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PubMed1986

Polyamine cytochemistry: comparisons between cytochemical, autoradiographic, immunocytochemical and chemical results in the prostate.

Results obtained with two newly developed fluorescence cytochemical methods for detecting the polyamines spermidine and spermine have been compared to autoradiographic localization of biosynthetically labelled polyamines, to immunocytochemical results obtained with antibodies directed against spermidine and spermine, and to chemical polyamine determinations using the rat prostate as a model tissue. Complete agreement between all five methods was obtained. Application of perchloric acid to formaldehyde-fixed sections of rat prostate strongly reduced the o-phthalaldehyde inducible and formaldehyde-fluorescamine inducible fluorescence characteristic of spermidine and spermine. Perchloric acid extracted 40% of tissue-bound polyamines from formaldehyde-fixed tissue sections, and molecules with the physicochemical characteristics of polyamines constituted 80-90% of all fluorescamine reactive molecules extracted. Our results therefore confirm the specificity of the o-phthalaldehyde and formaldehyde-fluorescamine methods for polyamine cytochemistry. As polyamines are strongly implicated in cellular growth regulation and cancer, simple and inexpensive techniques for polyamine histochemistry may be useful for interpreting the biological and pathophysiological roles of these molecules.

Animals↗
PubMed1983

Effect of ion concentration on phosphatidylethanolamine distribution in mixed vesicles.

The membrane of mixed phosphatidylcholine-phosphatidylethanolamine vesicles was found to be impermeable to the fluorescent label fluorescamine. Added to the vesicle solution, fluorescamine labels only the phosphatidylethanolamine molecules in the outer layer. The separation of labeled and free phospholipid by thin-layer chromatography permits the determination of the inner to outer phosphatidylethanolamine ratio. This ratio can be independently obtained by multiple sonication and the addition of fluorescamine, which results in a progressive increase of the labeled phosphatidylethanolamine. These two methods give identical results for the phospholipid distribution. The ratio of the outer to the total phosphatidylethanolamine decreases with the increase in the mole fraction of phosphatidylethanolamine, in agreement with the results published by Litman (Litman, B.J. (1973) Biochemistry 12, 2545-2554). It was also found that the lipid distribution is sensitive to changes of the electrolyte concentration in the aqueous phase. At high concentrations the distribution is close to the symmetrical one; a decrease in ionic strength results in preferential localisation of phosphatidylethanolamine molecules in the inner vesicle layer.

Fluorescamine↗
PubMed1986

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis.

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.

Anemia↗
PubMed1990

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions.

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.

Actins↗
PubMed1975

Proteolytic degradation of insulin and glucagon.

The degradation of insulin and glucagon by a highly purified enzyme isolated from rat skeletal muscle was investigated. A sensitive assay for proteolytic degradation of insulin and glucagon using fluorescamine to detect an increase in primary amine groups was established. As measured by an increase in fluorescamine reactive materials, insulin was rapidly degraded by this highly purified enzyme without requiring initial disulfide cleavage. Associated with the increase in fluorescamine reactive materials was a decrease in immunoassayable insulinmglucagon wal also proteolytically degraded by this enzyme but a number of other peptides and proteins including proinsulin, and A and B chains of insulin were not degraded. Thus, we have demonstrated that insulin (and glucagon) can be proteolytically degraded by an enzyme isolated from an insulin sensitive tissue, skeletal muscle. Proteolytic degradation by this enzyme requires the intact insulin molecule rather than separate A and B chains.

Animals↗
PubMed1986

Fluorometric determination of nanogram quantities of protein in small samples: application to calcium-transport adenosine triphosphatase.

A fluorometric micro protein assay based on fluorescamine-labelling of homogeneous proteins in solution has been developed which is capable of accurately quantitating as little as 25 ng protein at a concentration of 1.25 micrograms/ml. This micro assay uses a flow-through HPLC fluorescence detector. Typical micro assays measuring bovine serum albumin standards (0-25 mg/l) yielded linear regression coefficients of r = 0.999. Assays of purified Ca2+-ATPase solutions determined by the micro fluorescamine procedure correlated well with measurements made using the deoxycholate-TCA-precipitation modification of the Lowry assay: 1.0 microgram ATPase by Lowry method = 1.1 microgram protein by fluorescamine microassay (when both procedures were standardized with bovine serum albumin) (r = 0.995). The assay proposed offers a 100-fold increase in sensitivity, compared to the Lowry procedure.

