Pálfi Melinda, Halász Attila Sándor, Tábi Tamás, Magyar Kálmán, Szöko Eva
Department of Pharmacodynamics, Semmelweis University, Nagyvárad tér 4, H-1089 Budapest, Hungary.
Anal Biochem. 2004 Mar 1;326(1):69-77. doi: 10.1016/j.ab.2003.11.010.
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.
已开发出一种快速灵敏的高效液相色谱法，用于在荧光衍生化后测量生物样品中的氧化吡啶二核苷酸（NAD +、NADP +）。在强碱性条件下，烟酰胺部分的吡啶环与羰基化合物反应，生成稳定的荧光产物。随后加入浓甲酸并加热处理后，这种荧光会进一步放大并转移到更高波长区域。在所测定的酮类（丙酮、甲乙酮、苯乙酮）中，与苯乙酮的缩合产物具有最高的摩尔相对荧光，因此在我们的实验装置中检测最为灵敏（检测限：0.02 pmol/50微升进样体积）。使用0.1M柠檬酸（pH 3.2）/乙腈（92/8，v/v）作为流动相，在反相C-18柱上分离荧光产物。我们的方法适用于通过定量氧化辅酶产物来测定依赖NADH和NADPH的酶反应。例如，通过测量不同底物（L-精氨酸）浓度下的产物NADP +和L-瓜氨酸，评估了神经元型一氧化氮合酶（nNOS）的活性，该酶需要NADPH。nNOS解偶联的NADPH氧化速率可根据产生的NADP +/L-瓜氨酸的比率来估算。
