Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water in oil microemulsion

Theophylline is an effective bronchodilatator used in the treatment of asth ma which requires frequent control because of its narrow therapeutic index. Over the past decade much attention has been dedicated to the peculiar pro perties of the inner water pools of AOT (sodium 2-bishexyl-ethyl sulfosucci nate) microemulsions as enzyme microreactors, yet few analytical applicatio ns of the latter have been reported. We developed an original assay based o n the uncompetitive inhibition by theophylline of the reaction catalyzed by alkaline phosphatase from bovine liver (E.C. 3.1.3.1) of the ELF-97(R) flu orogenic substrate in berate buffer 20 mM (pH 8.6)/AOT/iso-octane-ethyl ace tate (95:5) at a temperature of 37 degrees C. Optimal activity of endogenou s plasmatic alkaline phosphatase isoenzymes approximate to pH 10.5, interfe ring activity of the serum are avoided. The assay is multiple point rate, m onitoring the appearance of the photostable fluorescence emission of the re action product (510-530 nm) out of the water pool. The influence of several parameters such as the amount of buffer (W degrees), the amount of alkalin e phosphatase, sample volume (10-30 pi), optimal run time (1-7 min) and the use of phosphorylating acceptor (2A2MP) are discussed. The method was comp ared to HPLC-UV and TDx methods. (C) 1998 Elsevier Science B.V. All rights reserved.