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Biomedical subjects

M DeLuca

Publications and source records attributed to M DeLuca.

At least 55 records · Page 3Linked to original sources
PubMed1983

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes.

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.

Bile Acids and Salts↗
PubMed1983

How to prevent losses of protein by adsorption to glass and plastic.

An improved procedure for reducing the loss of protein by adsorption to glass or plastic surfaces is reported. For working with proteins at the microgram level, the solvent is modified by adding glycerol (50% final concentration) or Triton X-100 (0.2 mM final concentration). Coating the plastic or glass surfaces with proteins such as bovine serum albumin or other materials is not as effective; adding proteins such as bovine serum albumin to the solvent is counterproductive.

Adsorption↗
PubMed1983

Bioluminescent immunoassays.

The procedures described here have been developed for only a few antigens at present. It seems very likely that they can be extended to any other antigen of choice. For sensitivities in the picomole range, the luciferase-antigen procedure is satisfactory. For increased sensitivity, the glucose-6-phosphate dehydrogenase-antigen is the method of choice. With some minor modifications the sensitivity of this assay can certainly be increased. The lower limits of detection will ultimately be determined by the affinity of the antibody for the antigen, not by the detection system.

Antigen-Antibody Complex↗
PubMed1982

Bioluminescence measurement of primary bile acids using immobilized 7 alpha-hydroxysteroid dehydrogenase: application to serum bile acids.

A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. The method is based on enzymatic dehydrogenation of bile acids using a bacterial 7 alpha-hydroxysteroid dehydrogenase that is co-immobilized on Sepharose 4B beads with NADH:FMN oxidoreductase and a bacterial luciferase. The assay is specific for 7 alpha-hydroxy bile acids and has a detection limit of 0.5 pmol/tube, with a linear range of 0.5-50 pmol/tube. The assay shows good precision (6-8% intra-assay; 8-10% inter-assay). The values obtained with the bioluminescence assay showed good agreement with those obtained by gas-liquid chromatography, radioimmunoassay, or endpoint enzymatic assays. When applied to the measurement of serum bile acids, there was no interference from serum albumin, and the effect of other dehydrogenase activity in serum could be eliminated by heating the sample prior to assay. Since the method is rapid (1 minute), extremely sensitive (requires only 10 microliters of serum), and specific, it appears to be the best method currently available for the measurement of serum primary bile acids.

Bile Acids and Salts↗
PubMed1981

The creatine kinase curve area and peak creatine kinase after acute myocardial infarction: usefulness and limitations.

We determined creatine kinase (CK) curve areas in 112 patients with acute myocardial infarction. Two-hour sampling was performed for the first 24 hours or until peak CK was reached, and a gamma density function was used to calculate curve areas from all available samples. Attempts to predict CK curve area by means of the portion of the curve prior to peak CK proved to be inaccurate; not until values 2 hours or more beyond peak CK were utilized did predicted and actual CK areas agree well. A good correlation (r = 0.93) was found between CK area and peak CK. To establish an approach for detecting peak CK in the clinical setting, a range of sampling intervals (4 to 24 hours) was assessed; 4- and 6-hour sampling intervals for 48 hours produced maximum CK values at or above 85% of true peak CK in 90% and 89% of patients, respectively, and average maximum CK at both sampling intervals exceeded 94% of that obtained with 2-hour samplings. We conclude that this simplified approach can provide a basis for estimating infarct severity in the individual patient.

Creatine Kinase↗
PubMed1979

Properties and uses of immobilized light-emitting enzyme systems from Beneckea harveyi.

Bacterial luciferase and NADH:FMN oxidoreductase have been immobilized onto arylamine glass beads. These immobilized enzymes can detect as little as 0.2 pmol of NADH per assay sample. Glucose-6-phosphate dehydrogenase has been co-immobilized with these enzymes, and with this system it is possible to quantitate 1 pmol of glucose 6-phosphate. By co-immobilizing a fourth enzyme, hexokinase, onto the glass beads, the system can reproducibly detect 20 pmol of glucose per liter. These immobilized enzyme systems are potentially superior to soluble enzymes by being reusable and much more stable. We compared the light-emitting properties of the immobilized enzyme systems with that of an equivalent mixture of the soluble enzymes. The most striking difference was the apparently more efficient conversion of NADH or glucose 6-phosphate to light by the immobilized enzymes. We used hydroxysteroid dehydrogenase in developing a soluble coupled system for the assay of androsterone and testosterone. The lower limit of detection was 100 pmol.

Enzymes, Immobilized↗