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PubMed1987

Membrane phospholipid organization in calcium-loaded human erythrocytes.

Intracellular Ca2+ levels in human erythrocytes were increased by incubating them with variable concentrations of Ca2+ in the presence of ionophore A23187. Experiments were done to confirm that the Ca2+ loading did induce changes in the cell shape and membrane protein composition. The effect of the increased cytoplasmic Ca2+ levels on the membrane phospholipid organization was analysed using bee venom and pancreatic phospholipases A2, Merocyanine 540 and fluorescamine as the external membrane probes. About 20% phosphatidylethanolamine (PE) and 0% phosphatidylserine (PS) were hydrolysed by the phospholipases in intact control cells, whereas in identical conditions these enzymes readily degraded, 20-30% PE and 7-30% PS, in Ca2+-loaded erythrocytes, depending on the cytoplasmic Ca2+ concentration. Also, Merocyanine 540 failed to stain the fresh or control erythrocytes, but it labeled the cells loaded with Ca2+. Furthermore, fluorescamine labeled approx. 20% PE in fresh or control erythrocytes while in identical conditions, significantly higher amounts of PE were modified in intact Ca2+-loaded cells. These results demonstrate that Ca2+ loading in human erythrocytes leads to loss of the transbilayer phospholipid asymmetry, and suggest that, together with spectrin, polypeptides 2.1 and 4.1 may also play an important role in maintaining the asymmetric distribution of various phospholipids across the erythrocyte membrane bilayer.

Bee Venoms↗

PubMed1987

Trypanosoma rhodesiense: antigenicity and immunogenicity of flagellar pocket membrane components.

A low density membrane fraction, isolated from the bloodstream stage of Trypanosoma rhodesiense and enriched in flagellar pocket membrane, was characterized with regard to antigenicity using antibodies raised against purified flagellar pocket membrane. Mild trypsinolysis of flagellar pocket membrane released two small peptides (Mr = 13-16 X 10(3)) separated by chromatofocusing (pI = 6.8 and 5.8) that were antigenic as monitored by fused rocket immunoelectrophoresis. Both of these antigenic peptides were enriched in relative fluorescence when flagellar pocket membrane was prepared from surface labeled (fluorescamine-beta-cyclodextrin) trypanosomes, indicating that cleaved peptides were on the external (luminal) side of the flagellar pocket membrane. More extensive release of fluorescamine labeled flagellar pocket membrane components was affected using mild detergent treatment (0.15% Zwittergent 3-12/0.4% Triton X-100), crossed immunoelectrophoresis separating two prominent antigens was more pronounced after incubation of flagellar pocket membrane with either porcine pancreas phospholipase A2 or umbilical cord sphingomyelinase. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent electroblotting to nitrocellulose also revealed two principal flagellar pocket membrane antigens (Mr approximately 60 and 66 X 10(3)), the latter showing greater release after exposure to sphingomyelinase or phospholipase, compared to mild detergent or 50 mM acetate, pH 5.0. Both antigens were glycoprotein as judged by electroblotting and the use of concanavalin A conjugated horseradish peroxidase as probe. Neither flagellar pocket membrane antigen was found to react with monoclonal antibodies prepared against T. rhodesiense variable surface antigen. The use of flagellar pocket membrane in the presence of Freund's complete adjuvant was found to protect mice against challenge infections with either the CP344 clone or uncloned CT Well-come isolate of T. rhodesiense.

Animals↗

PubMed1992

A sensitive fluorometric assay for reducing sugars.

A simple and rapid fluorometric assay for reducing sugars that is sensitive to the nanomolar range has been developed. The assay involves the derivatization of a given sugar with hydrazine at pH 3 to form a hydrazone, which is reacted with fluorescamine following adjustment of pH to first 9.4 and then 7.4. The amount of sugar in a sample is quantitated by measuring the fluorescence intensity at an excitation wavelength of 400 nm and an emission wavelength of 490 nm. The assay is precise and reproducible, as indicated by intra- and inter-run variations of at most 3% and 4%, respectively. In addition to reducing sugars, the assay can also be used to measure aliphatic and aromatic aldehydes, but not acetone. Compared with an existing fluorometric sugar assay, the assay reported here does not require chromatographic separation of the fluorescent derivative from unreacted fluorescamine. The assay can, however, be potentially adapted for postcolumn detection of aldehydes, reducing sugars, and hydrazones in HPLC.

Aldehydes↗

PubMed1995

Determination of tetrahydro-beta-carbolines in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry without interference from artifactual formation.

