New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter.

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

Characteristics of dibucaine inhibition of “usual” and “atypical” forms of BChE.

<p>DN values were calculated in assays performed at various dibucaine concentration at 25°C using 5mM BTC (A) or PTC (B) as substrate with two types of 400-fold diluted serum samples from heterozygous individuals with usual UU and UA phenotype. Dibucaine Number (DN) = (1 - (butyrylcholinesterase activity in presence of inhibitor/butyrylcholinesterase activity in absence of inhibitor)) x 100. Assays were performed in triplicate, standard error for all points is less than 3% of the values.</p

Optimization of the BChE assay linearity.

<p>Measurement of TNB ion concentration at 412 nm produced in Ellman's reaction in the presence of 5 mM BTC and 40 (5), 100 (2), 200 (1), 400 (0.5), 600 (0.3), 800 (0.25)-fold diluted serum. Volume (μl) of serum in the reaction mixture (200 μl total volume) is shown above in brackets. Assays were performed in triplicate, standard error for all points is less than 2.5% of the values.</p

The effect of serum dilution on BChE activity.

<p>The activity was estimated based on the mean of hydrolysis rate (OD/min) determined from at least 5 spectrophotometer reads (every 1 minute) to ensure linearity and regularity of slopes. Assays were done in triplicate, error bars represent mean ± standard error (Student’s t-test, p<0.05). <b>A.</b> The effect of BTC concentration on the BChE activity in 100, 400, 4000 -fold diluted serum samples. Inset shows a Lineweaver–Burk replot of the results presented in Fig 2A at [BTCh] = 0.1, 0.5 and 1 mM. <b>B.</b> The effect of 5mM and 2.5 mM BTC concentration on the BChE activity in variously diluted serum samples. <b>C.</b> BChE activity in 100, 400, 2000 -fold diluted eleven randomly selected serum samples from our serum collection samples.</p