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At least 1,261 records · Page 70Linked to original sources
PubMed2001

An improved method for the rapid assessment of persisting chylomicron remnant concentrations.

Persisting chylomicron remnant concentrations have been linked to premature atherosclerosis. The analysis of persisting chylomicron remnant concentrations by an oral triglyceride tolerance test, however, is time-consuming for the study subjects and requires large resources in the laboratory. Therefore, only small numbers of subjects have been studied in the past. We describe major improvements of the testing procedure in regard of composition of the fatty meal, of patient testing, and measurement of postprandial remnants. Shifting the time of the (ready-to-use) fatty drink from the morning hours to bedtime was well accepted by the study subjects and allowed the analysis of blood samples drawn at the morning with minimal impact on the participants' time and with minimal interferences by confounding factors (e.g. smoking, additional food intake, physical activity). Chylomicron remnants were measured by fluorometry of the supernatant after ultracentrifugation. This procedure was sensitive, was specific for chylomicron remnants, and was easy to perform. The biological validity of the improved procedure was evaluated by studying type III hyperlipoproteinemia patients and normolipemic apolipoprotein (Apo) E2 homozygotes. In conclusion, this improved test permits the rapid testing for persisting chylomicron remnants in the clinical routine and in large epidemiological studies.

Adult↗
PubMed1987

Validity of 5-aminolevulinate dehydratase activity (5-ALAD) for the discrimination of alcoholics and nonalcoholics with chronic liver disease.

Alcohol consumption decreases erythrocyte 5-ALAD activity. Our aim was to assess the diagnostic value of 5-ALAD in discriminating alcoholics from nonalcoholics among patients with chronic liver disease and to find the combination of 5-ALAD and other clinical chemical tests that best differentiates these two groups. Measurements of erythrocyte 5-ALAD activity by fluorometry and 10 other biochemical tests were performed in 205 subjects with biopsy-verified liver damage (88 alcoholics, 117 nonalcoholics). The highest six Chi-square (chi 2) test values out of the 11 tests performed were diagnostically evaluated individually and in combinations of two to five tests, according to the number of positive findings. Our results show that 5-ALAD provides the highest efficiency as an individual test, while combinations of three tests where 5-ALAD is involved, especially the combination of 5-ALAD, gamma-glutamyltransferase and mean corpuscular volume of erythrocytes, seem to be the best test combinations for discriminating alcoholics from nonalcoholics among patients with chronic liver diseases.

Chronic Disease↗
PubMed1988

Time-resolved fluoroimmunoassay of cortisol in serum with a europium chelate as label.

A non-isotopic heterogeneous competitive immunoassay of serum cortisol is described. Cortisol present in the sample competes with immobilised cortisol (cortisol-thyroglobulin conjugate) for binding to a monoclonal anti-cortisol biotinylated antibody. The amount of antibody bound is measured on the dry solid-phase by time-resolved fluorometry after adding streptavidin labeled with the Eu3+ chelate 4,7-bis(chlorosulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The assay is simple to perform, its characteristics are similar to those of radioimmunoassay techniques, and is suitable for routine clinical use.

Antibodies, Monoclonal↗
PubMed1989

Sensitive time-resolved immunofluorometric assay of thyrotropin in serum.

We have developed a time-resolved solid-phase immunofluorometric assay for thyrotropin (TSH). The assay is performed in white opaque microtitration wells which are coated with a monoclonal capture antibody. Serum TSH binds simultaneously to the solid phase and to a biotinylated monoclonal detection antibody. The degree of biotinylated antibody binding is quantitated with streptavidin conjugated to thyroglobulin which is heavily labelled with the Eu3+ chelator 4,7-bis [chlorosulfophenyl] -1,10-phenanthroline -2,9-dicarboxylic acid (BCPDA). The final fluorescent complex is measured on the solid phase with time-resolved fluorometry. The assay requires two incubation steps and can be completed in 5 hours. The detection limit is 0.03 milli-int. units/L. The present assay was compared with two immunoradiometric assays and gave satisfactory results.

Antibodies, Monoclonal↗
PubMed1989

A double monoclonal time-resolved immunofluorometric assay of carcinoembryonic antigen in serum.

A non-isotopic heterogeneous non-competitive immunoassay for carcinoembryonic antigen is described. CEA present in serum binds simultaneously to two monoclonal antibodies specific for different antigenic determinants of CEA. One antibody is immobilized on a solid-phase (microtiter well) and the other is biotinylated. The amount of biotinylated antibody bound is measured on the dried solid-phase by time-resolved fluorometry after adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10 phenanthroline-2, 9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The assay is simple to perform, its characteristics are similar to those of other isotopic and non-isotopic techniques, and it is suitable for routine clinical use.

