Highly sensitive assay for acetylcholinesterase activity by high-performance liquid chromatography with electrochemical detection.

A highly sensitive assay for acetylcholinesterase (AChE) activity was devised by high-performance liquid chromatography with electrochemical detection. It is based on the separation of acetylcholine and choline on an octadecylsilane reversed-phase column, followed by their enzymatic conversion into hydrogen peroxide through the post-column reaction with immobilized AChE and choline oxidase. The system is highly sensitive, and the relationship between the peak height and the amount of choline is linear over the range 5 pmol to 5 nmol. When homogenate of bovine caudate nucleus was used as enzyme, the Michaelis constant of the enzyme for acetylcholine was 0.4 mM. The regional distribution of AChE activity in rat brain was examined, and the order of the activity from the highest to the lowest agreed with the reported brain distribution of AChE: striatum, thalamus plus hypothalamus, pons plus medulla oblongata, cerebral cortex, olfactory bulb, and cerebellum.