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PubMedDate not supplied

Flow cytometry and sizing for routine andrological analysis.

Flow fluorometry and Coulter type sizing analysis of sperm have been applied separately in order to improve human semen analysis. Different methods of sample preparation were evaluated and a protocol involving prestaining pepsin treatment of sperm samples is proposed for fluorometric analysis. The data obtained with fluorometry and sizing analysis result in different kinds of information: Coulter counting allows to automate sperm counting and fluorometry yields more detailed information about normozoospermia and oligozoospermia by determining the proportion of mature spermatozoa and immature germ-cells. These two methods, together with light microscopy, may help to explore the correlation of fertility and pathology of spermatozoa. The aim of these investigations is to yield the preconditions for simultaneous two-parameter analysis of DNA content and cellular size distributions.

DNA↗
PubMed1992

[Future developments in ocular fluorophotometry instrumentation].

The scope of ocular fluorometry is to monitor exogenous and endogenous fluorophores in ocular tissues, in relation with ophthalmic and systemic diseases using the unique optical prospectives of the eye. The elderly population and the incidence of blindness are increasing rapidly due to more cases of diabetes, glaucoma, cataract and age-related macular degeneration. Monitoring changes in specific fluorophores in the eye may help identify the high risk groups in these diseases. New developments in instrumentation include differential fluorometry and introduction of confocal optics. Differential fluorometry has already achieved significant progress for the study of the autofluorescence of the lens and cornea and measurements in the aqueous. Improved spatial resolution obtained with improved optics opens interesting possibilities like measurement of corneal endothelial permeability and retinal vascular permeability. The results already obtained will be presented with particular incidence on measurements of lens fluorescence (normals--336.2 +/- 56.3; diabetes--659.9 +/- 123.9; age group--40-50 y) and corneal endothelial permeability (normals--3.14 +/- 60.10(-1) cm-1).

Fluorophotometry↗
PubMedDate not supplied

Interlaboratory study of blood selenium determinations.

Fifty-one laboratories from 14 countries participated in a survey on the determination of selenium (Se) in 8 bovine blood samples with Se concentrations ranging from 0.2 mumol/L (0.016 microgram/mL) to 14 mumol/L (1.1 micrograms/mL). The methods used (and the percentage of participants using each method) were fluorometry (61), hydride-generation atomic absorption spectrophotometry (AAS) (23), graphite-furnace AAS (6), gas chromatography (4), neutron activation analysis (4), and X-ray fluorometry (2). There was little difference in the mean Se results obtained by fluorometry or hydride-generation AAS (P greater than 0.05). Mean intralaboratory coefficients of variation (CVs) from known replicates ranged from 4 to 14% for all samples. Interlaboratory CVs were related to blood Se concentration and increased to 55% at Se levels below 0.4 mumol/L (0.032 microgram/mL). Laboratories that used quality control (QC) schemes had lower interlaboratory CVs than those that did not, but the advantage began to diminish at blood Se concentration below 0.4 mumol/L (0.032 microgram/mL). The high interlaboratory CVs, coupled with the false assurance from the low intralaboratory CVs and the ineffectiveness of the QC schemes at blood Se concentrations below 0.4 mumol/L (0.032 microgram/mL), are of concern in diagnosis of marginal Se deficiency in livestock where the concentrations of interest are in the range 0.15-0.5 mumol/L (0.012-0.039 microgram/mL).

Animals↗
PubMed1975

Microscope fluorometric investigations on the reticulocytic maturation distribution as diagnostic criterion of disordered erythropoiesis.

A microscope fluorometric technique is described which permits not only the visual identification of reticulocytes under the fluorescence microscope but also the determination of their relative stage of maturation to normocytes. The technique is based on a specific staining procedure which results in a fluorescent complex between the reticulocytic RNA and acridine orange. Thus, the relative mass of RNA in the individual reticulocytes can be measured by means of mciroscope fluorometry. As the reticulocytic RNA content decreases and finally disappears during the final maturation process of reticulocytes after their release into the peripheral blood stream, the fluorescence signal indicates the relative degree of this maturation. A characteristic frequency distribution of this parameter can be obtained for a given blood sample by microscope fluorometry measuring 200 to 300 reticulocytes. The preliminary use of this technique for following up the course of two cases of hemolytic anemia and one of pernicious megaloblastic anemia during their treatment demonstrates the potential diagnostic value of this technique of identifying the change of the reticulocyte maturation distribution in addition to the reticulocyte count. Satisfactory agreement between the microscope fluorometric results and those obtained by counting separately the four reticulocytic maturation stages according to Heilmeyer and Wesbäuser has been achieved. The possibility of obtaining quantitative and comparable results by use of this method may be considered a general advantage and a promising basis for the development of an automated technique.

