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At least 667 records · Page 37Linked to original sources
PubMed1989

A rapid and specific HPLC-electrochemical method for the determination of endogenous 5-methyltetrahydrofolic acid in plasma using solid phase sample preparation with internal standardization.

A rapid and specific HPLC-electrochemical method for determining endogenous 5-methyltetrahydrofolic acid (5MeTHF) in plasma is described. Quantitative solid phase extraction of 5MeTHF and internal standard, beta-hydroxyethyltheophylline, was carried out using proprietary phenyl bonded-silica columns (Bond Elut Phenyl cartridges, 1.0 mL capacity). Chromatographic separation was achieved using a mobile phase consisting of 15% (v/v) methanol in 0.05 M KH2PO4, pH 3.5 at a flow rate of 2.0 mL/min in conjunction with a Waters Assoc. radially compressed Nova-Pak phenyl column (10 cm x 8 mm, 4 microns bonded silica). The internal standard was measured by UV detection at 254 nm. A Bioanalytical Systems Inc. LC-17 glassy carbon oxidative flow cell with a potential held at +0.35 V vs Ag/AgCl using the LC-4A amperometric controller allowed levels of 1-2 ng/mL 5MeTHF to be measured in 500 microL of plasma. Daily appraisal of the ratio produced by authentic materials clearly demonstrated that quantitation using dual detection was not subject to problems of differential response. Inter-day variation of the differential detector response is cited. Comparison of the Lactobacillus casei bioassay with HPLC demonstrates good agreement between methods but at the same time highlights the drawback of using such non-specific methods to measure samples where more than one folylmonoglutamate may be present. Antoxidant free storage for three months at -70 degrees C in darkness resulted in no deterioration of 5MeTHF. A comparison of the means and range of values for plasma folate obtained using HPLC, L. casei bioassay and the radiometric binding assay is reported.

Chromatography, High Pressure Liquid↗
PubMed1998

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat.

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC-ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100x1 mm I.D. C8, 5 microm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)-methanol-dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 microl/min. For monoamines and metabolites we used a 150X1 mm I.D. C18 5 microm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 microM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-microm tissue slices. Tissue samples were homogenized in 50 or 100 microl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and gamma-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.

Amino Acids↗
PubMed1995

Laboratory assessment of von Willebrand factor. Use of different assays can influence the diagnosis of von Willebrand's disease, dependent on differing sensitivity to sample preparation and differential recognition of high molecular weight VWF forms.

Three separate laboratory assays for von Willebrand Factor (VWF), a standard "antigen" (antisera-ELISA-based) assay (VWF:Ag), a standard ristocetin-dependent-platelet-agglutination procedure (VWF:RCof), and an ELISA-based collagen-VWF binding assay (VWF:CBA), have been evaluated for their ability to detect alterations in VWF levels following differential processing of blood for testing, and specifically in (1) serum compared to plasma and (2) filtered plasma compared to nonfiltered plasma. Although all assays tended to detect some change, sensitivity of detection varied between assays, with the VWF:CBA most consistently able to detect large decreases in VWF levels in serum and filtered plasma. The authors propose that the increased sensitivity of the VWF:CBA assay to VWF depleted in these circumstances is that this assay selectively detects higher molecular weight forms (ie, those known to be more functionally relevant), and that assay results reflect the preferential incorporation of these forms in in the platelet-fibrin-gel during the clotting process, and onto the filter matrix during filtration. To confirm this, multimer analysis was performed and showed a reduction in high molecular weight forms of VWF in these cases. Finally, direct evidence that the VWF:CBA assay preferentially detects high molecular weight forms of VWF was obtained following fractionation of normal plasma VWF (separation according to molecular weight using size exclusion matrix; confirmed by specific multimer analysis) and assessment of eluted VWF. Using a standard VWF:Ag assay, detection of eluted VWF was unrelated to molecular size. In contrast, the VWF:CBA showed selective detection, and was able to preferentially discriminate high and intermediate forms of VWF from low molecular weight forms. The findings are of particular relevance to diagnostic pathology laboratories because filtered plasma or serum can be inappropriately (and unknowingly) provided for the clinically queried diagnosis of von Willebrand's disease (VWD). As outlined in this report, these samples can yield VWF results that closely mimic those of a Type 2A or Type 2B VWD individual, and thus, VWD may be incorrectly diagnosed.

