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At least 685 records · Page 38Linked to original sources
PubMed1989

Determination of plasma homocysteine by high-performance liquid chromatography with fluorescence detection.

Severe homocystinemia is frequently associated with vascular disease while the pathological consequences of moderate or slightly elevated plasma homocysteine are unknown. Cobalamin and folate deficiencies may result in an elevation of plasma homocysteine. A sensitive and reproducible assay for total plasma homocysteine has been developed. The essential steps in the assay include (i) conversion of homocysteine disulfides to free homocysteine with borohydride reduction; (ii) conjugation of homocysteine with monobromobimane; (iii) separation of homocysteine-bimane from other plasma thiol-bimane adducts by reverse-phase high-performance liquid chromatography; and (iv) detection and quantitation of homocysteine-bimane by fluorometry. The method has a sensitivity of 4.4 pmol of homocysteine and is highly reproducible (intra- and interassay coefficients of variation = 4.97 and 4.53%, respectively). The mean concentration of total plasma homocysteine in nonfasting adult males (n = 12) and females (n = 12) was 15.8 (range, 7.0-23.7) and 16.5 nmol/ml (range, 8.6-20.7), respectively. Markedly elevated levels of homocysteine were found in patients with cobalamin and folate deficiency. Total plasma homocysteine represents approximately 4% of borohydride-generated thiol reactivity in the plasma of normal individuals.

Chromatography, High Pressure Liquid↗
PubMed1990

A fluorometric method for measuring ethoxycoumarin O-deethylase activity by reversed-phase high performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method that uses an on-line change in the protonation state of the nonfluorescent product to yield a fluorescent derivative that is detected by fluorometry was developed for the determination of 7-ethoxycoumarin O-deethylase activity. Tissue samples (1-20 micrograms protein) were incubated with 7-ethoxycoumarin, and 7-hydroxycoumarin metabolite was extracted in chloroform. Following drying under nitrogen, the extract was resuspended in methanol (10-100 microliters) and an aliquot of 5-20 microliters was directly injected into a C8 Nova-Pak column. Isocratic separation of hydroxycoumarin was achieved using a mobile phase consisting of methanol:1% acetic acid, 35:65, v/v, pH 3.5, at a flow rate of 1 ml/min. Following chromatographic separation, samples were derivatized with 1.0 N NaOH prior to fluorescent measurements. The detection limit for 7-hydroxycoumarin was less than 1 pmol, with a mean recovery from the incubates of 96.4 +/- 2.3%. This HPLC-fluorometric method was linear up to at least 400 pmol of 7-hydroxycoumarin and could accurately detect metabolite formation in incubates containing control liver microsomes with less than 0.05 microgram total protein. The method also allowed determinations of cytochrome P450-dependent function in extrahepatic tissues of rats, including individual segments of gastrointestinal epithelium and brain, as well as in cultured cells, such as HepG2 cells, in which microsomal protein yield is very small. The wide range of linearity afforded by this method allows a reliable estimation of cytochrome P450-dependent function in samples containing varying concentrations of protein.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1993

Folding and dynamics of melittin in reversed micelles.

The main structural characteristics and the dynamic properties of melittin bound to the internal surface of reversed micelles, formed by sodium bis(2-ethyl-1-exyl)sulfosuccinate (AOT) in isooctane, were investigated by several spectroscopic techniques. Melittin has been found associated to reversed AOT micelles in a single state, thus indicating that this system behaves differently with respect to phospholipid vesicles where at least two forms of lipid associated melittin are observed. The dynamic properties of melittin in reversed AOT micelles at different water contents were examined by frequency domain fluorometry. The whole emission decay was analyzed in terms of lifetime distribution having a Lorentzian shape. The results indicated that the binding of melittin to inverted micelles determines an increase of emission heterogeneity compared to that observed for the fully extended helical monomer. This was explained in terms of a larger variety of microenvironmental conditions that the tryptophan residue experiences during its excited state. However, the conformation freedom of the peptide can be modulated by varying the micellar size.

Circular Dichroism↗
PubMed1986

Regulation of palmitoylcarnitine oxidation in isolated rat liver mitochondria. Role of the redox state of NAD(H).

