Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorometry”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,117 records · Page 62Linked to original sources
PubMed1994

Lipid chain order and dynamics at different bilayer depths in liposomes of several phosphatidylcholines studied by differential polarized phase fluorescence.

The influence of acyl chain length, double bond number and position on the structural and dynamic properties of phosphatidylcholine bilayers in the liquid crystalline state was studied. The range and rate of the rotation of 1,6-diphenyl-1,3,5-hexatriene and a set of n-(9-anthroyloxy)stearates(n = 2, 7 and 12), which are located at different depths in the lipid bilayers, were measured using differential polarized phase fluorometry in multilamellar liposomes of several unsaturated synthetic phosphatidylcholines. For the anthroyloxy-stearate probes, two rotational modes, 'in' and 'out' of the plane of the anthroyl aromatic ring, were partially resolved by measuring at different excitation wavelengths. The data obtained here indicate that in lecithins containing the same acyl chain in sn-1 and sn-2, the introduction of double bonds towards the deep interior of the lipid bilayer decreases the order in the external region of the bilayer and the viscous resistance to the rotation of all the probes tested. On the contrary, double bonds located towards the external region of the bilayer increase order and viscous resistance to the rotation of probes located in the external region. Increasing acyl chain length has different effects depending on the probe and on the rotational mode considered. Measurements in mixed chain lecithins indicate that the influence of increasing unsaturation in sn-2 has different consequences depending on the acyl group that is present in sn-1. Similarly, increasing acyl chain length in sn-1 influences differently the bilayer properties depending on the fatty acid that is present in sn-2.

Diphenylhexatriene↗
PubMed1976

Determination of quinidine in human serum in the presence of diuretics.

A new method for the determination of quinidine in human serum by fluorometry is described. The interference of triamterene encountered with other methods is greatly reduced, whereas a number of other diuretics do not interfere at all. The specificity is based on quenching of the fluorescence of cinchona alkaloids by chloride ions. The method has good reproducibility and linearity, and gives a recovery of 98%. A serum sample of 0.1 ml is sufficient for the assay. Comparison with two other methods showed that this procedure yields the total amount of cinchona alkaloids present in serum, which makes it extremely useful for routine clinical analysis.

Chlorides↗
PubMed1981

Sequential fluoroimmunoassay for measurement of pregnancy specific beta 1 glycoprotein using antibody coupled to magnetisable particles.

A direct, sequential fluoroimmunoassay has been developed for the determination of serum levels of pregnancy specific beta 1 glycoprotein (SP1). The method employs rabbit anti-SP1 serum coupled to magnetisable cellulose-iron oxide particles and fluorescein-labelled SP1. Serum samples or standards are incubated with magnetisable solid phase anti-SP1 for 30 min. After magnetic sedimentation of the particles, the supernate, which includes endogenous fluorophores and other interfering factors, is discarded. Fluorescein-labelled SP1 is then added and incubated for a further 45 min; the particles are again sedimented and the fluorescence of the labelled SP1 remaining in the supernate is estimated. This reading related directly to the SP1 content of the original sample. The entire procedure, including fluorometry, is performed within a single disposable polystyrene test tube and is sufficiently simple and reliable for routine application. The sensitivity, specificity and precision is very similar to that of radioimmunoassay, and the results correlate with those of the radioimmunoassay (r = 0.9887).

Female↗
PubMed1988

On the fluorometric assay of circulating lipoperoxides.

The methodology for the measurement of circulating thiobarbituric acid (TBA)-reactive products of lipid peroxidation in human plasma by means of fluorometry has been reinvestigated. The lipid precipitation of plasma with phosphotungstic acid, which step is laid down in the standard assay method, strongly increases the apparent TBA-reactivity. However, subsequent washing of the precipitate may reduce the apparent levels of lipoperoxide to values close to those obtained by using untreated plasma. Also, short-time storage of plasma at either 4 degrees C or -20 degrees C induces large day to day variations in assay results. This phenomenon may be prevented almost completely by the addition of glutathione plus EDTA. It is proposed to use untreated plasma and to perform the assay immediately after collection of plasma or to add both glutathione and EDTA before storage.