Calcium-Transporting ATPases↗
PubMed1991

Location of lysine-129 and lysine-40/41 with respect to retinylidene chromophore in bacteriorhodopsin.

Fluorescence properties of fluorescamine-modified bacteriorhodopsin (BR) have been studied. BR reacts with fluorescamine (1:2 ratio) to give protein (FLBR-I) modified at Lys-129. By making use of citraconic anhydride as a masking reagent, fluorescamine modification of Lys-40/41 in BR has been achieved. Forster's resonance energy transfer studies indicate that the distance between FL-Lys-129 and retinylidene chromophore is 11 A, whereas that between FL-Lys-40/41 and retinylidene chromophore is 24 A. These measured distances have been analysed in terms of BR structure.

Bacteriorhodopsins↗
PubMed1994

Determination of tetrahydro-beta-carbolines in urine by high-performance liquid chromatography with suppression of artefact formation.

A high-performance liquid chromatographic method has been developed for the determination of urinary tetrahydro-beta-carbolines. When standing tryptamine with formaldehyde and acetaldehyde under extraction conditions, the significant amounts of artefact 1,2,3,4-tetrahydro-beta-carboline (TBC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) were formed in a short time. Urine samples added with 2-ethyl-1,2,3,4-tetrahydro-beta-carboline (an internal standard) were treated with fluorescamine, and then with glycine, followed by serial solvent extractions. Such a pretreatment using two-step reactions removed a precursor (trypamine) by extracting its fluorescamine derivative, and enhanced the detection response by consuming excess fluorescamine. It solved the analytical problem that artefact TBC and MTBC are formed during analysis. Reversed-phase ion-pair chromatography using a C8-column and trifluoroacetic acid as a counter ion completed a base-line separation of three analytes within 10 min. The calibration graphs showed a good linearity in the range 0.1-50.0 ng ml-1 of urine samples spiked with standard TBC and MTBC. In the spike experiment, the recovery and relative standard deviation were almost 100% and less than 3.0%, respectively, for both TBC and MTBC. The proposed method enables the determination of the genuine urinary concentrations of TBC and MTBC without involving their artefacts.

Acetaldehyde↗
PubMed1978

Rapid detection of amphetamine in urine by micro thin-layer chromatography and fluorescence.

A rapid, inexpensive, and simple screening procedure for the detection of amphetamine abuse was developed for use by laboratories without sophisticated equipment. A small volume of extract from a pH-adjusted urine specimen is used to spot a high-resolution micro TLC plate. The developed TLC plate is sprayed with a solution of fluorescamine in dry acetone. When viewed under ultraviolet illumination, amphetamines and other compounds with a primary amino group complexed with fluorescamine appear as greenish or bluish-white fluorescent spots. Secondary or tertiary amines do not react with fluorescamine. About 20 min is required to perform the procedure; the lower limit of detectability is approximately 100 ng/ml urine.

Amphetamine↗
PubMed1980

Fluorescence energy transfer studies on normal and leukemic mouse lymphocytes.

Control and acute leukemic cells of AKR:Lac C3H mg:Lac/F1 hybride mice were labeled either with ethidium bromide or ethidium bromide plus fluorescamine. Theoretical and experimental evidence shows that the ethidium bromide binding ability of cells and the average efficiency of Förster-type fluorescence energy transfer between fluorescamine and ethidium bromide inside the cells depend on both the cell and dye concentrations. It is shown further that fluorescamine treatment enhances the uptake of ethidium bromide by both normal and leukemic cells. A sevenfold difference was found in ethidium bromide binding ability between normal and leukemic cells at optimal dye concentration.

Animals↗
PubMed1980

Membrane studies in Huntington's disease: steady-state fluorescence studies of intact erythrocytes.

Recent evidence suggests that a membrane abnormality may be present in Huntington's disease (HD), including peripheral tissues such as erythrocytes. Steady-state fluorescence spectroscopy was performed on intact erythrocytes from patients with HD and from normal age- and sex-matched controls. Fluorescent probes with known specificity for certain membrane areas were used to survey membrane dynamics; these probes included fluorescamine, which binds to primary amines on the cell surface, and both 12(9)-anthroyl stearate, 12(9)AS, and 2(9)-anthroyl stearate, 2(9)AS, which insert at different depths in the hydrocarbon core of membranes. In the intact HD erythrocyte, fluorescamine revealed decreased polarization, 12(9)AS showed increased polarization, and 2(9)AS showed no difference as compared to normal erythrocytes. These studies suggest that fluorescamine, attached to primary amines of the cell surface in intact HD erythrocytes, has increased rotational mobility whereas the anthroyl moiety of 12(9)AS in the hydrocarbon interior has decreased rotational mobility. These studies thus support the concept of a membrane abnormality in HD.

