Rate assay for determination of serum pseudo-cholinesterase activity

A simple and reproducible method for the determination of serum pseudo-cholinesterase activity was developed by making use of a stable substrate, p-hydroxybenzoylcholine, with p-hydroxybenzoate hydroxylase as a linked enzyme. The method is based on spectrophotometric measurement of the decrease of NADPH. p-Hydroxybenzoate released from p-hydroxybenzoylcholine is hydroxylated by the action of p-hydroxybenzoate hydroxylase in the presence of NADPH and O2 to produce 3,4-dihydroxybenzoate and NADP+. This method is superior to the conventional methods in that this substrate is extremely stable up to pH 9.0, which is close to the optimum pH for the assay (pH 8.0). Serum interference was resolved by the use of p-hydroxybenzoate hydroxylase as a linked enzyme. The Km value of pseudocholinesterase for p-hydroxybenzoylcholine is 1 X 10(-5) M. The results of our method and Garry's method (Clin. Chem. 11, 91-96, 1965) correlated well (r = 0.962). The within-run and between-run C.V. values were 2.1 and 2.7, respectively.