Blood↗
PubMed2006

Shear mechanical anisotropy of side chain liquid-crystal elastomers: influence of sample preparation.

We study the mechanical anisotropy of a series of uniaxial side chain nematic elastomers prepared with the same chemical composition but with different preparation protocols. For all the compounds, the experiments performed as a function of temperature show no discontinuity in both G' (//) and G' ( perpendicular) (the labels // and perpendicular stand for the director parallel, respectively perpendicular to the shear displacement) around the nematic-isotropic (N-I) phase transition temperature determined by DSC. They also all show a small decrease in G' (//) starting at temperatures well above this temperature (from approximately 4( degrees ) C to approximately 20( degrees ) C depending on the compound studied) and leading to a small hydrodynamic value of the G' ( perpendicular)/G' (//) ratio. The measurements taken as a function of frequency show that the second plateau in G' (//) and the associated dip in G (//)" expected from dynamic semi-soft elasticity are not observed. These results can be described by the de Gennes model, which predicts small elastic anisotropy in the hydrodynamic and linear regimes. They correspond to the behavior expected for compounds beyond the mechanical critical point, which is consistent with the NMR and specific heat measurements taken on similar compounds. We also show that a reduction in the cross-linking density does not change the non-soft character of the mechanical response. From the measurements taken as a function of frequency at several temperatures we deduce that the time-temperature superposition method does not apply. From these measurements, we also determine the temperature dependence of the longest relaxation time tau(E) of the network for the situations where the director is either parallel or perpendicular to the shear velocity. Finally, we discuss the influence on the measurements of the mechanical constraint associated with the fact that the samples cannot change their shape around the pseudo phase transition, because of their strong adherence on the sample-bearing glass slides.

Anisotropy↗
PubMed2003

Bioprocess considerations for expanded-bed chromatography of crude canola extract: sample preparation and adsorbent reuse.

Compared to the conventional microbial and mammalian systems, transgenic plants produce proteins in a different matrix. This provides opportunities and challenges for downstream processing. In the context of the plant host Brassica napus (canola), this work addresses the bioprocessing challenges of solid fractionation, resin fouling by native plant components (e.g., oil, phenolics, etc.), hydrodynamic stability, and resin reuse for expanded bed adsorption for product capture. Plant tissue processing and subsequent protein extraction typically result in an extract with a high content of solids containing a wide particle-size distribution. Without removal of larger particles, the column inlet distributor plugged. The larger particles (> 50 microm) were easily removed through centrifugal settling comparable to that attainable with a scroll decanter. The remaining solids did not affect the column performance. Less than 4% of the lipids and phenolics in the fed extract bound to STREAMLINE trade mark DEAE resin, and this small proportion could be satisfactorily removed using recommended clean-in-place (CIP) procedures. Hydrodynamic expansion and adsorption kinetics of the STREAMLINE trade mark DEAE resin were maintained throughout 10 cycles of reuse, as was the structural integrity of the resin beads. No significant accumulation of N-rich (e.g., proteins) and C/O-rich components (e.g., oil and phenolics) occurred over the same period.

Brassica napus↗
PubMed1991

High performance liquid chromatographic determination of Picumast and two active metabolites in plasma using on-line sample preparation.

A method for determining Picumast, an antiallergic drug, in plasma by HPLC and column switching has been developed. The system consisted of two precolumns, an analytical column, three pumps, an autosampler and a fluorescence detector. The precolumns (17 x 4.6 mm i.d.) were packed with LiChroprep RPR (a moderately polar reversed phase) and the analytical column with Nucleosil ODS (RP 18, 5 microns). The columns were connected according to the alternating precolumn technique. The mobile phase consisted of 30% CH3CN/70% 0.05 M KH2PO4, pH 2.5, with a flow gradient. Detection wavelengths were 333 nm for excitation and 383 nm for emission. The retention times of Picumast, M1 and M2 were 12, 3.6 and 4.0 min, respectively. Total run time was 15 min. The limit of detection was 3 ng/mL for M1 and 1 ng/mL for M2 and Picumast using an injection volume of 150 microL. The recoveries vary between 89% and 97% with standard deviations between 2.4 and 3.3%.