The redox-mediated regulation of palmitoylcarnitine oxidation was studied in isolated rat liver mitochondria in which the mitochondrial free NADH/NAD+ ratio was controlled by graded concentrations of acetoacetate and ketomalonate in a rotenone and malonate-inhibited system in the presence of ADP. The NADH/NAD+ ratio was buffered kinetically by adjusting the concentrations of the hydrogen acceptor substances and determined by calibrated NAD(P)H fluorometry of the mitochondrial suspension. A two-fold variation in the beta-oxidation rate and a five-fold variation in the free NADH/NAD+ ratio was obtained in the presence of rotenone. A non-linear negative correlation was found between the acetyl-CoA concentration and the beta-oxidation rate and a negative correlation between the long-chain acyl-CoA concentration and the beta-oxidation rate. The data indicate that the redox state is a partial controller of the beta-oxidation rate in liver mitochondria. The contribution of acetyl-CoA, a putative regulator of beta-oxidation at the acyl-CoA thiolase step is small under the conditions used.

Acetoacetates↗
PubMed1986

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin.

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.

Cysteine↗
PubMed1995

Sterol carrier protein-2 stimulates intermembrane sterol transfer by direct membrane interaction.

It is unclear how the cytosolic sterol carrier protein-2 (SCP-2) binds sterols and enhances sterol transfer between membranes. Therefore, human recombinant SCP-2 was used in conjunction with phase fluorometry, dialysis, and chemical labeling techniques to show if a direct membrane effect accounted for this activity. SCP-2 directly interacted with L-cell fibroblast plasma membrane vesicles as determined by increased fluorescence anisotropy of coumarin-labeled protein (CPM-SCP-2). Furthermore, a new fluorescence lifetime component due to plasma membrane-bound CPM-SCP-2 was observed. Dialysis studies with 3H- cholesterol loaded plasma membranes indicated that SCP-2, added to the donor compartment, stimulated sterol transfer whether or not the dialysis membrane was permeable to SCP-2. Nevertheless, ligand-binding experiments indicated that chemically blocking the SCP-2 sterol binding site inhibited the ability of SCP-2 to enhance sterol transfer between plasma membrane vesicles. SCP-2 did not stimulate plasma membrane fusion. Addition of SCP-2 to plasma membranes increased the anisotropy plasma membrane proteins covalently reacted with CPM, but not that of lipids labeled with the fatty acid analogue octadecyl rhodamine B. In conclusion, the data are consistent with SCP-2 stimulating intermembrane sterol transfer by direct interaction with sterol in the membrane and enhancing its desorption from the membrane.

Animals↗
PubMed1990

Methods of successive multiparametric cytochemistry and microfluorometry on identical cells with special reference to cell cycle phases in a chick embryo.

The study of cell cycle kinetics in relation to regulatory mechanisms in embryonic development is considered quite important, but many technical difficulties still remain owing to the complexity of embryonic systems. To facilitate such study, a novel method of multiparametric microfluorometry was developed and is presented here. In many other multiparametric methods, various cytochemistries and fluorometries are conducted at the same time, whereas in our system only one type of cytochemistry and its microfluorometry are performed first and then other types subsequently. Our system is composed of two parts, a method for combining various types of quantitative cytochemistries to be performed in succession on identical cells and a method that permits the implementation of cytochemistries and microfluorometries in succession using in combination a microfluorometer, TV camera, video recorder, digital Telopper, and microcomputers. A method for the evaluation and normalization of microfluorometric data on DNA content is described. The cell cycle phases of each cell can be accurately distinguished on the basis of DNA content and BrdU uptake, using a statistical method. A cell cycle traverse study using embryonic avian scale dermal cells was carried out in which the four parameters of DNA content, double labeling with BrdU and [3H]AdR, and time lapse between the two labelings were included. As an example of its application to molecular cytochemistry, in situ nick translation was conducted on developing scale dermal cells. The special features and scope of application of the present system are discussed.

Animals↗
PubMed1981

A sensitive and quantitative fluorescence assay for cell surface antigens.

