Determination of low-abundant metabolites in plant extracts by NAD(P)H fluorescence with a microtiter plate reader

This article describes a method for the enzymatic detection of low-abundant metabolic intermediates in plant extracts via NAD(P)H fluorescence using a microtiter plate reader. The detection of changes in NAD(P)H fluorescence (excitation 340 nm, emission 465 nm) exhibits a high signal-to-noise ratio and is as sensitive (greater than or equal to 20 pmol per well) as absorban ce measurements with dual-wavelength photometers. Since up to 96 reactions can be initiated, monitored, and evaluated simultaneously, this method migh t be suitable for high-throughput screening programs on metabolite profiles . However, in contrast to absorbance measurements, fluorescence detection o f NAD(P)H yields relative data, which can be impaired by the quench charact eristics and the basic fluorescence of the extracts. Hence, extensive calib ration is required to gain reproducible results. Calibration of the assay s ystem was performed using leaf or root material (equivalent to 2-35 mg of f resh weight per well) extracted with perchloric acid, chloroform/water/meth anol, or hot ethanol. Extraction with perchloric acid was found to be super ior for metabolite quantification. Examples of the kinetics of individual m etabolite determinations are presented and the contents of 3-phosphoglycera te, hexose phosphate, triose phosphates, pyruvate, and phosphoenolpyruvate in illuminated and darkened spinach leaves as well as leaf rosettes of Arab idopsis thaliana and leaf segments of the inducible crassulacean acid metab olism plant Mesembryanthemum crystallinum were measured via NAD(P)H fluores cence and, where possible, compared to reported data determined with dual-w avelength photometers. (C) 2000 Academic Press.