Biophysical Phenomena↗
PubMed2006

A sequential injection fluorometric procedure for the determination of procaine in human blood and pharmaceuticals.

An automated procedure for the assay of procaine hydrochloride in human blood and pharmaceuticals was developed using a sequential injection (SI) technique with fluorometric detection and fluorescamine as the fluorescence probe. A few microliters of fluorescamine and procaine hydrochloride solutions were used in the SI system leading to the formation of a derivative, which was then excited by a 400-nm LED and whose emitted fluorescence was monitored at a wavelength of 494 nm. A linear calibration graph was obtained with 10-200 ng mL(-1) (procaine) by loading 10.0 microL of sample solution and 5.0 microL of fluorescamine solution (both 0.125 % m/v). A detection limit of 2.6 ng mL(-1), defined as 3 times the blank standard deviation (3sigma), was achieved along with a sampling frequency of 25 h(-1) and a precision of 2.1 % RSD at the 50.0 ng mL(-1) level. Procaine contents in injection solutions from various pharmaceutical manufactures were analyzed and reasonable agreement was achieved between the values obtained by using the present procedure and the documented spectrophotometry, and both were coincident with the nominal concentrations. In addition, the degradation of procaine in human blood was investigated. A fast degradation of procaine in human blood was observed for the first 30 min, while afterwards the degradation was retarded.

Buffers↗
PubMed1988

Effect of nitrogen dioxide on surface membrane fluidity and insulin receptor binding of pulmonary endothelial cells.

Nitrogen dioxide (NO2), an environmental oxidant pollutant, is known to peroxidize membrane lipids of lung cells. We evaluated the ability of NO2 to alter the surface membrane fluidity, lipid composition, and insulin receptor binding of porcine pulmonary artery endothelial cells in culture. After 3- to 24-hr exposure to 5 ppm NO2, cells were labeled with either 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), a cationic fluorescent aromatic hydrocarbon that anchors at the lipid-water interface, or fluorescamine, a fluorescent molecular probe that covalently binds with amino groups of surface phospholipids and proteins. Membrane fluidity was measured by monitoring changes in the steady-state fluorescence anisotropies (rs) for TMA-DPH and fluorescamine. Insulin specific receptor binding was monitored by measuring time-dependent binding of 125I-insulin. Following NO2 exposure, rs values for TMA-DPH and fluorescamine were increased significantly in a time-dependent fashion, with maximum increases at 24 hr (P less than 0.001). Similar increases in rs values were observed in isolated plasma membranes as well as in lipid vesicles prepared from total lipid extracts of endothelial cells or their plasma membranes. Phosphatidylethanolamine plus phosphatidylserine content in lipid extracts from 24-hr but not 3- to 12-hr NO2-exposed cells was increased significantly (P less than 0.01) compared to control cells. Specific binding of 125I-insulin to cells exposed to NO2 for 12 and 24 hr (but not 3 and 6 hr) was reduced significantly (P less than 0.05) compared to binding in control cells. Scatchard analysis of the binding data indicated that NO2 exposure caused a 5-fold reduction in insulin receptor binding sites in endothelial cells. Recovery was achieved 24 hr after NO2 exposure with, but not without, changing culture medium. These results indicate that NO2 exposure causes reversible changes in the physical state of lipids in the superficial lipid domains of the pulmonary endothelial cell plasma membrane, and these alterations may interfere with plasma membrane-dependent functions such as receptor-ligand interaction.

Amino Acids↗
PubMed2002

Synthesis of aromatic nitrogen mustard agents and analysis of their alkylation activity at physiological pH and temperature.