Antibodies, Monoclonal↗
PubMed1997

Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA.

OBJECTIVE: To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA. METHODS: PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry. RESULTS: The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). CONCLUSIONS: A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

Humans↗
PubMed2000

In vivo histamine release from brain cortex: the effects of modulating cellular and extracellular sodium and calcium channels.

The in vivo mechanisms underlying the actions of modulating Na(+)- and Ca(2+)-sensitive channels and its effect on basal histamine release in the cerebral cortex of freely-moving unanesthetized rats was investigated. Basal histamine release in the cerebral cortex was determined by in vivo microdialysis coupled with high-performance liquid chromatography (HPLC) fluorometry detection. Basal levels of histamine were 0.67+/-0.02 pmol/10 microl of dialysate. Diltiazem, a Ca(2+) channel antagonist, produced a dose-dependent decrease in dialysate basal histamine concentration. Elevated K(+) (100 mM) in the perfusion medium increased basal histamine to a maximum of 223% of the baseline value. Similarly, diltiazem (60 mM) reduced the K(+), veratridine (100 microg/ml) and ouabain (100 microM)-evoked increase in dialysate histamine. Basal histamine decreased by 48% when the perfusate contained 3 microM of voltage dependent Na(+) antagonist tetrodotoxin. The results of these studies indicate that the release of histamine in rat cerebral cortex can be induced by modulating Na(+) and Ca(2+) channels and that the L-type voltage-dependent sensitive Ca(2+) channels are involved in this release process.

Animals↗
PubMed2003

GABAergic modulation of hippocampal glutamatergic neurons: an in vivo microdialysis study.

We have demonstrated the effects of activation of presynaptic gamma-aminobutyric acid (GABA) receptors on glutamate release using in vivo brain microdialysis. A dialysis probe inserted into the hippocampus CA2 area of freely moving rats was perfused with Ringers solution containing 100 mM potassium chloride (KCl) or 0.05 mM veratridine for 20 min. Extracellular concentrations of amino acids were monitored by measuring their levels in dialysates by high performance liquid chromatography (HPLC) fluorometry. Perfusion with depolarizing agents, such as KCl or veratridine, increased extracellular glutamate levels in the hippocampus. Pretreatment with 1 mM GABA, before perfusion with depolarizing agents, significantly suppressed the depolarizing agent-induced increase in glutamate levels. The GABA(B) receptor agonist baclofen (1 mM) also significantly inhibited the depolarizing agent-induced increase in glutamate levels, whereas the GABA(A) receptor agonist, muscimol, had no affect. Similarly, baclofen (0.5 mM) decreased the KCl (13.5 mM)-induced 45Ca(2+) influx into cortical synaptosomes to 57% of the level induced in the absence of baclofen. On the other hands, GABA did not affect the increases in glycine and taurine level by depolarizing agents. These results suggest that GABA modulates depolarization-evoked glutamate release in the hippocampus by inhibiting Ca(2+) entry into neurons, an effect mediated by presynaptic GABA(B) receptors.

Animals↗
PubMed1996

Halothane counteracts acetylcholine-induced increase in Ca2+ sensitivity of the contractile apparatus in airway smooth muscle.

The direct relaxing effect of halothane on airway smooth muscle has been reported to involve the reduction of the cytosolic Ca2+ concentration ([Ca2+]i) and the [Ca2+]i-independent inhibitory mechanism. To clarify the extent of the contribution of these mechanisms, the effect of halothane on the [Ca2+]i-tension relationship in porcine tracheal smooth muscle strips was evaluated, using fura-2 fluorometry. The control [Ca2+]i-tension relationship was constructed from data of [Ca2+]i and tension during the contractions induced by the stepwise increment of extracellular Ca2+ concentrations under high K+ depolarization. In the presence of acetylcholine (1 microM), the [Ca2+]i-tension relationship shifted upward, which indicated the acetylcholine-induced increase in the Ca2+ sensitivity of the contractile apparatus. Halothane (0.034 mM), in the absence of acetylcholine, did not alter the increases in either [Ca2+]i or tension, hence no change in the [Ca2+]i-tension relationship. However, in the presence of acetylcholine, halothane did attenuate the acetylcholine-induced upward shift of the [Ca2+]i-tension relationship. Halothane proved to have a potent attenuating effect on the acetylcholine-induced increase in Ca2+ sensitivity of the contractile apparatus with little influence on [Ca2+]i. This desensitization of the contractile apparatus to [Ca2+]i may play a major role in the direct airway relaxing effect of halothane.

Acetylcholine↗
PubMed1998

Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery.

