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Fluorescence reactions of fluorescamine with levodopa and its derivatives: fluorescence assay of 3-methoxy-4-hydroxyphenylalanine in levodopa dosage forms.

A simple fluorometric method for the quantitation of 3-methoxy-4-hydroxyphenylalanine, either alone or in the presence of levodopa, is presented. Fluorescence is developed by reaction with fluorescamine. The interactions of this reagent with levodopa and a number of its derivatives under various experimental conditions were studied. Negligible fluorescence was obtained with levodopa, dopamine, levodopa benzyl ester, and 6-hydroxydopamine.

Chemical Phenomena↗

Fluorescamine use in high-performance liquid chromatographic determination of aminocaproic acid in serum.

A sensitive and specific high-performance liquid chromatographic determination of aminocaproic acid in serum is described. omega-Aminocaprylic acid is used as an internal standard. To 10 microliter of serum, 10 microliter of the internal standard solution and 50 microliter of ethanol are added. After centrifugation, a portion of the supernate is evaporated. The residue is dissolved in 750 microliter of 50 mM dibasic sodium phosphate, and then 250 microliter of fluorescamine in acetonitrile (35 mg/100 ml) is added. The reaction mixture is chromatographed using a column of octadecylsilane bonded to silica and 44% acetonitrile in 0.5 mM phosphoric acid as the eluent. Quantitation is achieved by monitoring either the absorbance of the effluent at 405 nm or the fluorescence of the compounds with a fluorometer equipped with a flowcell. The method is reproducible, simple, and fast and has a precision of 4.4%.

Aminocaproates↗

Protein oxidation and aging. II. Difficulties in measuring alkaline protease activity in tissues using the fluorescamine procedure.

A current hypothesis explaining the aging process implicates the accumulation of oxidized protein in animal tissues. This is primarily based on a series of reports showing an age-dependent increase in protein carbonyl content and an age-dependent decrease in the activities of enzymes, especially of alkaline proteases, which preferentially degrade oxidatively modified protein. Recently, this hypothesis was strongly supported by the report of a novel effect of the spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) in reversing these age-dependent changes. However, we found that the reactive protein carbonyls could not be reliably measured in tissues by using the 2,4-dinitrophenylhydrazine procedure described in the PBN study. We now focus on the alkaline protease activity assay and show that alkaline protease activity cannot be reliably measured in crude tissue extracts by using the fluorescamine procedure also described in the PBN study. We were, however, able to reliably measure a protease activity in crude tissue extracts at alkaline pH by using a synthetic fluorogenic peptide substrate, but no effect of aging or PBN treatment was found on the protease activity in rat brain cortexes. Thus, the reported age-dependent changes in protein carbonyl formation and alkaline protease activity remain to be confirmed.

Aging↗

Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method.

Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.

Breast Neoplasms↗

Polyamine cytochemistry. Use of a novel o-phtalaldehyde method for visualizing spermidine and spermine. Comparisons to the formaldehyde-fluorescamine method.

o-Phtalaldehyde (OPT) reacts with a number of biologically important molecules, including the polyamines, spermidine and spermine. By systematically varying reaction conditions with respect to temperature, pH, concentration and length of exposure to the reagent, using both model systems and tissues, we have succeeded in constructing a cytochemical OPT-method specific for spermidine and spermine. The method detects cell types known to contain these polyamines, including growing and neoplastic cells. The staining pattern obtained with the OPT method is identical to that obtained with the formaldehyde-fluorescamine (FF) technique recently shown to be specific for spermidine and spermine. In contrast to the FF technique, the OPT method can be used for staining suspensions of isolated cells and may hence be employed in studies using fluorescence-activated cell sorting (FACS). Preliminary such studies show a pronounced decrease in cellular OPT-induced fluorescence, paralleled by a decrease in content of polyamines, after treatment with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine (alpha DFMO). In contrast, cells simultaneously treated with alpha DFMO + spermidine show pronounced increases in their spermidine content and parallel increases in their OPT-induced fluorescence. Availability of methods selectively demonstrating polyamines at the cellular and subcellular level is expected to aid our understanding of polyamine functions in normal growth and cancer.

