Peter M. Hammond

Development and validation of an automated enzyme assay for paracetamol (acetaminophen).

A rapid, enzymatic assay for serum or plasma paracetamol has been developed with the potential for adaptation to a wide range of clinical analysers. The method involves the action of an amidase enzyme to produce 4-aminophenol from paracetamol, which in turn reacts with 8-hydroxyquinoline in the presence of manganese ions to form a blue dye. Two stable reagents are used and excellent precision is achieved over the drug concentration range 0-2.5 mmol/l. The method, which is complete within 6 min, has been validated using a Monarch centrifugal analyser and shows no significant interference from endogenous serum compounds, drugs or paracetamol metabolites.

The measurement of serum paracetamolusing a discrete analyser

Introduction The current trend in clinical biochemistry towards the use of discrete analysers capable of performing a wide variety of analyses on a single sample, at the discretion of the analyst, suggests that this type ofequipment may have an important role to play in the emergency laboratory. There are, however, few analysers possessing the facility for stat discretionary testing. Recently, a specific method for the measurement of paracetamol has been described [1]. The method is a two-stage reaction involving, firstly, the enzymatic hydrolysis ofthe parent compound to p-aminophenol and acetate; and, secondly, the specific colorimetric detection of the p-aminophenol by the formation of a blue indophenol dye [2]. This assay has been adapted for use on a microcentrifugal analyser. In performing the adaptation the main objectives were to investigate (1) the limitations imposed by a two-stage reaction sequence; and (2) the performance ofsuch an automated system.

Purification and Characterisation of Gentamicin Acetyl Transferase

Homogeneous gentamicin acetyl transferase from Escherichia coli JR225 was isolated using four major steps: ion-exchange chromatography on DEAE-Sepharose, dye ligand chromatography on Procion Scarlet H-2G Sepharose, ion exchange by FPLC MonoQ HR 10/10 and gel-filtration by FPLC Superose 12 10/30. The purified enzyme had a specific activity of approximately 34 units/mg of protein and was shown to exist as a dimer of subunit molecular weight 30,000. The isoelectric point of the enzyme was calculated to be 5.34. The enzyme was maximally active at pH8.5 and at a temperature of 50°C. The enzyme shows Michaelis-Menten kinetics with Km values of: gentamicin (µM) apramycin (83µM), kanamycin (202µM), neomycin (31µM), netilmicin (2µM), ribostamycin (190µM)soframycin (21µM).The enzyme shows some activity towards 2-deoxystreptamine.