Edetic Acid↗
PubMed1991

Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex.

Previous methods to quantitate urobilinogen lack precision due to either incomplete reduction of urobilin or to losses of pigment before the use of Ehrlich's aldehyde reaction or due to pigment precipitation, as occurs in Schlesinger's fluorescent assay. The present procedure modifies the latter assay to obviate described problems as it is based on direct spectrophotometry (or spectrofluorometry) of a zinc complex of urobilin in dimethylsulfoxide. The sample is extracted with dimethylsulfoxide to increase recovery of urobilinogen from samples of various origin (feces, urine, bile, serum etc.) and to prevent the precipitation of proteins. After oxidation of urobilinogen with iodine, the concentration of the resulting urobilin is directly determined from the absorption (or fluorescent) spectrum. High sensitivity and high specificity for the procedure result from the high value of absorption coefficient and by the characteristic absorption spectrum of zinc complex of urobilin, respectively. Within-day and day-to-day coefficients of variation of stool and bile samples range from 1.6 to 9.2%. The smallest concentration of urobilinogen measurable by spectrophotometry is approximately 0.5 mumol/l, by fluorometry it is 0.25 mumol/l. The recovery varies from 82.2 to 93.8% depending on re-extraction of the sample. The method is linear in the range of 1 to 35 mumol/l and of 0.5 to 17.5 mumol/l for spectrophotometric and fluorescent determinations, respectively. The results obtained with the present method correlated well with Ehrlich's determination (r2 = 0.912), but are approximately two-fold higher. Storage of the samples at -20 degrees C or extraction with dimethylsulfoxide prior to storage are good ways for sample preservation. Twenty stool samples from healthy adults were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Optimized determination of malondialdehyde in plasma lipid extracts using 1,3-diethyl-2-thiobarbituric acid: influence of detection method and relations with lipids and fatty acids in plasma from healthy adults.

We investigated the influence of different concentrations of Fe3+, phosphoric acid, butylated hydroxytoluene and glutathione on the production of the malondialdehyde-1,3-diethyl-2-thiobarbituric acid adduct in plasma lipid extracts. Following organic solvent extraction the stable product was analyzed by spectrophotometry (537 nm), fluorometry (547 nm) and high-performance liquid chromatography with fluorometric detection. Using optimized reaction conditions there was good agreement between the three methods, with slightly higher values for the spectrophotometric method. Plasma total lipid malondialdehyde reference values for 24 healthy adults amounted to 1.30 +/- 0.23 mumol/l (spectrophotometric method) and 1.11 +/- 0.31 mumol/l (fluorometric method). Plasma lipid malondialdehyde concentrations correlated significantly with plasma triglycerides (r = 0.527), total cholesterol (r = 0.612) and total fatty acids (r = 0.810) and with the total number of double bonds present in plasma fatty acids with three or more double bonds (r = 0.923).

Adult↗
PubMed1992

Antibodies to the p53 tumor suppressor gene product quantified in cancer patient serum with a time-resolved immunofluorometric technique.

We have developed new methodology for quantifying antibodies to the p53 tumor suppressor gene product in human serum. The assay involves solid-phase immobilization of a monoclonal anti-p53-specific antibody that is then reacted with a tumor cell line lysate containing mutant p53. The immunopurified p53 antigen acts as an immunosorbent for the serum p53 antibodies that are then detected by reaction with a goat anti-human immunoglobulin G antibody labeled with alkaline phosphatase (ALP). ALP activity is then measured with enzymatically amplified time-resolved fluorometry. The developed assay has many advantages over the radioactively labeled techniques previously used. In a preliminary clinical study involving 790 patient sera, we have identified 16 positive samples (2%). Highest titers were observed in a patient with melanoma and two breast cancer patients. Further studies are needed to improve the sensitivity of this test and to evaluate its possible use for cancer diagnosis, prognosis or monitoring of therapy.

Alkaline Phosphatase↗
PubMed1992

An enzymatic method for measuring serum mannitol and its use in hemodialysis patients.

