Development and application of serum cholinesterase activity measurement using benzoylthiocholine iodide.
BACKGROUND
Measurement of cholinesterase activity in serum is important to identify substantial liver disease and damage by pesticides, and to assess the degree of development of fatty liver and preoperative risk. Many procedures using various artificial substrates have been developed but suffer from problems with substrate specificity and interference by endogenous substances.


METHODS
An assay pseudocholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) activity was developed using a stable substrate specific to ChE, benzoylthiocholine iodide (BZTC). The thiocholine generated by hydrolysis of BZTC by ChE activity reacts with 2, 2'-dipyridildisulfide (2-PDS) to produce 2-thiopyridine (2-TP), which is measured at 340 nm. Optimum pH, buffer types and concentrations, substrate concentrations, and optimum conditions of the color reaction were investigated. The substrate specificity, test interferences, correlation with other measurement methods, and reference interval were evaluated.


RESULTS
The optimum pH of this method was 7.8, and 3-[4-(2-hydroxyethyl)-1-piperazinyl] propanesulfonic acid (EPPS) buffer solution was selected. Constant activity was shown at buffer concentrations >200 mmol/l, and the maximum activity was shown at a substrate concentration of 0.2 mmol/l. When a Hill plot was utilized, the Hill number was 1.08 and 1.09. The reaction velocity at this substrate concentration was 94% of V(max). The K(m) of ChE to BZTC was between 1.2 x 10(-2) and 1.3 x 10(-2) mmol/l. The range was 0-300 U/l. The coefficients of variation (CV) for 20 measurements of serum containing 53.1, 96.6, and 270.7 U/l of ChE were 0.82%, 0.76%, and 0.54%, respectively. The relative reactivity of acetylcholinesterase (AChE) to this substrate was 2%. The correlation factors of this method to three other methods were between 0.993 and 0.998.


CONCLUSIONS
This method provides excellent specificity, reproducibility, a wide measurement range, and minimal interference from endogenous substances to common serum analytes. Correlation of this method with conventional methods was good. Because the reagents are stable after preparation, this assay is useful for routine analysis.
Osawa
Susumu
S
School of Medicine, Biological Sciences and Technology, Department of Health Sciences, Kyushu University, Higashi, Fukuoka City 812-8582, Japan. osawas@shs.kyushu-u.ac.jp
Kariyone
Kazuko
K
Ichihara
Fumio
F
Arai
Kenji
K
Takagasa
Nobuyuki
N
Ito
Hiromi
H
D000293:Adolescent; D000328:Adult; D000368:Aged; D001322:Autoanalysis; D002021:Buffers; D002794:Choline / Q000031:analogs & derivatives*; D002802:Cholinesterases / Q000097:blood*; D004228:Dithionitrobenzoic Acid; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D011725:Pyridines / Q000737:chemistry; D012016:Reference Values; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents
D002021:Buffers; D007202:Indicators and Reagents; D011725:Pyridines; D013439:Sulfhydryl Reagents; C499395:benzylthiocholine iodide; D004228:Dithionitrobenzoic Acid; D002802:Cholinesterases; C029347:2-thiopyridine; D002794:Choline