This paper describes a quantitative method for neuroactive alkaloids, 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) and 1,2,3,4-tetrahydro-beta-carboline (TBC), in rat brain by gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NICIMS). After addition of tetradeuterated MTBC and TBC (internal standards), the samples were subjected to deproteinization, reaction with fluorescamine, solvent extractions, trifluoroacetylation and GC-NICIMS analysis. In contrast to the other previous methods, the artifactual formation during analysis did not interfere with the determination of MTBC and TBC because their precursor tryptamine was removed as a fluorescamine derivative from the analytical system at the first step of pretreatment. MTBC and TBC were specifically and reliably determined in the range of pg-ng/sample. Application of the proposed method has revealed that the MTBC and TBC contents in rat brain significantly increase after intraperitoneal administration of MTBC and TBC, indicating their ability to easily cross the blood-brain barrier.

Animals↗

PubMed2004

An HPLC method for the determination of lisinopril in human plasma and urine with fluorescence detection.

A selective, sensitive and precise HPLC method with fluorimetric detection has been developed for the assay of lisinopril in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction, firstly with C18-cartridge and secondly with a silica-cartridge. After a pre-column derivatization with fluorescamine, the reaction mixture was chromatographed on C18-column with gradient elution, using methanol and 0.02 M phosphate buffer (pH=3.2). The fluorescamine-lisinopril derivative was detected fluorimetrically by monitoring the emission at 477 nm, with excitation at 383 nm. Linear quantitative response curve was generated over a concentration range of 5-200 ng/ml and 25-1000 ng/ml for plasma and urine samples, respectively. The mean recovery of lisinopril from plasma and urine was 63.41 and 74.08%, respectively. Intra-day and inter-day R.S.D. and R.M.E. values at three different concentrations were assessed. The method was applied for pharmacokinetic study in a healthy volunteer after a single oral dose of 20 mg of the drug.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1980

Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography.

A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

Animals↗

PubMed1987

Determination of trace levels of oxytocin in pharmaceutical solutions by high-performance liquid chromatography.

A high-performance liquid chromatographic assay has been developed for quantitating oxytocin in common large volume parenteral intravenous solution matrices. Separation is accomplished by a reversed-phase mechanism using a C18 column. The analyte is detected fluorimetrically after post-column derivatization with fluorescamine. Reaction efficiency is controlled by the use of a reaction buffer which is added separately from the fluorescamine via a dual pump reactor system. Given the low analyte concentration [40 parts per billion (10(9)) or less], the samples are concentrated on-line through the use of a trapping column and switching valve. To improve productivity, pre-concentration and analysis of adjacent samples is timed to occur concurrently. Performance of the assay is characterized by a high degree of accuracy, precision and ruggedness; the system is capable of distinguishing between the analyte, matrix components, impurities and common degradates.

Chromatography, High Pressure Liquid↗

PubMed1996

Multiphoton-excited fluorescence of fluorogen-labeled neurotransmitters.

Fluorescence detection of fluorogen-labeled neurotransmitters is demonstrated using 100 fs pulses from a titanium-sapphire mode-locked laser to achieve molecular excitation by simultaneous absorption of two and three photons of near-IR radiation. Two-photon excitation spectra are determined for the naphthalene-2,3 dicarboxaldehyde derivative of glycine and the fluorescamine derivative of leucine enkephalin, with the peak excitation cross section (o2) approximately equal to 1 x 10(-50) cm4 s/photon for both species. Three-photon-excitation fluorescence is demonstrated for o-phthaldialdehyde-labeled glutamate using excitation wavelengths between 965 and 1012 nm. The three-photon excitation cross section (o3) remains nearly constant in this wavelength range, with an absolute value of approximately 10(-84)-10(-85) cm6 s2/photon 2. Rapid cycling of analytes through the fluorescent excited state and detection that is free from background caused by Rayleigh and Raman scatter combine to make multiphoton-excited fluorescence a highly sensitive approach for detecting trace amounts of neurotransmitters. Measurements of two-photon-excited fluorescence of fluorescamine-labeled bradykinin and analysis of multiphoton-excited background reveal the potential of this method to detect fewer than 1000 neurotransmitter molecules.

Fluorescent Dyes↗

PubMed1982

Progress in isolation and purification of an inhibitor of sodium transport obtained from dog urine.

Among the potential modulators of transtubular sodium transport is the putative natriuretic hormone. Widespread efforts are underway to isolate this substance in pure form. The present studies describe a series of experiments directed to this goal. Urine samples in the amount of 150 liters were taken from normal, mineralocorticoid hormone "escape" dogs and were chromatographed through Sephadex G-25. The active fraction of the eluate (that is, the fraction containing the inhibitor of sodium transport) was then subjected to high pressure liquid chromatography (HPLC) in four consecutive steps using three different resins. Approximately 5% of the each column eluate was diverted by use of a stream-splitting apparatus, and the fluorescent pattern was measured and recorded, in most instances following the addition of fluorescamine. Based on the respective fluorescent patterns obtained from the eluates of the successive chromatographic steps, the residual portion (95%) of each eluate was divided into fractions, and each fraction was bioassayed. In each instance only the biologically active fraction was subjected to further purification. In the first step involving HPLC and a cation exchange resin, six fractions were obtained. Only one was active. When this fraction was subjected to reverse-phase chromatography, seven new fractions emerged. Again only one was active. When it was chromatographed using a second cation exchange resin, two fluorometrically detectable peaks, termed N ad H, were identifiable. N exhibited spontaneous fluorescence; H exhibited fluorescamine-dependent fluorescence. In the final step, N and H were separated and bio-assayed individually. N was inactive; H proved to be a potent inhibitor of sodium transport. Accumulation of H in sufficient quantity will determine whether it is a single compound and will permit analysis of its chemical nature.