We evaluated two chemical methods for quantifying mannitol in serum, based on the oxidation of mannitol by periodate, and measurement of the formaldehyde formed with chromotropic acid (colorimetry) or acetylacetone (fluorometry). We found interference in these methods by serum glycerol. Additionally, a high-performance liquid chromatography (HPLC) method was evaluated and found to be specific but impractical for routine use. We therefore, developed an enzymatic fluorometric procedure, based on the oxidation of mannitol by beta-NAD to fructose and NADH, in the presence of the enzyme mannitol dehydrogenase (MD). MD is not commercially available and was partially purified from cultures of Leuconostoc mesenteroides. This new method is specific, sensitive, simple, and accurate and is proposed as the method of choice for measuring mannitol in the serum of patients who received this sugar alcohol during routine hemodialysis treatment.

Chromatography, High Pressure Liquid↗
PubMed1995

Quantification of creatine kinase BB isoenzyme in tumor cytosols and serum with an ultrasensitive time-resolved immunofluorometric technique.

OBJECTIVES: To develop a highly sensitive immunofluorometric procedure for creatine kinase BB isoenzyme and use it to measure CK-BB in tumor cytosolic extracts and serum of cancer patients and healthy volunteers. DESIGN AND METHODS: For assay development, we used two monoclonal antibodies in combination with time-resolved fluorometry and the biotin-avidin system. We measured CK-BB in breast tumor cytosols and studied its association with steroid hormone receptors. We also measured CK-BB in the serum of healthy subjects and patients with prostate cancer. We have examined the molecular weight of CK-BB in serum using high performance liquid chromatography. RESULTS: The evaluation of the method revealed good precision and accuracy. Study of 336 breast tumor cytosols and 9 normal breast cytosols has shown that CK-BB is overexpressed by 95% of breast tumors and that CK-BB is present in its 80 kDa form. A close association between CK-BB and estrogen but not progesterone receptors was found, suggesting that CK-BB overexpression is another marker of estrogen sensitivity of these tumors. Previous studies, using CK-BB radioimmunoassay could not detect CK-BB in the serum of about 50% of healthy subjects. We have assessed CK-BB levels in 80 male volunteers, detected CK-BB in all sera and provided a detailed distribution of values. We further demonstrated that 30% of prostate cancer patients in remission (PSA < 0.4 microgram/L) post radical prostatectomy and 50% of patients with active prostate cancer (PSA > 20 microgram/L) have elevated serum CK-BB levels. The patients with highly elevated CK-BB also had highly elevated serum PSA. We have demonstrated that some patients who have elevated serum CK-BB also have macromolecular CK complexes in their serum with molecular weights of 700 and 350 kDa as well as the 80 kDa CK-BB isoenzyme. Only the latter was recognized by the assay developed. CONCLUSIONS: CK-BB is a marker of estrogen sensitivity in breast cancer; Patients with prostate cancer have elevated CK-BB in their serum; The new highly specific and sensitive assay may be further used to study the role of CK-BB in various malignancies.

Age Factors↗
PubMed1991

Effects of trimebutine on cytosolic Ca2+ and force transitions in intestinal smooth muscle.

The effects of trimebutine maleate on cytosolic free Ca2+ and force transitions in the guinea-pig taenia cecum were studied by fura-2 fluorometry and tension recording. The addition of 80 mM K+ induced a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) and tension, followed by a sustained increase. Trimebutine (10 microM) suppressed both [Ca2+]i elevation and tension development. The tonic responses were more potently inhibited than the phasic responses. Phasic components gradually increased as the added K+ increased (10-40 mM). The relationship between the peak increases in [Ca2+]i and tension was not affected by trimebutine (10 microM). This means that trimebutine does not affect the Ca2+ sensitivity of contractile elements. In a high K+ and Ca(2+)-free medium, carbachol (10 microM) or caffeine (30 mM) caused transient [Ca2+]i elevation and tension development in the smooth muscle. Trimebutine (10 microM) decreased the amplitude of both responses. Trimebutine (10 microM) inhibited the spontaneous fluctuations in [Ca2+]i and motility of taenia cecum in the presence of tetrodotoxin (TTX; 0.3 microM). These results suggest that trimebutine has two types of inhibitory actions on intestinal smooth muscle; one, the inhibition of Ca2+ influx through voltage-dependent calcium channels, and the other, the inhibition of Ca2+ release from intracellular storage sites.

