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PubMed2004

Cortisol and free thyroxine determination by time-resolved fluorometry in canine serum.

Validation for canine serum of 2 commercially available time-resolved fluoroimmunoassays (TR-FIAs) designed for analysis of cortisol and free thyroxine (fT4) in human serum was carried out. Included was the study of interference by hemolysis, lipemia, and bilirubinemia. With the dissociation enhancement lanthanide fluoroimmunoassay kits, the intra-assay coefficient of variation (CV) ranged from 6.4% to 8.7% for cortisol and from 5.3% to 9.8% for fT4; the interassay CVs ranged from 5.8% to 10.8% and from 3.9% to 14.1%, respectively. Accuracy was evaluated by comparing cortisol and fT4 results obtained with TR-FIA and those obtained with a validated enzyme-linked immunosorbent assay (ELISA) and an equilibrium dialysis (ED) assay, respectively. The regression equations obtained were y = 0.57x + 1.18 (r2 = 0.90) for cortisol and y = 0.87x + 0.82 (r2 = 0.93) for fT4. The limits of detection for cortisol and fT4 were 4.84 nmol/L and 2.68 pmol/L, respectively. The results of adrenocorticotropin-stimulation and dexamethasone-suppression tests were similar to those published previously; likewise, serial dilution of a canine serum sample with a high cortisol content demonstrated that the TR-FIA was immunologically specific. Serial dilution of a serum sample with a high fT4 concentration showed a methodologic bias, a dependence on serum binding capacity, which indicates that the results obtained with this method should be interpreted with caution. Finally, hemolysis and lipemia significantly interfered with cortisol and fT4 measurements, whereas bilirubinemia did not affect the results.

Animals↗
PubMed1992

Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry.

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.

Chorionic Gonadotropin↗
PubMed1990

[Study on the specific diagnosis of meconium aspiration syndrome (MAS) with fluorometry].

Porphyrins are found in fetal urine, neonatal urine, amniotic fluid, and meconium. Zn-coproporphyrin (Zn-CP), which we identified in meconium, has two fluorescent peaks at 580 nm and 630 nm, and Coproporphyrin (CP), found in fetal urine, has one fluorescent peak at 620 nm, when both porphyrins are excited at 405 nm. In this study, the fluorescent peak at 580 nm was used to detect Zn-CP in neonatal urine, using the new index, that is the Urinary Fluorescence Meconium Index (UFMI); UFMI = F580-(F580 + F600)/2 [F: fluorescence intensity]. We obtained urine samples from neonates in these three groups: Group-I: Meconium Aspiration Syndrome (MAS; n = 3), Group-II: non-MAS with meconium stained amniotic fluid at delivery (n = 18), and Group-III: non-MAS with clear amniotic fluid at delivery (n = 25). The UMI (Urinary Meconium Index) was also measured by conventional optical absorbance. The results show that many false positive cases were observed in UMI, and that no false positive cases were observed in UFMI. These results lead one to speculate that Zn-CP, which is specific in meconium, is used in UFMI measurement, while UMI measurement by absorbance cannot exclude contamination by CP in neonatal urine, so Zn-CP and CP are measured all together. UFMI values reflected the clinical course of neonates. It is concluded that UFMI is a more sensitive method than UMI for the diagnosis of MAS.

Coproporphyrins↗
PubMed1977

Histotoxic properties of carbon monoxide in the living organ I. Fluorometry of oxidation-reduction state of the rabbit kidney in situ.

Effect of carbon monoxide on the redox state and blood flow in the renal cortex of the rabbit was examined by the fluorometric method. By a short-period (1-3 min) inhalation of CO in concentrations over 1%, the following were revealed: (1) Reduced pyridine nucleotide was increased, (2) oscillatory change of the reduced pyridine nucleotide, which is characteristic for the histotoxic reaction, developed, and (3) extent of the fluorescence changes tended to be in proportion to the concentration of CO, but no change was caused by inhalation of CO in lower concentrations (0.1%, 0.5%) even if the inhalation was continued for 30 min. As to the blood flow, CO in concentrations over 0.5% decreased it, whereas anoxia due to nitrogen increased it. Some implications were discussed from toxicological points of view on the results obtained.

Animals↗
PubMed1988

Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine.

A new fluorometric assay for determining dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) was developed. The synthetic substrate glycyl-L-proline-4-methoxy-2-naphthylamide (20 mmol/L), Tris buffer (50 mmol/L, pH 8.3), and serum (20 microL) are mixed and incubated. The reaction is stopped with citrate (100 mmol/L, pH 4.0) and the released 4-methoxy-2-naphthylamine is measured fluorometrically. The mean value of DPP IV activity in serum for 64 healthy subjects was 58 (SD 16) mumol of 4-methoxy-2-naphthylamine released per liter of serum per minute. The proposed procedure is sensitive, rapid, and accurate and can easily be automated.

2-Naphthylamine↗
PubMed1987

[The use of laser fluorometry of NADH in cardiology].

In this paper the first results of experimental studies of myocardial energy metabolism conducted in situ by laser fluorimetry are described. With this method the myocardial oxygen uptake is determined by measuring the fluorescence of NADH (reduced nicotinamide adenine dinucleotide). The equipment required consists of pulsed laser beams in the ultraviolet (337 nm) and visible (586 nm) spectra, very thin catheters with a single optic fibre, and an original digital data processing technique. The first applications envisaged concern: 1. pharmacological studies, notably evaluation of the protective effect of drugs against ischaemia; 2. evaluation of revascularization procedures in emergency surgery, notably assessment of reversibility of the ischaemic lesions; 3. monitoring of myocardial protection during cardiac surgery.

Cardiovascular Agents↗
PubMed1988

Glucose-induced early changes in cytoplasmic calcium of pancreatic beta-cells studied with time-sharing dual-wavelength fluorometry.

The cytoplasmic calcium concentration (Ca2+i) was measured in suspensions of fura-2-loaded mouse pancreatic beta-cells by recording the 340:380 nm fluorescence excitation ratio. Exposure to 20 mM glucose resulted in an initial reduction and later increase of Ca2+i irrespective of preincubation in medium containing 0.5 or 1.28 mM Ca2+ and 0 or 3 mM glucose. When elevating the Ca2+ concentration to 5 or 10 mM only 5 min before raising glucose to 20 mM, the sugar-induced reduction of Ca2+i became more pronounced like the subsequent increase. However, when the Ca2+ concentration was increased from 1.28 to 10 mM 2 min after stimulation with glucose, there was a sudden pronounced Ca2+i transient, which was followed by a decrease and a slower secondary rise. After preincubation in 20 mM glucose the glucose-induced initial reduction of Ca2+i was only seen in a Ca2+-deficient medium. Reintroduction of the sugar in the presence of extracellular Ca2+ resulted in an immediate rise of Ca2+i, the rapidity of which depended on the transmembrane Ca2+ gradient. The results emphasize the role of a saturable beta-cell pool of Ca2+ in glucose-induced reduction of Ca2+i and indicate that the first phase of insulin release depends on an influx of extracellular Ca2+.

Animals↗