New thiocholine ester substrates for the assay of human serum cholinesterase

Background: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after be ing subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443-50 a nd Arch Biochem Biophys 1959;82:70-7). Methods: We synthesized thiocholine ester iodides by the method of Renshow et al. (J. Am Chem Soc 1938;60: 1765-70). We examined solubility in H2O sub strate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme. Results: isobutyryl and cyclohexane-carboxyl esters showed the best efficie ncy for the specific and stable assay of human serum cholinesterase. Aqueou s solubility of each was > 10 mmol/L, and the reactivity with acetylcholine sterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, res pectively, spontaneous hydrolysis in the aqueous phase was similar to1/25 a nd similar to1/175 slower than the enzymatic hydrolysis, and assays with th ese substrates were linear to 1800 and 3000 U/L, respectively. The Km value s of these acylthiocholines with human cholinesterase were almost equivalen t (6.9 X 10(-3) mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 degreesC. Conclusions: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase. (C) 2001 Am erican Association for Clinical Chemistry.