Acridines↗
PubMed1995

Cholesterol interaction with recombinant human sterol carrier protein-2.

The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols. Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with a Kd of 0.3 and 1.7 microM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime, and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption.

Acrylamide↗
PubMed1976

Separation of mouse epidermal basal and differentiating cells for microflow fluorometric measurements: a methodologic study.

The DNA content of lymphocytes and of basal cells from normal hairless mouse epidermis was measured by microflow fluorometry (MFF). To obtain a relatively pure suspension of epidermal basal cells a combined mechanical and enzymatic method was used. The admixture of differentiating cells into the basal cell fraction after cell separation was 13%. The results were compared with those obtained with conventional Feulgen microspectrophotometry applied to basal cells and dermal lymphocytes in histologic sections. The results from both cytophotometric methods were in good agreement and clearly demonstrated the improved resolution obtained by using microflow fluorometry. When the lymphocytes were not treated with pepsin before being stained with ethidium bromide for MFF, the modal DNA value was consistently below that of the basal cells from the same specimen. Pepsin treatment of lymphocytes, however, increased their fluorescence intensity to the value of epidermal basal cells. The modal DNA value of Feulgen-stained dermal lymphocytes in histologic sections was consistently below that of epidermal basal cells from the same section. The advantage of pepsin treatment for obtaining higher resolution of DNA measurements of basal and differentiating epidermal cells and of lymphocytes was evaluated. The cell cycle distribution of basal cells from epidermis in different states of proliferative activity was determined. Changes in the proportion of cells in S phase were parallel to changes in the 3H-Tdr labeling index.

Animals↗
PubMed1993

Membrane heterogeneity in isolated rat hepatocytes and liver plasma membrane subfractions: a comparative study using DPH and its cationic derivative TMA-DPH.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) has been studied in hepatocytes isolated from rat liver and in isolated plasma membrane subfractions (cLPM, canalicular membranes and bLPM, basolateral membranes) using frequency domain fluorometry. The decay has been analyzed either by using a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values in order to study different aspects of membrane heterogeneity. The results obtained by the two analyses are practically superimposable but the distributional approach allows an evaluation of membrane heterogeneity through the width of the distribution that has shown particularly significant differences when freshly hepatocytes are compared with in vitro aged hepatocytes. Moreover, the comparison of the distributional analysis of the two probes has shown in cLPM a tendency to higher values of the main lifetime component and a narrower distribution width with respect to bLPM. These results indicate changes of membrane domain organization that have been discussed in relation with the specific lipid composition that characterizes the two membrane subfractions. Our results indicate that frequency domain fluorometry may be used to study membrane heterogeneity in intact cells and isolated membranes.

Animals↗
PubMed1995

Serial fluorometric assessments of skin perfusion in isolated perfused human skin flaps.

The applicability of serial skin surface fluorometry for repeated assessments of skin flap perfusion was investigated using the isolated perfused human transverse paraumbilical (TP) skin flap model. The flow rate, perfusion pressure and skin surface temperature were kept constant in seven human TP skin flaps and a low dose of fluorescein (3 x 10(-5) M) was used for each assessment. It was observed that the mean values for total dye fluorescence measured by a fluorometer and the maximum distance of perfusion estimated by dye fluorescein index remained consistent in five repeated assessments at 15 min interval. The variation in the maximum distance of perfusion within each TP skin flap over 5 repeated assessments was also relatively small, as judged by the mean coefficient of variation (6.1%; SEM 0.4%). Furthermore, a highly significant correlation between microsphere (15 microns) radioactivity index and dye fluorescence index was observed at corresponding locations in these seven TP skin flaps (r = 0.81; p < 0.001, n = 75). Taken together, these observations indicate that serial skin surface fluorometry provided consistent repeated assessments of skin perfusion in human skin flaps in vitro and the dye fluorescence index provided a consistent assessment of skin perfusion distance along the length of the TP skin flap. These observations lead us to speculate that critical (threshold) dye fluorescence index determined at various postoperative time points should be useful for prediction of skin viability in clinical skin flaps; thus, a clinical investigation is recommended.

