This invention concerns an improvement in the lipase determining method and reagent of Kurooka et al., U.S. Pat. No. 3,986,930, and Kurooka et al., J. Biochem. 81,361-369 (1977) and Kurooka, Japanese Patent Application No. Sho. 51-68342, the disclosures of which are hereby incorporated by reference. In this method and reagent, an S-acyl compound is provided as a substrate. Suitable S-acyl compounds are described in the above references. Lipase in a biological fluid specimen such as serum or plasma catalyzes the hydrolysis of the S-acyl compound, releasing sulfhydryl groups. The release sulfhydryl groups are reacted with a chromogenic sulfhydryl reagent, such as 5,5'-dithiobis(2-nitrobenzoic acid) ["DTNB" as abbreviated by Kurooka et al.] and the resulting color is measured to provide a measurement of lipase activity. Suitable chromogenic sulfhydryl reagents are disclosed by Kurooka et al. and by Ellman, U.S. Pat. No. 3,119,668, the disclosure of which is hereby incorporated by reference.
The above method and reagent has many desirable features. However, as disclosed in the Kurooka Japanese patent application, it suffers from a lack of sensitivity, which Kurooka attributed to inhibition by serum albumin. It also lacks specificity for lipase, and does not always measure true lipase activity. Additionally, the method requires addition of acetone to precipitate protein prior to measurement, requiring centrifugation or a similar protein removal step prior to measurement.
Eserine, also named as physostigmine, is a plant alkaloid extracted from calabar beans.