Affordable Access

Publisher Website

Determination of rat serum esterase activities by an HPLC method usingS-acetylthiocholine iodide andp-nitrophenyl acetate

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
381
Issue
1
Identifiers
DOI: 10.1016/j.ab.2008.06.019
Keywords
  • Rat Serum Esterases
  • S-Acetylthiocholine
  • P-Nitrophenyl Acetate
  • On-Line Radiochemical Chromatography
  • Ion Pair Chromatography
  • Mass Spectrometry
Disciplines
  • Biology
  • Chemistry
  • Physics

Abstract

Abstract Establishing esterase assays allows the determination and comparison of esteratic activities of tissues of one organism and between organisms. We have developed a high-performance liquid chromatography (HPLC) assay for the determination of S-acetylthiocholine (ATC) and p-nitrophenyl acetate (NPA) hydrolyzing activities of rat serum esterases based on ion pair chromatography with on-line radiochemical and ultraviolet (UV) detection. ATC is a substrate for cholinesterases, whereas NPA is cleaved by a variety of esterases and other proteins (e.g., cholinesterases, paraoxonase, carboxylesterase, albumin). Both substrates were incubated, simultaneously or separately, with rat serum to explore potential interferences between the enzymatic hydrolyses of the compounds. The ratio of the peak area of the 14C-labeled substrates to the total peak area of the substrates and their corresponding cleavage products was compared with the UV quantitation of ATC and p-nitrophenolate (NP), the cleavage product of NPA, measured at 230 and 350 nm, respectively. The peak identity of ATC and NP was confirmed by electrospray ionization–tandem mass spectrometry (ESI–MS/MS). The reaction rates of the assays using one substrate or both, as well as using radiochemical or UV detection, were equal. Moreover, the correlation between rat serum volumes and reaction rates was shown for both substrates. In conclusion, one can (i) choose between the two detection methods reliably, (ii) take advantage of monitoring both substrate and product by using radiochemical detection, and (iii) combine both substrates to determine esterase activities in rat serum and probably other biological matrices.

There are no comments yet on this publication. Be the first to share your thoughts.