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Clinical Chemistry 47: 1962-1966, 2001;
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(Clinical Chemistry. 2001;47:1962-1966.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

New Thiocholine Ester Substrates for the Assay of Human Serum Cholinesterase

Magohei Yamada1a, Yoji Marui2, Chozo Hayashi2, Yasuyoshi Miki3 and Shoji Takemura3

1 International Reagents Co., Ltd., 4-3-2 Takatukadai, Nishiku, Kobe 651-2271, Japan.

2 Laboratory for Clinical Investigation, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan.

3 Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-0818, Japan.

aAddress correspondence to this author at: 2-5-7 Nakagaito, Daito, Osaka 574-0013, Japan. Fax 81-72-889-7305; e-mail yama53{at}viola.ocn.ne.jp

Background: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443–50 and 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, substrate 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 efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was >10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was ~1/25 and ~1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The Km values of these acylthiocholines with human cholinesterase were almost equivalent (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 °C.

Conclusions: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.






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