Animals↗
PubMed1983

Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum.

In Physarum polycephalum, microplasmodia differentiated into spherules when cultures were aged for 8-10 days. Respiration rates of the microplasmodia decreased rapidly with ageing to a 90% decrease in oxygen consumption over 9 days. We studied this phenomena by isolating and characterizing mitochondria from microplasmodia and spherules at different stages of spherulation. Oxygen uptake by the isolated mitochondria decreased with spherulation. Morphological and biochemical analyses showed that mitochondrial differentiation to inactive state was characterized by a decrease not only in dimension but also of content (DNA, RNA and protein). Diminutive mitochondria contained small particle-shaped mitochondrial nuclei. The DNA content, measured by microscopic fluorometry, was about 1.15 and 0.58 X 10(-10) g, which corresponded to about 16 and 8 genome copies, respectively (e.g., 32 genome copies per mitochondrion at mitochondrial G1). Restriction endonuclease analysis showed that the physical structure and methylation pattern of the mtDNA had not changed although the DNA content per mitochondrion had decreased remarkably with spherulation. This showed that changes in the ploidy level of the mitochondrial nucleus during spherulation were due to reduction in the number of whole mitochondrial genomes.

Cell Fractionation↗
PubMed1988

Specific binding of lectins with the nucleus of the sea urchin embryo and changes in the lectin affinity of the embryonic chromatin during the course of development.

(1) Embryonic cells of sea urchins were made permeable by treating them with glycerol solution for the purpose of allowing penetration of macromolecules into the cell. With the use of such permeabilized cells, several kinds of fluorescent dye-labeled lectins were introduced into the cell, and it was found that some lectins showed notable affinity with the nucleus as compared with cytoplasmic structures. (2) Isolated chromatin was incubated with several kinds of fluorescent dye-labeled lectins in vitro, and the amount of lectins bound with the chromatin was measured by fluorometry. By means of this method, the lectin-binding capacity of chromatin was estimated and compared at various stages of development. It was found that lectins could be classified into three groups according to the mode of binding with the chromatin: (a) Extent of binding increased notably at the gastrula stage (Con A and RCA-120); (b) extent of binding showed a temporary decrease at the gastrula stage (TTA); and (c) very low level of binding was maintained throughout all stages, and no particular change was observed at any stage of development (WGA, SBA, and UEA-I). (3) These facts seem to suggest that lectin-binding components are contained in sea urchin chromatin, and that drastic changes occur in these components of chromatin at the stage of gastrulation. It was proposed that the lectin-binding components such as proteoglycans and glycoproteins may play regulatory roles in embryonic chromatin at early stages of development.

Age Factors↗
PubMed1984

A simple method of measuring aqueous humor flow with intravitreal fluoresceinated dextrans.

Fluoresceinated dextran was injected into the vitreous cavity of rabbits and its concentration was followed in the vitreous and aqueous humors for a period of weeks by slip-lamp fluorometry. The concentrations in both fluids fell in parallel and in an exponential manner where the time constant was determined by the molecular weight of the dextran. The rate of movement of the dextran from the vitreous into the anterior chamber is controlled by passive diffusion and may be assumed to be relatively steady; accordingly, the faster the rate of flow in the aqueous humor the lower will be the concentration of dextran in it. Circadian variations in the aqueous flow were observed, a drop at night time being noted. These were small enough for changes due to systemic influences to be determined; in particular, it was found that general anesthesia with ketamine had no significant effect. The ratio of the concentration in the anterior chambers of the two eyes of one animal normally remains constant with time. Changes in the ratio can be used to determine the influence of unilateral interventions. This was illustrated by the use of adrenergic drops which led to a brief rise in flow rate and then a longer-lasting fall, and by X-ray irradiation with 1000 rads, which caused an immediate fall in flow rate interrupted for a time by a phase of increased flow. An absolute estimate of the aqueous flow can be derived from measurements of the total amount of dextran in the eye and its concentration in the anterior chamber. A definite value has not yet been established owing to ambiguities in determining the former quantity.

