title: New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.
abstract: Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman's method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Jońca
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Żuk
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Wasąg
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Janaszak-Jasiecka
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Lewandowski
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Wielgomas
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Waleron
authors.affiliation: Medical University of Gdańsk, Gdańsk, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Jasiecki
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002091:Butyrylcholinesterase / Q000097:blood* / Q000737:chemistry*; D002092:Butyrylthiocholine / Q000737:chemistry; D002800:Cholinesterase Inhibitors / Q000737:chemistry; D006801:Humans; D007201:Indicator Dilution Techniques; D010575:Pesticides / Q000737:chemistry
chemical_list: D002800:Cholinesterase Inhibitors; D010575:Pesticides; D002092:Butyrylthiocholine; D002091:Butyrylcholinesterase