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An Efficient In Vitro Assay for Acetylcholinesterase Reactivators Using Immobilized Enzyme (1984)

Trammel, Andy M. ; Simmons, John E. ; Borchardt, Ronald T.

Springer

Pharmaceutical research 1 (1984), S. 115-120

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ISSN: 1573-904X

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract A new and efficient in vitro assay for evaluating reactivators of organophosphate-inhibited acetylcholinesterase has been developed. Low density polyethylene beads (4 mm) were func-tionalized to terminal aldehydes and used to immobilize acetylcholinesterase (AChE, Electrophorus electricus, E.G. 3.1.1.7) via a stable Schiff base link. AChE activity in columns containing immobilized enzyme could be continuously monitored spectrophotometrically in a closed loop flow system using acetylthiocholine and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). Immobilized enzyme exhibited good esterase activity (0.5 units/bead), which could be retained on storage at −16°C for four months. The kinetics for substrate hydrolysis were flow-rate dependent below substrate saturation levels. This system allowed for independent inhibition and reactivation of the enzyme. Immobilized enzyme could be inhibited with diisopropylfluorophosphate (DFP) and 20–90% of original activity restored with several oximes in less than 20 minutes. The extent of reactivation was dependent on the concentration of the reactivators. This system has advantages over previously reported procedures, because hydrolysis of substrate due to reactivator is minimized and inhibitor-reactivator interactions are eliminated, subsequently minimizing the need to correct experimental results.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1016323919445

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Capillary Gas Chromatographic (GC) Analysis of Nitroglycerin and Its Denitration Products in Plasma (1988)

Carlin, Alan S. ; Simmons, John E. ; Shiu, Gerald K. ; [et al.]

Springer

Pharmaceutical research 5 (1988), S. 99-102

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ISSN: 1573-904X

Keywords: nitroglycerin ; metabolites ; capillary gas chromatography with electron capture detection (GC-EC) ; nanogram ; beagles

Source: Springer Online Journal Archives 1860-2000

Topics: Chemistry and Pharmacology

Notes: Abstract A convenient, specific, and sensitive capillary gas chromatographic (GC) assay for analyzing nanogram concentrations of nitroglycerin and its dinitro- and mononitrometabolites in plasma has been developed. Using a bonded-phase (DB-1) 30-m, 1-µm-thick film capillary column and a 1-m, 5-µm-thick film precolumn, separation of nitroglycerin and all four partially nitrated metabolites was achieved in less than 15 min. On-column injection, electron capture detection, and isothermal operation at 100°C yielded a linear extraction curve over a 300-ng/ml range without any need to concentrate sample extracts. Using methyl t-butyl ether as extraction solvent and o-chloronitrobenzene as internal standard, recoveries from plasma spiked at levels greater than 10 ng/ml approximated 35% for the 1-monometabolite, 40% for the 2-monometabolite, and greater than 90% for all others. The method was employed in a pharmacokinetic study of nitroglycerin administered intravenously to beagle dogs. Plasma samples were collected at various time points and analyzed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1015988117588

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