A procedure is described for the assay of cell surface antigens based on quantitative fluorometry. Fluorescent immunospheres are coupled with sheep anti-mouse immunoglobulins or Protein A and used to detect specific antibody bound to target cells. The fluorescent sphere assay described here offers 16--128-fold greater sensitivity than complement mediated lysis or Protein A radioimmune assays and comparable sensitivity to rosetting assays. In addition, the assay is simple to perform, uses commercially available reagents and is completely objective in that a common laboratory fluorometer is used to obtain fluorescence measurements.

Animals↗
PubMed1982

Histamine contamination of pokeweed mitogen.

The histamine content of commercial preparations of pokeweed mitogen was measured by amino acid analysis technique, automated fluorometry, and bioassay employing the guinea pig ileum. Ten samples from 5 companies were examined and found to contain between 0.026 micrograms and 167.5 micrograms of histamine per ml of solution. The protein content of 9 of these putative 5 mg samples measured by folin assay and by amino acid analysis varied from 0.56 to 4.4 mg. Their amino acid compositions were similar, except for notable variations in 3 of the 16 residues quantitated.

Amino Acids↗
PubMed1987

A method for the quantification of a colored or fluorescent signal in enzyme immunoassays by photodensitometry.

A colored or fluorescent signal is generally evaluated with the naked eye, or by means of different more or less sophisticated and costly instruments. Photodensitometry is an additional technique which is both inexpensive and simple to perform. This technique can satisfactorily quantify a signal without the use of either a spectrophotometer or a fluorometer. In this study we compared readings obtained by spectrophotometry, fluorometry and photodensitometry in 96-well ELISA plates and in Terasaki plates. In ELISA plates, it is possible to detect 1220-300,000 femtograms (fg) of peroxidase by spectrophotometry and 4800-125,000 fg by photodensitometry. In Terasaki plates, we were able to measure between 3.8 and 8000 fg of beta-galactosidase per sample by spectrofluorometry, and from 30 to 8000 fg by photodensitometry. Using a sandwich procedure in Terasaki plates we were able to measure between 100 and 10,000 fg of IgE per sample by spectrofluorometry and from 500 to 10,000 fg by photodensitometry. Photodensitometry is the least expensive technique for the reliable detection of enzyme or enzymatic marker in small sample volumes treated with a fluorogenic substrate.

Chromogenic Compounds↗
PubMed1994

Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay.

Cell Line↗
PubMed1987

Effects of oral and intravenous administrations of dopamine and L-dopa on plasma levels of two isomers of dopamine sulfate in man.

The levels of two isomers of dopamine sulfate, dopamine-3-O-sulfate (DA3S) and dopamine-4-O-sulfate (DA4S), in human plasma were measured by HPLC-fluorometry. The basal plasma levels of DA3S and DA4S in the early morning were 13.8 +/- 1.9 and 3.2 +/- 0.5 pmoles/ml, respectively (means +/- S.E.M.). Oral administrations of dopamine (50 mg/body) and 1-dihydroxyphenylalanine (L-DOPA, 250 mg/body) increased the plasma levels of these dopamine sulfates almost 100-fold to 1807 +/- 266 and 1674 +/- 195 pmoles/ml of DA3S, and 466 +/- 83 and 321 +/- 76 pmoles/ml of DA4S. Intravenous dopamine infusion (5 micrograms/kg/min for 30 min) markedly increased the plasma free dopamine concentration, as expected, but increased the levels of DA3S and DA4S only slightly to 110 +/- 32 and 25 +/- 9 pmoles/ml, respectively. In contrast, intravenous L-DOPA (25 mg/body) resulted in a slight increase of free dopamine followed by marked increases of DA3S and DA4S to 691 +/- 219 and 139 +/- 40 pmoles/ml, respectively. These data indicate that O-sulfation of dopamine, especially 3-O-sulfation, is the main pathway for metabolism of intravenously and orally administered L-DOPA and orally ingested dopamine. This sulfation is suggested to occur in the gut wall.

Administration, Oral↗
PubMed1990

Interrelationship between the concentrations of some elements in the organs of Japanese with special reference to selenium-heavy metal relationships.

