Application of the measurement of oxidized pyridine dinucleotides with high-performance liquid chromatography-fluorescence detection to assay the uncoupled oxidation of NADPH by neuronal nitric oxide synthase

A rapid and sensitive high-performance liquid chromatography method has been developed for the measurement of oxidized pyridine dinucleotides (NAD+, NADP+) in biological samples following fluorescence derivatization. Under strongly alkaline conditions the pyridinium ring of the nicotinamide moiety reacts with carbonyl compounds, resulting in stable fluorescent products. Upon subsequent addition of concentrated formic acid and treatment with heat, this fluorescence is further amplified and is shifted to higher-wavelength regions. From among the ketones assayed (acetone, ethylmethyl ketone, acetophenone) the condensation product with acetophenone possesses the highest molar relative fluorescence, thus allowing the most sensitive detection in our experimental setup (limit of detection: 0.02pmol/50 microliter injected volume). The fluorescent products have been separated on a reverse-phase C-18 column using 0.1M citric acid (pH 3.2)/acetonitrile (92/8, v/v) as mobile phase. Our method is suitable for assaying NADH- and NADPH-dependent enzyme reactions by quantifying oxidized coenzyme products. As an example, the activity of neuronal nitric oxide synthase (nNOS), a NADPH-requiring enzyme, has been assessed by measuring the products NADP+ and l-citrulline at various substrate (l-arginine) concentrations. The rate of the uncoupled NADPH oxidation by nNOS can be estimated from the ratio of NADP+/l-citrulline produced.