Acetylcholine↗
PubMed1996

Time-resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand.

A time-resolved fluorometric assay for the measurement of natural killer cell activity against target cells labelled with the acetoxymethyl ester of the fluorescence enhancing ligand 2,2':6',2"-terpyridine-6,6"-dicarboxylic acid (TDA) is described. The hydrophobic esterified form of TDA (bis(acetoxymethyl) 2,2':6',2"-terpyridine-6,6"-dicarboxylate, BATDA) diffuses readily through the cell membrane of viable cells. BATDA is hydrolysed by intracellular esterases resulting in accumulation of membrane impermeable TDA inside the target cells. After incubation of labelled K-562 cells with effector cells the TDA released from lysed cells into the supernatant is chelated with Eu3+. The natural killer cell activity is then quantified by measuring the intense fluorescence of the EuTDA chelates formed. Target cells are rapidly labelled when incubated with BATDA, TDA is released from target cells faster than 51Cr, the spontaneous release permits a short-term release assay to be set up and the detection of EuTDA is fast (5 min/96 well plate). Furthermore, this non-radioactive method permits the use of complex culture media since, in contrast to methods based on prompt fluorometry, the problem with autofluorescence can be avoided by the use of time-resolved fluorometry.

2,2'-Dipyridyl↗
PubMed1987

Fluorometric documentation of increased cutaneous blood flow after topical application of a PGE2 analog in man.

The present study employed fiberoptic fluorometry, a noninvasive means of documenting delivery and removal of fluorescein dye, to evaluate the local circulatory changes elicited by topical application of DHV-PGE2 ME, an investigational PGE2 analog. On Day 1, inactive vehicle was applied to a 5 X 4 cm study site on each thigh of healthy volunteer subjects (n = 12). Symmetrical perfusion was confirmed by similar determinations of dye delivery and removal at each site. On Day 2, DHV-PGE2 ME, 30 or 120 micrograms, was applied to one site while inactive vehicle again was applied to the other. After administration of 120 micrograms in a petrolatum vehicle, fluorometry detected a pronounced increase in nutritive perfusion. There was significant acceleration of dye delivery and removal (p less than 0.05 by ANOVA). Less pronounced changes were noted after the lower dose of DHV-PGE2 ME and when the drug was applied in a triethyl citrate vehicle. The local circulatory changes were not accompanied by systemic effects; there were no changes in vital signs or in fluorometric indices at remote sites.

Adolescent↗
PubMed2005

The usefulness of urine fluorescence for suspected antifreeze ingestion in children.

PURPOSE: To evaluate urine fluorescence as a diagnostic tool. PROCEDURES: Using a Wood lamp, 60 physicians, assigned to group 1 or 2, independently rated 150 urine specimens from nonpoisoned children as fluorescent or nonfluorescent. Interobserver and intraobserver agreements were assessed. Physician ratings were compared with fluorometry results. The prevalence of urine fluorescence was determined by fluorometry. MAIN FINDINGS: Group 1 reported fluorescence in 80.7% (95% CI 73.4%-86.6%) of urine specimens; group 2 reported fluorescence in 69.3% (95% CI 61.3%-76.5%). Interrater agreement was poor (72.5%, kappa = 0.25, 95% CI 0.13-0.37); intrarater agreement was good (physician group 1: 97.9%, kappa = 0.93, 95% CI 0.77-1.00; physician group 2: 93.3%, kappa = 0.85, 95% CI 0.69-1.00). The prevalence of urine fluorescence was 100% (95% CI 98.1%-100%). CONCLUSION: Our data suggest that determination of urine fluorescence using a Wood lamp is a poor screening tool for suspected antifreeze ingestion in children.

Adolescent↗
PubMed2006

Cellular redox state predicts in vitro corneal endothelial cell proliferation capacity.