A Structure Activity Relationship (SAR) study was accomplished with six aromatic compounds which have a nitrogen mustard (N-mustard) substituent. N-mustard agents are very important for the clinical treatment of many types of cancers. All N-mustard agents synthesized alkylated a nucleophilic primary amine (p-chloroaniline) in aqueous solvent at pH 7.4 and 37 degrees C. Rate constants and rate equations were determined for the alkylation reactions by monitoring the formation of a fluorescent complex formed when fluorescamine complexes the unreacted p-chloroaniline. Fluorescamine complexation of the unreacted primary amine halts the alkylation reaction and allows the determination of remaining unreacted primary amine, which in turn permits the determination of rate constants and rate equations. The fluorescamine-amine complex shows a strong absorbance peak at a wavelength of 400 nanometers in aqueous solvent. The molar absorptivity (epsilon) was calculated to be 18.37 L/(mole x cm). First order rate constants ranged from 0.513E-2 minute(-1) to 1.32E-2 minute(-1) with second order rate constants from 2.85E-2 (M x minute)(-1) to 4.78E-2 (M x minute)(-1). The aromatic compounds included benzoic acid, m-chlorobenzoic acid, hydrocinnamic acid, m-toluic acid, and 3,4-dimethoxybenzoic acid. The synthesis procedure produced the N-mustard agents at > or = 90% yield and high purity. Partition coefficient Log(Kow)Log P values ranged from 2.66 to 4.18. The N-mustard agents consisted of greyish-yellow crystals which retained alkylation activity for more than ten weeks when stored at -10 degrees C and were soluble in water at 25 degrees C and 37 degrees C.

Alkylation↗
PubMed1988

Biliary proteins: assessment of quantitative techniques and comparison in gallstone and nongallstone subjects.

Although protein is the third most abundant solid in bile and is important in cholesterol crystal formation, methods for quantitating the concentration of total protein in bile have not been systematically evaluated. To establish a reliable protein assay for bile, we evaluated three protein assays (Lowry's method and the fluorescamine and Coomassie blue methods), and employed amino acid analysis as a reference technique. Large protein-to-protein variations were observed with the fluorescamine and Coomassie blue methods. Although all assays were affected by interfering substances, Lowry's method and the fluorescamine technique (after trichloroacetic acid precipitation and delipidation of bile) and the Coomassie blue method with native bile showed excellent correlations (P less than 0.0001) with those obtained by amino acid analysis. Using these reliable protein assays, we examined gallbladder bile obtained at surgery from subjects with and without gallstones. No differences in the concentrations of total biliary proteins were observed among patients with cholesterol (n = 23) or pigment (n = 7) gallstones and subjects without gallstones (n = 10). Protein values obtained by amino acid analysis also did not differ among groups. As expected, bile from patients with cholesterol gallstones was supersaturated with cholesterol while bile from nongallstone subjects and those with pigment stones was unsaturated. These results indicate that it is not possible to separate patients with and without gallstones on the basis of the total protein concentration of gallbladder bile.

Animals↗
PubMed1994

[Comparison of several fluorescence HPLC methods for fumonisin analysis].

Fumonisin has no particular cromophore groups in its chemical structure applicable for trace analysis, and therefore requires derivatization prior to measurement. Currently, the analysis of fumonisin B1 (FB1), B2 (FB2) is generally carried out after derivatizing FB1 mainly with o-phthalaldehyde to make a fluorescent compound (OPA-F). The OPA-F is, however, not sufficiently stable for precise analysis, and we have thus examined other fluorescent derivatives including fluorescamine, FMOC, NBD-F and DBD-F. The analytical method using FMOC as fluorescent agent required column chromatography after the derivatizing reaction, while reaction of FB1 with fluorescamine gave two chromatographic peaks. FMOC and fluorescamine are accordingly not desirable as fluorescent agents for analysis of small quantities of FB1 and FB2 in foods. In conclusion, for precise analysis of such trace amounts of FB1 and FB2, NBD-F or DBD-F should be used as fluorescent agents.

4-Chloro-7-nitrobenzofurazan↗
PubMed2004

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection.

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

Calibration↗
PubMed1990

Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides.

New indirect spectrophotometric methods of analysis of tobramycin using the derivatizing agents o-phthaldialdehyde, fluorescamine, and dansyl chloride were developed and compared with existing methods. Optimum reaction conditions were established for the complex formation between tobramycin and each reagent. The sensitivity and reproducibility of these new procedures were compared with results obtained using published methods. Of the new methods, the o-phthaldialdehyde and fluorescamine procedures were more sensitive and reproducible than the dansyl chloride procedure. Further, these procedures were subsequently applied to the analysis of aqueous solutions of other aminoglycosides including amikacin, neomycin, gentamicin, and kanamycin.

Anti-Bacterial Agents↗