Anesthesia, General↗
PubMed1985

Selected positions of acyl chains are affected differently by antibody binding which results in decreased membrane fluidity.

We have studied the interaction between monoclonal anti-trinitrophenyl antibodies (IgGl and IgG2a) and haptenated phospholipid vesicles using stopped-flow fluorometry. Conformational changes of the antibodies were induced very rapidly (within 0.1 s) after binding to lipid haptens (TNP-Cap-DPPE) on the membrane surfaces. Conversely, after that, the bound antibody molecules decreased the degree of molecular motion at different depths in the bilayer, ranging from the polar head group to the terminal methyl groups of the fatty acyl chains. Such an effect reaches all places of the bilayer within 40 s at 25 degrees C.

Acylation↗
PubMed1986

Stopped-flow spectrophotometry monitoring the initial processes of platelet activation by platelet-activating factor.

The initial processes of platelet activation by platelet-activating factor (PAF) were observed by stopped-flow light scattering and fluorometry. The binding of PAF to rabbit platelets did not change the membrane fluidity, though it caused the removal of calcium from internal stores and induced concomitantly platelet shape changes. These results were quite in contrast to those by thrombin and ADP activation, where the membrane fluidity increased before the calcium release from the internal stores. The increased membrane fluidity in the latter system seemed to be used to transmit an external signal to a GTP-binding protein.

Animals↗
PubMed1986

Dolichol alters dynamic and static properties of mouse synaptosomal plasma membranes.

Dolichols are isoprenologues that are found in almost all tissues and whose biochemical function, aside from dolichol phosphate precursors, is not known. In addition, an understanding of the organizational and dynamic properties of dolichols in biological membranes has not been forthcoming. The purpose of the experiments reported here were to examine the effects of dolichol on the physical properties of mouse synaptic plasma membranes (SPM). Differential polarized phase fluorometry indicated that dolichol both fluidized and rigidified SPM. Membrane areas detected by diphenylhexatriene and trans-parinaric acid were selectively fluidized and rigidified, respectively. It also was found that the spin label, 5-doxyl stearic acid indicated that dolichol reduced membrane fluidity. These results report for the first time a structural effect of dolichol on a biological membrane.

Animals↗
PubMed1988

Increased membrane heterogeneity in stimulated human granulocytes.

TMA-DPH fluorescence decay in human PMN before and after stimulation with FMLP was studied using frequency domain fluorometry. Membrane heterogeneity was assessed by the width of the continuous distributions of lifetime values of Lorentzian shape used to describe the fluorescence decay. In non-stimulated granulocytes TMA-DPH fluorescence decay is characterized by two distributions of lifetime values centered at 6.5 and 1.0 ns and full width at half maximum of 0.3 and 1.2 ns, respectively. Within 15 min after stimulation, the center values of the two distribution components were 5.1 and 0.8 ns and the distribution width was 0.8 and 0.6 ns, respectively. These results indicate changes of membrane domain organization which can be ascribed to compositional changes and redistribution of membrane components.

Cell Membrane↗
PubMed1989

Glucose-induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans.

Intracellular Ca2+ levels were monitored in single, acutely isolated mouse islets of Langerhans by dual emission Indo-1 fluorometry. High-frequency (3.1 min-1) [Ca2+]i oscillations with a brief rising time (1-2 s) and 10 s half-width ('fast' oscillations) were detected in 11 mM glucose. Raising the glucose concentration to 16.7 mM increased the duration of these oscillations, which were otherwise absent in 5.5 mM glucose. [Ca2+]i waves of lower frequency (0.5 min-1) and longer rising time ('slow' oscillations) were also recorded. The data indicate that "fast" oscillations are directly related to beta-cell bursting electrical activity, and suggest the existence of extensive networks of electrically coupled cells in the islet.

Animals↗