Characterization of four BCHE mutations associated with prolonged effect of suxamethonium.

Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a "silent" BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower kcat value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (Ω-loop) can explain the modification of the kinetic parameter of the mutated protein.

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system.

OBJECTIVE: To evaluate the analytical performance of the Diazyme ADA assay on the Cobas® 6000 system for pleural fluid samples analysis. DESIGN AND METHODS: Imprecision, linearity, calibration curve stability, interference, and correlation studies were completed. RESULTS: The Diazyme ADA assay demonstrated excellent precision (CV<4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 µmol/L and haemoglobin above 177 µmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r(2)=0.93) but exhibited a negative bias (~ -30%). CONCLUSIONS: The Diazyme ADA assay on the Cobas® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.