Cellular redox state using the non-invasive mitochondrial autofluorescence technique of redox fluorometry was evaluated as a predictor for corneal endothelial proliferative capacity in vitro. Human corneal endothelial cells (HCEC) harvested from eye bank corneas were cultured in plates with two different coating substrates; type I collagen and poly-D-lysine. Cellular autofluorescence was measured with both DAPI (excitation: G365, emission: bandpass 445/50) and FITC (excitation: bandpass 450-490, emission bandpass 515-565) filter sets on days 3, 5, 7, and 14. The redox fluorometric ratio was calculated as net "DAPI" signal intensity divided by net "FITC" signal intensity. Normalized redox ratio was calculated as redox ratio divided by individual cell size. Cellular proliferation was analyzed by live cell count on days 2, 7, and 14. Mitochondrial staining was performed on days 4 and 14. The poly-d-lysine substrate decreased the proliferation capacity of HCEC in comparison to type I collagen out to 2 weeks (p=0.045). The cellular redox fluorometric ratio decreased significantly as the cells proliferated (p<0.001). The cells cultured on type I collagen coated plates exhibited significantly lower redox fluorometric ratios than cells cultured on poly-D-lysine coated plates at day 7 (p=0.015). Normalized redox ratio showed significantly lower value in type I collagen coated plates at days 7 (p=0.015) and 14 (p=0.039). Correlated cell proliferation capacity was significantly higher on type I collagen coating at days 7 and 14 (p=0.045 and p=0.049 respectively). HCECs showed different growth potential in vitro on different culture surface coating agents. This difference was well correlated with cellular redox ratios determined using redox fluorometry. Cellular redox ratio can be a potential predictor of cellular proliferation capacity.

Cell Culture Techniques↗
PubMed2001

Association between homocysteine and neopterin in healthy subjects measured by a simple HPLC-fluorometric method.

OBJECTIVES: Neopterin and homocysteine promote vascular smooth muscle cell proliferation through the activation of nuclear factor(kappa) B. The aim of this study was to investigate the relation between these two compounds in healthy subjects by a rapid HPLC-fluorometric method which simplifies sample pretreatment for the measurement of neopterin in serum. DESIGN AND METHODS: In 40 healthy subjects (45.9 +/- 2.1 yr, mean +/- SEM, 10 males, 30 females) serum neopterin concentrations were measured by HPLC-fluorometry and enzyme-linked immunusorbant assay-ELISA and the results were compared. Urinary neopterin and plasma total homocysteine concentrations were assayed by HPLC-fluorometry. RESULTS: Serum neopterin concentrations measured by HPLC and ELISA were 7.5 +/- 0.4 and 7.4 +/- 0.3 nmol/L, respectively, r = 0.92, p < 0.01. Urinary neopterin level was 163.9 +/- 11.0 nmol/mmol creatinine and plasma total homocysteine 7.6 +/- 0.4 micromol/L. A significant positive correlation was observed between serum neopterin and plasma total homocysteine (r = 0.59, p < 0.01). CONCLUSIONS: A simple and rapid sample pretreatment for the measurement of neopterin in serum has been introduced. The significant positive correlation between neopterin and homocysteine implies that, interference with leukocyte function might be a new possible mechanism for the deleterious effects of homocysteine on vascular function.

Adult↗
PubMed1997

Oxidant generation with K(+)-induced depolarization in the isolated perfused lung.

This study evaluated whether cell membrane depolarization can induce oxidant generation in the isolated perfused rat lung as has been demonstrated with bovine pulmonary artery endothelial cells. Depolarization was produced by perfusing the lungs with high [K+] or with glyburide and was evaluated with bis-oxonol lung surface fluorometry. Lung surface bis-oxonol fluorescence increased above baseline (at 5.9 mM K+) by 18.5% with 24 mM K+, 35% with 48 mM K+, and 67% with 96 mM K+, indicating graded membrane depolarization, and by 75% during perfusion with 10 microM glyburide. Oxidant generation was evaluated with hydroethidine lung surface fluorometry, and with assay of tissue thiobarbituric acid reactive substance (TBARS), conjugated dienes, and perfusate H2O2. Depolarization by high K+ or glyburide led to significant increases in generation of tissue oxidants and lipid peroxidation. Bodipy-FL-glyburide microfluorography showed localization of glyburide binding primarily to vascular endothelial cells vascular and airway smooth muscle cells, alveolar type II cells, and to nonciliated cells of the airway epithelium. These results indicate that cellular depolarization is associated with oxidant generation by the lung and suggests a role for K(+)-channels in these events.

Animals↗
PubMed1996

An NMR, CD, molecular dynamics, and fluorometric study of the conformation of the bradykinin antagonist B-9340 in water and in aqueous micellar solutions.

