Determination of lactate by the intrinsic fluorescence of lactate oxidase

A fluorimetric method for the determination of lactate based on its enzymatic reaction with lactate oxidase (LOx) is proposed. The method relies on the relationship between the decrease in the enzyme intrinsic fluorescence during the reaction and the lactate concentration, which is shown to be the result of a primary internal filter effect on the enzyme by the pyruvate formed in the reaction. A mathematic model for predicting the effect of some parameters on the magnitude of the analytical signal was developed that provided good correlations with the experimental results. All those experimental variables affecting the analytical signal were optimized. Under the best possible conditions, the method features a linear determination range from 4 to 25 mgl lactate and a reproducibility of 3.5%, as the relative standard deviation. The proposed method allows lactate and pyruvate to be determined simultaneously. The predictions of the proposed model were also quite consistent with the experimental results obtained for synthetic samples containing both species.