Interactions of the organophosphates paraoxon and methyl paraoxon with mouse brain acetylcholinesterase.

The mechanism of acute toxicity of the organophosphorus insecticides has been known for many years to be inhibition of the critical enzyme acetylcholinesterase (EC 3.1.1.7), with the resulting excess acetylcholine accumulation leading to symptoms of cholinergic excess. The bimolecular inhibition rate constant k(i) has been used for decades to describe the inhibitory capacity of organophosphates toward acetylcholinesterase. In the current study, a new approach based on continuous systems modeling was used to determine the appk(i)s of paraoxon and methyl paraoxon towards mouse brain acetylcholinesterase over a wide range of oxon concentrations. These studies revealed that the bimolecular inhibition rate constants for paraoxon and methyl paraoxon appeared to change as a function of oxon concentrations. For example, the appk(i) found with a paraoxon concentration of 1000 nM was 0.16 nM-1h-1, whereas that for 0.1 nM paraoxon was 1.60 nM-1h-1, indicating that the efficiency of phosphorylation appeared to decrease as the paraoxon concentration increased. These data suggested that the current understanding of how these organophosphates interact with acetylcholinesterase is incomplete. Modeling studies using several different kinetic schemes, as well as studies using recombinant monomeric mouse brain acetylcholinesterase, suggested the existence of a second binding site in addition to the active site of the enzyme, to which paraoxon and methyl paraoxon bound, probably in a reversibly manner. Occupation of this site likely rendered more difficult the subsequent phosphorylation of the active site by other oxon molecules, probably by steric hindrance or allosteric modification of the active site. It cannot be ascertained from the current study whether the putative second binding site is identical to or shares common elements with the well-characterized propidium-specific peripheral binding site of acetylcholinesterase.

Development and validation of a high-performance liquid chromatography method for the determination of cocaine, its metabolites and lidocaine.

Street cocaine varies in purity and is often adulterated with various compounds. Some of these additives, such as lidocaine, may increase the toxicity of cocaine. A new precise, accurate and sensitive reversed-phase high-performance liquid chromatography method for the determination of cocaine, its metabolites and lidocaine in plasma samples has been developed and validated. This assay employed a phosphate-buffered aqueous mobile phase (pH 6.0) with an organic component consisting of acetonitrile and methanol and a C-18 column as the stationary phase. Minimum detection limits were 1 ng ml-1 for cocaine, 2.5 ng ml-1 for ethylcocaine and 5 ng ml-1 for benzoylecgonine, norcocaine, norethylcocaine and lidocaine. Linearity was demonstrated over a broad range of concentrations in plasma, with good sensitivity for cocaine and cocaine derivatives.