Multi-element analyses were conducted on internal organs (cerebrum, cerebellum, heart, spleen, liver and kidney) of Japanese obtained from 45 forensic medical autopsy cadavers. Elements analyzed and analytical methods were as follows: Ca, Cd, Cu, Fe, K, Mg, Na, P and Zn by inductively coupled plasma atomic emission spectrometry; total Hg (T-Hg) and inorganic Hg (I-Hg) by cold vapour atomic absorption spectrometry; methyl Hg (MeHg) by gas chromatography; and Se by fluorometry. A significantly positive correlation between Se and T-Hg was observed in kidney, liver, heart and spleen. The T-Hg/Se molar ratio was less than 1 in all the organs examined. The correlation between Zn and Cd was significant in liver and kidney. Multiple regression analyses using Se as a dependent and I-Hg, MeHg, Zn, Cd, Cu as independent variables were conducted on each organ. Inorganic Hg was a significant independent variable in kidney, whereas in liver and spleen it was MeHg. Cadmium was significant in explaining the variations in Se in liver and kidney medulla, indicating Cd-Se co-accumulation in humans.

Adolescent↗
PubMed1988

Urinary catecholamines: comparison between HPLC with electrochemical detection and fluorophotometric assay.

Comparisons between catecholamines determined by HPLC (with electrochemical detection) and fluorometry in urine samples from healthy adults and children, respectively, showed high correlations. In agreement with greater specificity for HPLC, mean values were higher for the fluorometric assay. However, it was concluded that fluorometric assays provide as valid and sensitive indices of stress-induced changes in catecholamine excretion in humans as HPLC.

Adult↗
PubMed1995

Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing.

We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 microM Ca2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.

Calcium↗
PubMed1991

Cholecystokinin increases intracellular Ca2+ concentration in cultured striatal neurons.

Although it has been established that pancreatic cholecystokinin (CCK) receptors are coupled to phosphatidylinositol turnover, the events which follow activation of CCK receptors in the central nervous system have not received much attention. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]1) in response to CCK peptides were measured in single cultured rat striatal neuron by fura-2 fluorometry. CCK peptides dose-dependently increased [Ca2+]i in a monophasic manner. The order of the potencies of CCK peptides to increase [Ca2+]i was as follows: caerulein greater than CCK-8 greater than desulfated CCK-8 greater than CCK-4. The effect of caerulein was completely blocked in a Ca2(+)-depleted medium. In addition, omega-conotoxin GVIA completely inhibited the effect of caerulein, while neither nifedipine nor verapamil affected it. Our results indicate that CCK receptors couple to N-type voltage-sensitive Ca2+ channels in cultured rats striatal neurons.

Animals↗
PubMed1991

Sensitive, reproducible and convenient fluorometric assay for the in vitro evaluation of anti-cytomegalovirus agents.

Fluorescein diacetate (FDA), a non-fluorescent diacetyl fluorescein ester that becomes fluorescent upon hydrolysis by cytoplasmic esterases, permitted the easy distinction by fluorometry between non-infected and human cytomegalovirus (CMV)-infected HEL cell cultures. As a result of enhanced cytoplasmic esterase activity after CMV infection, FDA-derived fluorescence intensity was brighter for infected than non-infected HEL cells. A similar increase in fluorescence intensity was observed after loading the cells with Indo-1/AM, a non-fluorescent ester of Indo-1 that becomes fluorescent upon cleavage by cytoplasmic esterases. The 50% effective concentrations of a number of anti-CMV agents as determined by the fluorometric assay were very similar to those obtained by the conventional and more time-consuming microscopic evaluation. The fluorometric assay appears very suitable for an automated evaluation of anti-CMV compounds, and also allows rapid determination of the cytotoxicity of potential antiviral compounds.

Acyclovir↗
PubMed1987

Does the fluorescence quencher acrylamide bind to proteins?

We have studied the protein concentration dependence of the acrylamide quenching of the fluorescence of the proteins, human serum albumin and monellin, and we have found no such dependence for the concentration range of 0.5-20 mg/ml. These quenching studies were performed by fluorescence lifetime measurements using phase/modulation fluorometry. We have also performed equilibrium dialysis studies, which show no large degree of association of acrylamide with serum albumin, and we have found that acrylamide has only a small effect on the activity of selected enzymes. These various studies do not indicate the existence of strong acrylamide-protein interactions and are in discord with a recent report by Blatt et al. in this journal (Blatt, E., Husain, A. and Sawyer, W.H. (1986) Biochim. Biophys. Acta 871, 6-13).

Acrylamide↗