A detailed NMR, CD, fluorometry, and molecular modeling study of a novel bradykinin antagonist B-9340, containing a novel amino acid D-Igl (alpha-(2-indanyl)glycine) at position 7, was carried out. The sequence of B-9340 is D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D- Igl7-Oic8-Arg9, where Hyp is hydroxyproline, Thi is beta-(2-thienyl)alanine, and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid. The CD results exhibit a striking effect of SDS on the spectrum of the BK antagonist, indicating that interaction with the surfactant induces a folded peptide structure. The interaction of this antagonist with phosphatidylinositol was monitored by fluorometry, indicating that the interaction of the peptide with the lipid is cooperative, and gives a Hill coefficient of 2.3. The two-dimensional proton NMR measurements indicate that B-9340 has no stable secondary structure in water solution and contains about 10-15% cis peptide bonds arising from Pro2, Hyp3, and Oic8. In SDS micelles, NMR reveals the existence of two beta-turns based on a number of medium-range connectivities that were useful for molecular modeling. The actual molecular modeling and dynamic runs were performed on B-9340 in an environment consisting of a layer of octyl sulfate anions and water. Ther results indicate that the structure of B-9340 in a micellar environment is characterized by a nonideal betaII-turn comprising residues Pro2 to Thi5, a nonideal betaII'-turn comprising residues Ser6-Arg9, and broad folding in the middle part of the molecule. The structure is stabilized by several hydrogen bonds and by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9, whereas the middle part of the peptide is buried in the micelle. The structure is deposited as Brookhaven PDB file 1 BDK.

Amino Acid Sequence↗
PubMed1982

Increased total concentration of amino acids in the cerebrospinal fluid of patients with purulent meningitis.

Total concentrations of amino acids, as measured by fluorometry of primary amino nitrogen with the use of fluorescamine, were determined in the cerebrospinal fluid (CSF) of 50 patients with purulent meningitis, 40 patients with aseptic meningitis, and 36 control subjects. On admission total concentrations of amino acids in the CSF were significantly higher in patients with purulent meningitis (mean +/- SEM, 2.27 +/- 0.27 mM) than in patients with aseptic meningitis (1.07 +/- 0.03 mM, P less than 0.001) or in control subjects (1.16 +/- 0.04 mM, P less than 0.001). This value was higher when the patients with purulent meningitis were reexamined one to two days later and reached a maximum after three to four days of illness. The fluorometry method proved to be simple, rapid, and precise and may be used as an additional test in diagnosing bacterial meningitis, especially in patients who are treated with antibiotics before admission. For patients with bacterial meningitis, a high concentration of amino acids in the CSF on admission may indicate a poor outcome.

Adolescent↗
PubMed2004

Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance.

The development of chlorophyll (Chl) a fluorescence imaging systems has greatly increased the versatility of Chl a fluorometry as a non-invasive technique for the investigation of photosynthesis in plants and algae. For example, systems that image at the microscopic level have made it possible to measure PSII photochemical efficiencies from chloroplasts within intact leaves and from individual algal cells within mixed populations, while systems that image over much larger areas have been used to investigate heterogeneous patterns of photosynthetic performance across leaves and in screening programmes that image tens or even hundreds of plants simultaneously. In addition, it is now practical to use fluorescence imaging systems as real-time, multi-channel fluorometers, which can be used to record continuous fluorescence traces from multiple leaves, plants, or algal cells. This paper discusses some of the theoretical and practical issues associated with the imaging of Chl a fluorescence and with Chl a fluorometry in general. This discussion includes a review of the most commonly used Chl a fluorescence parameters.

Chlorophyll↗
PubMed1992

Metabolic changes in the corneal epithelium resulting from hard contact lens wear.

The metabolic state of rabbit corneas was monitored in vivo using the noninvasive method of corneal redox fluorometry. The autofluorescence signals of reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) were measured in the corneal epithelium with and without contact lens wear. The PN/Fp ratio, which is related to the metabolic status of the tissue, was then calculated for each of these conditions. After application of polymethylmethacrylate (PMMA) contact lenses having an oxygen transmissibility (Dk) of less than 0.1, the PN signal increased and the Fp signal decreased. The PN/Fp ratio, generally a more precise indicator of metabolic state than either of these two quantities alone, was 1.93 +/- 0.78 without contact lenses, and increased to 2.78 +/- 0.86 (p less than 0.0001) with contact lenses. When oxygen-permeable silicon contact lenses (Dk = 12.5) were placed on the corneas, the PN/Fp ratio was found to increase slightly, but not as much as with the PMMA lenses. Newly developed highly oxygen-permeable contact lenses (Dk = 58.8) did not increase this ratio. Our findings indicate that redox fluorometry can be valuable in determining the effects of contact lens wear on corneal metabolism.

Animals↗