantitation of brain metabolites by 1H-MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water-suppressed and one water-unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times. Copyright © 2009 John Wiley & Sons, Ltd. [source] Quantitation of the Effect of Hydroxylamine on Rhodopsin Palmitylation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Wesley Jackson Rhodopsin (the photosensitive rod visual pigment) has been a model for photobiologic studies of the opsins as well as a structural model for G-protein-coupled receptors. The two palmitate groups attached to cysteines 322 and 323 are thought to serve as membrane anchors for the rhodopsin C-terminus, but the absence of the palmitates does not alter membrane localization. However, removal of the palmitates affects rhodopsin function. Therefore, it is important to quantitate the stability of rhodopsin palmitates to hydroxylamine, which is a widely utilized reagent in biochemical preparations of the apoprotein. We have developed a mass spectrometric method to quantitate the resulting opsin palmitylation. Our data show that both of the bovine rhodopsin palmitates are labile to hydroxylamine, with significant depalmitylation occurring at concentrations of ,100 mm, with an EC50 of 220 mm L,1. The palmitate at position 322 is the more stable to hydroxylamine. Samples prepared in the presence of >50 mm should therefore be considered to be at least partially depalmitylated and the results interpreted accordingly. [source] Characterisation of Plasmodium invasive organelles; an ookinete microneme proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009Kalpana Lal Dr. Abstract Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion. [source] Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detectionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2008Dennis J. Dietzen Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20,min. The dynamic range of each amino acid was generally 1,1000,µmol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88,125%. Deming regression with the Beckman 7300 yielded slopes from 0.4,1.2, intercepts from ,21 to 65,µmol/L, correlation coefficients from 0.84,0.99 and Syx from 2,125,µmol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, , -alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors. Copyright © 2008 John Wiley & Sons, Ltd. [source] | ||||||||||||||||||||||||||||||||||