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PubMed1983

Characterization of bacteria by mixed-dye fluorometry.

We describe an indirect detection method for bacterial identification and differentiation, based on selective adsorption of several fluorescent dyes. The lipid, protein, and nucleic acid components of fixed whole cells are stained with two mixtures, each containing two fluorochromes. The unadsorbed dyes were measured simultaneously with a video fluorometer [Clin Chem 22: 1483, 1976]. A dye-absorption matrix of the response can be generated, and we did so for each of nine bacteria. These responses were compared to a control or "complete" response matrix, and the response ratios of each bacterial species for each of the four dyes were calculated and plotted to obtain a characteristic pattern. From the response-ratio plots, plus simple pattern-recognition techniques, we could differentiate among all the bacteria. This rapid, sensitive technique is potentially applicable to a wide variety of bacteria.

Acridine Orange↗
PubMed1984

Digital cutaneous fluorometry: correlation between blood flow and fluorescence.

Central to the use of fluorescein in vascular diagnosis is the requirement that the intensity of evoked fluorescence be proportional to blood flow. With the introduction of the digital dermofluorometer, a device that quantitates cutaneous fluorescence, establishment of this relationship has become possible. After experimentally producing measured reductions in the distal aortic flow of eight rabbits, the ratio of fluorescence in the flow-restricted and unrestricted areas was obtained by measuring hind- and forelimb fluorescence. At any time between 20 and 60 minutes following a bolus injection of sodium fluorescein (1 mg/kg body weight), there was a significant linear relationship (p less than 0.05, r greater than 0.75) between residual aortic flow and the ratio of hind-/forelimb fluorescence. Simultaneously obtained plasma fluorescein concentrations decayed rapidly by first-order kinetics with a half-life of 12.5 minutes, regardless of the degree of distal aortic occlusion. The time course of the rise and fall of cutaneous fluorescence was slower than that of the plasma fluorescein concentration, proving that interstitial rather than intravascular fluorescein was responsible for the measured fluorescence. We conclude that the intensity of tissue fluorescence is linearly related to blood flow and that conclusions regarding perfusion may be drawn from relative fluorescence at any time between 20 and 60 minutes following a bolus injection of fluorescein. Furthermore, the passage of fluorescein into the interstitium is dependent on a time-limited diffusion process, which along with flow, establishes the time to peak and the absolute amplitude of the tissue fluorescence curve.

Animals↗
PubMed1978

Determination of 8-methoxypsoralen in plasma by scanning fluorometry after thin-layer chromatography.

A rapid and sensitive method is described for determining 8-methoxypsoralen in plasma. Plasma samples are acidified with 6 mol/liter and heated in a boiling water bath to release the plasma-bound drug nondestructively. It then is extracted into a solvent mixture consisting of benzene/ethyl acetate (9/1 by vol). The solvent phase is separated, evaporated, and an aliquot of the dissolved residue is thin-layer chromatographed, with benzene/ethyl acetete (9/1) as developing solvent. The plate is dried and the spots, made visible under ultraviolet light (320-400 nm), are scanned. The smallest amount detectable is 20 ng; the overall analytical recovery from plasma is 84%. We used the method to determine the drug in the plasma of rabbits after intravenous and oral administration of 10 mg, and in one patient after an oral dose of 30 mg.

Animals↗
PubMed1983

Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and nonsmokers.

We developed a simple, sensitive enzymatic assay involving the fluorogenic substrate naphthol AS-MX phosphate [(3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) phosphate] to measure heat-stable alkaline phosphatase (EC 3.1.3.1), the Regan isoenzyme, in human serum. The day-to-day CV was 5.7% for a serum activity of 0.080 arbitrary units/L. Measurable amounts of enzyme were detected in most normal individuals. The mean for 51 nonsmokers was 0.068 (SD 0.037) arb. units/L; for 25 smokers it was 0.440 (SD 0.360) arb. units/L. Activity of this isoenzyme in smokers was as much as 10-fold the upper normal limit for nonsmokers. Activation of this tumor marker by smoking has not received attention hitherto. We conclude that a truly normal range can only be established among nonsmokers. The isoenzymes in smokers, nonsmokers, and pregnant women were similar in their heat stability, immunologic cross reactivity, and inhibition by L-phenylalanine.

Alkaline Phosphatase↗