A novel assay to determine acetylcholinesterase activity: The application potential for screening of drugs against Alzheimer's disease

Abstract

Acetycholinesterase (AChE) that regulates hydrolysis of acetylcholine (ACh) in the brain, is an important target for treatment of Alzheimer's disease (AD), a feature of which is ACh deficiency. However, the methods to precisely determine AChE activity are still under development. We developed a new method to exploit acetylcholine-d as a surrogate substrate of ACh and measure product choline-d via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay detected activity of AChE present in the normal mouse brain, which is consistent with the standard Ellman assay that determines products spectrophotometrically. In AD mouse models, the result of LC-MS/MS assay showed significant higher AChE activity than that seen in control normal mice, while treatment of AD mice with an AChE inhibitor, huperzine A, led to partial decreases in AChE activity. Our results suggest that this surrogate-based LC-MS/MS method is a new, sensitive and convenient assay for the determination of AChE activity, providing a useful means for screening active compounds that target AChE.

Bioguided Isolation and Structure Identification of Acetylcholinesterase Enzyme Inhibitors from Drynariae Rhizome

“…A comparative study between this UPLC-MS/MS method and the classic spectrometry method was performed. We analyzed the inhibitory activity of galantamine and extract of Drynariae Rhizome via this novel method and modified Ellman's method [23]. We found that both assays displayed identically dose-dependent effects of the two inhibitors on AChE activity.…”

“…Acetonitrile at 0°C demonstrated better repeatability than methanol and 1M HCL in terminating enzyme reaction. At the given concentration of AChE in reaction solution, the starting concentration of ACh-I was investigated, and 400 μg/mL was selected, which matched the Km value (Figure 3) [23].…”

A Simple 3D-Printed Enzyme Reactor Paper Spray Mass Spectrometry Platform for Detecting BuChE Activity in Human Serum

“…The implementations of 3D printing technique in biochemical analyses have attracted enormous attention. , For instance, Salentijn et al developed a 3D-printed cartridge for paper spray ionization that can be used almost immediately after solvent introduction . With a careful design of the cartridge, spray lasted for tens of minutes, without any external pumping for mass spectrometry tests.…”

“…To date, several analytical approaches have been developed for BuChE activity assay, such as electrochemical sensors, , fluorometry, ,, colorimetry, and chemiluminescent methods . MS analysis of BuChE was also investigated with pepsin-based immobilized enzyme microreactors. , Generally, the main obstacles of BuChE detection come from the tedious pretreatment and denaturing trend of BuChE activity during the detection.…”

“…40 MS analysis of BuChE was also investigated with pepsin-based immobilized enzyme microreactors. 25,41 Generally, the main obstacles of BuChE detection come from the tedious pretreatment and denaturing trend of BuChE activity during the detection.…”