Chromatography, High Pressure Liquid↗
PubMed1996

Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.

Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.

Acetyl-CoA C-Acyltransferase↗
PubMed2004

Alternative sample preparation prior to two-dimensional electrophoresis protein analysis on solid lipid nanoparticles.

The proteins adsorbing onto the surface of intravenously injected drug carriers are regarded as a key factor determining the organ distribution. Depending on the particle surface properties, certain proteins will be preferentially adsorbed, leading to the adherence of the particle to cells with the appropriate receptor. Therefore, the knowledge of the protein adsorption pattern and the correlation to in vivo behavior opens the perspective for the development of intravenous colloidal carriers for drug targeting. After incubation in plasma, the adsorbed proteins were analyzed using two-dimensional polyacrylamide gel electrophoresesis (2-D PAGE, 2-DE). The purpose of the present study was to develop an alternative separation method to separate solid lipid nanoparticles (SLN) carriers from plasma by gel filtration prior to 2-D PAGE. Via the specific absorption coefficients and a two-equation system, elution fractions were identified being practically plasma-free. This allows protein analysis on SLN which are typically in density too close to the density value of water to be separated by the standard centrifugation method. The SLN used for establishing the gel filtration were prepared in a way that they had a sufficiently low density to be additionally separated by centrifugation. The adsorption patterns obtained after separation with both methods were qualitatively and quantitatively identical, showing the suitability of the gel filtration.

Adsorption↗
PubMed1988

Controlled environment vitrification system: an improved sample preparation technique.

The controlled environment vitrification system (CEVS) permits cryofixation of hydrated biological and colloidal dispersions and aggregates from a temperature- and saturation-controlled environment. Otherwise, specimens prepared in an uncontrolled laboratory atmosphere are subject to evaporation and heat transfer, which may introduce artifacts caused by concentration, pH, ionic strength, and temperature changes. Moreover, it is difficult to fix and examine the microstructure of systems at temperatures other than ambient (e.g., biological systems at in vivo conditions and colloidal systems above room temperature). A system has been developed that ensures that a liquid or partially liquid specimen is maintained in its original state while it is being prepared before vitrification and, once prepared, is vitrified with little alteration of its microstructure. A controlled environment is provided within a chamber where temperature and chemical activity of volatile components can be controlled while the specimen is being prepared. The specimen grid is mounted on a plunger, and a synchronous shutter is opened almost simultaneously with the release of the plunger, so that the specimen is propelled abruptly through the shutter opening into a cryogenic bath. We describe the system and its use and illustrate the value of the technique with TEM micrographs of surfactant microstructures in which specimen preparation artifacts were avoided. We also discuss applications to other instruments like SEM, to other techniques like freeze-fracture, and to novel "on the grid" experiments that make it possible to freeze successive instants of dynamic processes such as membrane fusion, chemical reactions, and phase transitions.

Microscopy, Electron↗
PubMed1992

Sample preparation for electron microscopy of internal cell structure.

Methods are reviewed for examination of internal cell structure by high-resolution scanning electron microscopy and compared with the rapid-freeze deep-etch replica technique used in transmission electron microscopy. Rapid freezing of fresh material, followed by freeze-fracture, provides a theoretically attractive approach in ultrastructure studies, but the high protein and solute content of most cells prevents a deep three-dimensional view for material frozen without some form of extraction. After discussion of other methods it is concluded that the most useful general approach, at least for cultured cells, is to first permeabilize or break open the cells in a medium which preserves the structure under study in a functional state as, for example, the movement of chromosomes along the division spindle, or transport of proteins within the Golgi region. After permeabilization, with attendant partial extraction, the preparation can be fixed, then viewed by either deep-etch replication, or by high-resolution scanning electron microscopy, with structure of interest revealed in deep view.

Animals↗