Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer’s disease versus controls

Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetyl coenzyme A to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease, therefore a specific, sensitive and reliable method for detecting ChAT enzyme activity of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, due to difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and rectify the problem, we have developed a chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including acetylcarnitine. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. We successfully detected several contaminants, especially in assays using human brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimer's disease compared to controls.