The mechanism of thrombin-induced endothelium-dependent relaxation was investigated using fura-2 front-surface fluorometry. Thrombin induced an endothelium-dependent relaxation during U46619-induced contractions in pig coronary arterial strips. The relaxation consisted of two components: the early phasic component with a transient decrease in [Ca2+]i of smooth muscle and the subsequent sustained tonic component without [Ca2+]i decrease. The phasic relaxation was inhibited by a combination of N(omega)-nitro-L-arginine and K+-depolarization, while the tonic component was inhibited by either indomethacin or K+-depolarization. Thrombin induced a transient [Ca2+]i increase and nitric oxide (NO) production in pig aortic valvular endothelial cells, which expressed NO synthase as determined by reverse transcription and polymerase chain reaction. Thus, it was concluded that NO and hyperpolarizing factor were involved in the phasic component of thrombin-induced relaxation and that hyperpolarizing factor and prostacyclin were involved in the tonic component.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1998

Biphasic elevation of plasma histamine induced by water immersion stress, and their sources in rats.

The effect of water immersion stress on the plasma concentration of histamine, in Wistar and mast cell-deficient (Ws/Ws) rats, was investigated. The histamine content of the plasma, skin and gastric mucosa, as well as the level of activity of histidine decarboxylase in the gastric mucosa, were determined by high performance liquid chromatography (HPLC)-fluorometry. In Wistar rats exposed to water immersion stress for a total of 6 h, an initial, acute, four-fold, transient increase in the plasma histamine level, followed by a sustained, though lower, elevation of the plasma histamine level, was observed. The initial acute increase in plasma histamine level was also seen in gastrectomized Wistar rats exposed to water immersion stress, but not in Ws/Ws rats exposed to stress. The sustained elevation of the plasma histamine level was observed in the Ws/Ws rats. However, in both the gastrectomized Wistar rats and gastrectomized Ws/Ws rats, the sustained elevation in plasma histamine level was not observed. The histamine content of the skin of Wistar rats after 15 min or more exposure to water immersion stress, was 20% lower than that of control rats. The mucosal histamine content of both Wistar rats and Ws/Ws rats, was 20% lower, whereas histidine decarboxylase activity in the gastric mucosa was enhanced by two-fold, during exposure to stress for 4 h. These findings indicate that water immersion stress causes a biphasic increase in plasma histamine concentration in Wistar rats; the initial acute increase in plasma histamine level originates from mast cells, and the second, sustained increase is attributed to enterochromaffin-like cells.

Animals↗
PubMed1987

The rate of diffusion of fluorophores through the corneal epithelium and stroma.

The time taken to cross the rabbit corneal epithelium and stroma was estimated for fluorescein (F), carboxyfluorescein (CF), rhodamine B (RB), and sulforhodamine B (SRB). Paired corneas were mounted in vitro; one was intact and the dye solution was kept in continuous contact with its epithelial surface; the epithelium was scraped from the other and the dye was applied as a pulse to the bare stroma. The time course of the dye appearing in a solution rapidly passing over the endothelial surface was determined by fluorometry. This rate of appearance was compared in the two cases and used to estimate the diffusional lag time introduced by the epithelium. For the very hydrophilic CF and SRB, the delay was too short to measure; this is compatible with the passage of these dyes taking place through the paracellular spaces. For the very lipophilic RB, the delay was about 2 min; this was rather too slow for it to be explained as being controlled entirely by diffusion in the cytosol. For the intermediate F, the delay was 5 min; it is suggested that this is a result of it partitioning between the spaces and the cytosol during its passage. The experiments also led to determinations of the permeability of the epithelial and endothelial layers to the dyes. In both cases lipophilicity was a strong determinant of penetration, but not the only one. The permeability of the endothelium to F was unchanged from its in vivo value in these experiments, but that of the epithelium was increased four-fold. The diffusion rate of the dyes across the stroma could also be determined. There was no clear relationship with molecular size or partition coefficient. The rate of diffusion of F across the tissue was about half that in its plane, as determined in previous experiments. This is possibly a result of the anisotropic structure of the tissue.

Animals↗
PubMed1988

Glycation of cataractous lens in non-diabetic senile subjects and in diabetic patients.

Early- and advanced-stage products in the Maillard reaction, glycation, were measured in patients with diabetic or senile cataracts. Early-stage products were measured by means of furosine, which is an acid-hydrolysis product derived from fructose-lysine. Advanced-stage products were measured by fluorometry using high-performance liquid chromatography. Furosine levels were high (listed in descending order) in capsule, cortex and nucleus in both diabetic and senile cataracts. The advanced-stage products were also high (listed in descending order) in nucleus, cortex and capsule in both diabetic and senile cataracts. These results suggest that advanced-stage products might accumulate in larger amounts in the nucleus and cortex than in the capsule, resulting in the formation of cataracts. The study also revealed that the Maillard reaction plays an important role in causing not only diabetic cataracts but also senile cataracts.