Aldehydes↗

Microspectrography of formaldehyde and fluorescamine-induced fluorescence in rabbit pulmonary neuroepithelial bodies: demonstration of a new, probably polypeptide intracytoplasmic substance.

The microspectrographic analysis of the fluorescence emitted by NEB's in gaseous formaldehyde-fixed lung tissue, posttreated with fluorescamine, revealed the presence of numerous primary amino groups which are clearly different from the serotonin identified in our earlier studies and correspond to a new, probably a polypeptide intracytoplasmic substance.

Amines↗

An evaluation of fluorometric proteinase assays which employ fluorescamine.

The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37 degrees C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.

Animals↗

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay.

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.

Apolipoproteins B↗

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria.

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.

Adenosine Triphosphatases↗

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis.

4-Phenylspiro [furan-2(3H),1-phtalan]3,3'-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.

Amines↗

Liquid chromatographic determination of urinary dopamine and norepinephrine as fluorescamine derivatives.

Dopamine (DM) and norepinephrine (NE) with 3,4-dihydroxybenzylamine as internal standard in normal human urine were extracted with alumina, labeled with fluorescamine and determined fluorometrically by liquid chromatography. The method is simple and in the case of excretion of DM in urines of subjects receiving 1-dopa, even the alumina treatment was unnecessary. The C.V. of method was 4.3% and 4.5% for DM and NE, respectively. The mean contents of DM and NE in 7 normal urines were 182 and 35.5 ng/mg creatinine, respectively.

Buffers↗

Fluorescamine-induced membrane permeability in mitochondria.

1. Addition of fluorescamine (75 microM) to mitochondria induced an increase in membrane permeability. 2. The leakiness of the inner mitochondrial membrane is characterized by extensive release of accumulated Ca2+, collapse of the transmembrane potential, mitochondrial swelling and efflux of matrix proteins, among them, malate dehydrogenase. 3. These effects were diminished by supplementing the media with 1 mM phosphate, and partially prevented by Mg2+. 4. These results indicate that the primary amino groups of membrane components contribute, partially, to the maintenance of the permeability barrier in mitochondria.

Animals↗

Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.

Animals↗

Isolation and HPLC of N-epsilon-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives.

N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL). The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum. NELL was isolated on a reversed phase Sep-Pak C18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors. The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate. Using this procedure we have demonstrated the presence of TBL in native histones.

Chromatography, High Pressure Liquid↗

Improved determination of the bisphosphonate pamidronate disodium in plasma and urine by pre-column derivatization with fluorescamine, high-performance liquid chromatography and fluorescence detection.

An improved method for the determination of 1-hydroxy-3-aminopropylidene-1,1-bisphosphonate (pamidronate) in human urine and plasma is described. The procedure is based on a co-precipitation of the bisphosphonates (pamidronate and 6-amino-1-hydroxypentilidene-bisphosphonate, used as internal standard) with calcium phosphate. After centrifugation the precipitate is redissolved in hydrochloric acid, followed by a second precipitation. Then the bisphosphonates are dissolved in ethylenediaminetetraacetic acid, derivatized with fluorescamine, and separated by high-performance liquid chromatography. Using fluorescence detection, the limit of quantitation for pamidronate was 0.8 mumol/l in plasma and 0.7 mumol/l in urine.

Chromatography, High Pressure Liquid↗

Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine.

A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.

Animals↗

Rapid and highly sensitive high-performance liquid chromatographic method for the determination of histamine and 3-methylhistamine in biological samples using fluorescamine as the derivatizing agent.

A highly sensitive and rapid high-performance liquid chromatographic assay for the determination of histamine and 3-methylhistamine in biological samples using 1-methylhistamine as the internal standard is described. Samples were purified and concentrated on cation-exchange columns and derivatized with fluorescamine. The lower detection limit was 20 pg on-column. Linearity was demonstrated up to 20 ng on-column. The samples could be derivatized simultaneously before injection and were stable for 7 days. The method was used for the determination of histamine and related compounds in coronary perfusates, extracts of homogenized rat hearts, and supernatants of stimulated peritoneal mast cells.

Animals↗