Animals↗

PubMed2000

Investigation of amine and polyol functionality in extracts of polyurethane wound management dressings using MALDI-MS.

Polyurethane (PU) foams used in wound management are produced by a reaction between aromatic diisocyanates and polyether polyols. There is concern that residues of these starting materials, which may contain aromatic amine functionality, may leach from the finished polymer during in vivo applications. Furthermore, oligomers and additives may be leached from the PU system after the polymerization process is complete. Finished polymers have, therefore, been extracted with a range of solvents, such as water, diethyl ether and dilute HCl. The extracts were subjected to MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analysis in an attempt to determine the amine and polyol functionality. Direct MALDI-MS analysis of the wound dressing extracts indicated the presence of components based on the polyols [corrected] used in the formulation of the foam. The spacing between the peaks identified the base monomer used in the polyol. MALDI-MS analysis of the fluorescamine derivatives of model amine compounds has demonstrated the anticipated increase in mass (278 for monoamines and 278 and 556 for diamines). Similar results were obtained from the derivatization of model polyols with phenyl isocyanate, where the mass shift (n x 119) was a direct measure of the number of active hydroxyl groups. Fluorescamine labelling of PU foams shows the colour change which could be [corrected] indicative of the presence of an amine, but the subsequent MALDI-MS analysis was unable to demonstrate the anticipated increase in mass.

Amines↗

PubMed1998

m-Xylylenediamine determination in the official EU aqueous food simulants.

European Union directive 90/128/EEC prescribes a specific migration limit of 0.05 mg/kg for the aliphatic diamine m-xylylenediamine (m-XDA) into food or food simultants, but there is no generally accepted method of analysis available for compliance testing with the given restriction. A method is described for the determination of m-XDA monomer in the following food simulants: distilled water, 3% (w/v) acetic acid, and 15% (v/v) ethanol. The method is appropriate for the quantitative determination of m-XDA at a minimum level of 0.020 mg/kg in these food simulants. Detection limits are in the range of 0.004 to 0.010 mg m-XDA per kilogram food simulant (depending on the type of food simulant). The method should also be applicable to other aqueous food simulants. m-XDA in aqueous simulant test samples is determined by high-performance liquid chromatography with fluorescence detection following derivatization with fluorescamine. Quantitation is relative to external standards. The identity of m-XDA may be confirmed by the presence of a second peak in the chromatograms obtained from samples derivatized with less fluorescamine or by comparison with authentic samples.

Food Analysis↗

PubMed1979

Proteins exposed on the outer surface of the plasma membranes of AH-66 hepatoma ascites cells.

The cell surface proteins of AH-66 hepatoma ascites cells were examined both by fluorescent labeling with cycloheptaamylose-fluorescamine complex and by radioiodination with lactoperoxidase. The labeled proteins were analyzed by measuring the distribution of fluorescence or radioactivity in polyacrylamide gels after electrophoresis. Although the degree of fluorescent labeling with cycloheptaamylose-fluorescamine complex was substantially different from that obtained by lactoperoxidase-catalyzed iodination, proteins with molecular weights of 130,000, 90,000, 86,000, 50,000--60,000, and 37,000 were strongly labeled in common by both reagents. It is suggested that at least these proteins are exposed on the surface of the plasma membranes of AH-66 cells. One of these proteins labeled strongly by both reagents is the major glycoprotein, with an apparent molecular weight of 130,000 in 10% polyacrylamide gels.

Animals↗

PubMed1985

A sensitive enzymic assay for benzylpenicillin.

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3.3-33 micrograms/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4. 7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open beta-lactam ring form of each compound shows little fluorescence with fluorescamine at pH 4 but shows strong fluorescence in the pH range 7-9. 6-Aminopenicillanic acid and its open beta-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7.8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.

Hydrogen-Ion Concentration↗

PubMed1996

Transbilayer phosphatidylethanolamine movements in the yeast plasma membrane. Evidence for a protein-mediated, energy-dependent mechanism.

Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP-dependent, protein-mediated process. To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharomyces cerevisiae (A184D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes. The levels of external PtdEtn in the intact cells were reduced to about 50% by pretreatment of the cells with inhibitors of mitochondrial ATP synthesis, ATPase inhibitors or protein-sulfhydryl-group-modifying reagents, or by depletion of the cells of ATP by metabolic starvation. The levels of external PtdEtn could be restored to normal by repletion of the energy-depleted cells with ATP. Furthermore, treatment of the energy-depleted cells with sulfhydryl-modifying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells. These results demonstrate an involvement of an ATP-dependent, protein-mediated process(es) in the regulation of the PtdEtn distribution across the plasma-membrane bilayer of yeast cells. The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane.

Adenosine Triphosphatases↗

PubMed1995

Direct and Rapid Analysis of the Adhesion of Bacteria to Solid Surfaces: Interaction of Fluorescently Labeled Rhodococcus Strain GIN-1 (NCIMB 40340) Cells with Titanium-Rich Particles.

A fluorimetric assay which enables direct and accurate analysis of the adhesion of bacteria to solid particles was developed. The assay is based on labeling of the bacteria with fluorescamine, which reacts with primary amino groups on the cell surface to yield a yellow fluorescence that is easily detectable by both fluorescence microscopy and spectrofluorimetry. As an example, fluorescent labeling of Rhodococcus strain GIN-1 (NCIMB 40340) cells enabled the detection and quantitative determination of their adsorption to TiO(inf2) and coal fly ash particles. Exposure of the cells to 10% acetone during the labeling reaction affected neither their viability nor their ability to adhere to these particles. Only a small fraction (;sim2%) of the total cell protein was labeled by fluorescamine upon staining of intact bacterial cells, which may indicate preferential labeling of certain proteins. Specificity studies carried out with the fluorescence assay confirmed previous findings that Rhodococcus strain GIN-1 cells possess high affinities for TiO(inf2), ZnO, and coal fly ash and low affinities for other metal oxides. In principle, the newly developed fluorimetric assay may be used for determination of cell adhesion to any solid matrix by either microscopic examination or epifluorescence measurements. In the present work, the adhesion of several other microorganisms to TiO(inf2) particles was tested as well, but their ability to adhere to these particles was significantly lower than that of Rhodococcus strain GIN-1 cells.

Journal Article↗

PubMed1983

Medium-sized peptides in the blood of patients with uremia.

We have carried out high performance liquid chromatographic analysis of serum and ultrafiltrate of blood obtained from uremic patients and normal subjects to elucidate the presence of medium-sized peptides unique to uremia. Many fluorescamine-positive substances, excluding amino acids and guanidine compounds, were increased in uremic serum compared with normal serum. At the 0.5-1.0 pmol/ml serum level, several peaks were unique to uremia. The retention time, fluorescamine reactivity, molecular weight distribution and the result of enzymatic digestion revealed that these peaks are peptidic substances.

Chromatography, High Pressure Liquid↗

PubMed1991

Tussock moth dermatitis: evidence for histamine involvement.

Moth hairs shed by the few tussock moths caught during the first documented outbreak of pruritic dermatitis in Singapore were investigated to provide evidence for the presence of histamine. Aqueous extract of the moth hairs was used for the test and analyses. In the isolated guinea pig ileum, the extract, like histamine, produced responses that were blocked by mepyramine. The high performance liquid chromatographic (HPLC) analysis of the fluorescamine derivative of the moth extract resulted in a fluorescent peak with exactly the same retention. time as the histamine fluorescamine derivative. The mass spectrum of the pentafluoropropionic acid (PFP) derivative of the extract was also identical to that obtained from the reference histamine-PFP derivative. We concluded that histamine was present in the tussock moth hairs and was involved as one of the inflammatory mediators responsible for the pruritic skin rashes.

Animals↗

PubMed2005

[Critical and methodological analyses on the determination of oxygen and nitrogen reactive species in skeletal muscle cells during contractions].

An excessive level of these species may be harmful as it may overcome the antioxidant capacity. Most evidences for ROS and NO formation in muscle cell are indirect whereas direct evidences are scarce. The present review suggests as an alternative method either fluorescent DCFH, DAF-2 or fluorescamine toward H2O2, NO and O2*- measurements, respectively in muscle cell. In addition, it was concluded that: 1) the "indirect" measurements of ROS and NO in blood sample do not reflect oxidative attack occurred in muscle cells during contractions; 2) isolated muscles and homogenates may contain a large amount of endothelial cells, red blood cells and leukocytes which may generate ROS and NO; 3) the fluorescent DCFH-DA/DCFH, DAF-2-DA/DAF-2 and fluorescamine are sensitive in detection of H2O2, NO and O2.- respectively, in muscle cell during contractions; 4) muscle cell culture and isolated muscle fiber are indicated as experimental models to study ROS and NO formation in muscular tissue.

Animals↗