Aged↗
PubMed1988

Glucose flux and the redox state of pyridine dinucleotides in the rat lens.

The concentration of alpha-glycerophosphate (GP) appeared to increase and readily reached a new steady state in lenses incubated with KCN or under hyperglucosic condition. This increase can be explained by the change in NADH/NAD ratio under each condition. The relative ratio of pyridine dinucleotides (NADH/NAD) was calculated from equilibrium equations of two NAD-linked enzymes, lactate dehydrogenase (LDH) and alpha-glycerophosphate dehydrogenase (GPDH). The change in NADH/NAD ratio based on biochemical assays correlates well with that estimated from GPDH reaction. This indicates that measurements of pyridine dinucleotides (e.g. in vivo redox fluorometry) can be used to demonstrate lens metabolic status.

Animals↗
PubMed2001

The effect of molecular confinement on the conformational dynamics of the native and partly folded state of apomyoglobin.

Inclusion in agarose gel significantly affects the conformational dynamics of native and acidic partly folded states of tuna apomyoglobin, a single tryptophan containing protein, as documented by frequency domain fluorometry investigations. The heterogeneity of the tryptophanyl emission decay increases on gel inclusion compared to that observed for free-in-solvent protein at both neutral and acidic pH, thus suggesting that the interconversion rate among conformational substates is somewhat reduced. The observation that this effect is much more pronounced for the partly folded state suggests that confined environments such as those existing in the living cells might favor the sequential folding process avoiding that structured intermediates rapidly convert into less structured ones.

Animals↗
PubMed1994

Inhibition of nonlysosomal calcium-dependent proteolysis by glycine during anoxic injury of rat hepatocytes.

BACKGROUND/AIMS: The mechanism by which glycine protects against hepatocyte death during anoxia remains unclear. Nonlysosomal proteolysis, including calpain proteolysis, has been implicated as a mechanism of lethal cell injury. However, the effect of glycine on nonlysosomal proteolysis is unknown. The aim of this study was to ascertain if glycine cytoprotection is associated with inhibition of nonlysosomal proteolysis. METHODS: Rat hepatocyte suspensions were rendered anoxic using an anaerobic chamber. Cell viability was measured by propidium iodide fluorometry. Nonlysosomal protease activity was quantitated by the release of trichloroacetic acid-soluble free amines or tyrosine. Calpain protease activity was measured using a fluorogenic substrate. RESULTS: Glycine and alanine (but not valine) markedly improved cell viability during anoxia in a concentration-dependent manner. During anoxia, the majority of nonlysosomal proteolysis (60%) was dependent on extracellular Ca2+. Glycine only inhibited that portion of nonlysosomal proteolysis that was dependent on extracellular Ca2+. Amino acids inhibited the anoxia-stimulated increase in calpain protease activity with the same specificity and concentration-dependence observed for cytoprotection. Glycine was more potent in directly inhibiting purified m-calpain as compared with mu-calpain protease activity. CONCLUSIONS: Glycine may exert its cytoprotective activity during lethal anoxic hepatocyte injury, in part by inhibiting Ca(2+)-dependent degradative, nonlysosomal proteases, including calpains.

Animals↗
PubMed1975

Inorganic saltsin the fluorometric detection of presticides.

A method is described for the detection and quantification of pesticides on silica gel ghromatograms by in situ fluorometry. Simple inorganic salts are used to induce fluorescence in a number of pesticidal compounds. Intensified fluorescence is shown for one naturally fluorescent pesticide. Spectral data and instrumental limits of detection are given.

Chromatography, Thin Layer↗
PubMed2002

Band broadening caused by the multiple labeling of proteins in micellar electrokinetic chromatography with diode laser-induced fluorescence detection.

When a labeling reagent is used, in the determination of proteins by capillary electrophoresis with laser-induced fluorescence detection, the multiple labeling of proteins frequently occurs, which can degrade the separation efficiency. In order to understand the influence of the multiple labeling of proteins on separation efficiency, the band broadening caused by a labeling reaction between bovine serum albumin (BSA) and a cyanine fluorescent dye (Cy5) was investigated using micellar electrokinetic chromatography in conjunction with diode laser-induced fluorometry. With the aid of an internal standard, methylene blue, the height equivalent to the theoretical plate (HETP) ratio of BSA to methylene blue was used as an indicator for band broadening under optimum separation conditions. Labeling conditions, including reaction buffer pH, reaction time, and initial concentration of Cy5 to bovine serum albumin, were found to influence the HETP ratio. The separation efficiency for the labeled protein was degraded by experimental conditions employed in the labeling, which indicates an increase in the heterogeneity of the final products.

Chromatography, Micellar Electrokinetic Capillary↗