Choline determination based on the intrinsic and the extrinsic (chemically modified) fluorescence of choline oxidase

This paper describes two methods for the fluorometric determination of choline in serum by its reaction with the enzyme choline oxidase (ChOx). The first method, is based on changes in the intrinsic fluorescence of the enzyme (lambda(excitation)=28 nm and lambda(emission)=336 nm). The second method follows changes in the fluorescence intensity of a chemical modificant bonded to the enzyme (ChOx-FS), with excitation and emission maxima at 492 and 516 nm, respectively. Both methods have a similar response range (from 5 x 10(-7) to 10(-5) M or to 5 x 10(-5) M, depending on the analytical parameter) and precision (about 5%). The origin of the enzyme fluorescence changes has been elucidated and a mathematical model explaining the analytical signal is presented. This model can be applied to other enzymatic reactions based on flavin-containing enzymes and enables prediction of the sensitivity of new methods based on the Km values. Both methods have been applied to choline determination in synthetic serum samples. Moreover, the use of ChOx-FS avoids sample pretreatment.