Determination of Reduced Nicotinamide Adenine Dinucleotide Phosphate Concentration Using High-Performance Liquid Chromatography with Fluorescence Detection: Ratio of the Reduced Form as a Biomarker of Oxidative Stress
Determination of Reduced Nicotinamide Adenine Dinucleotide Phosphate Concentration Using High-Performance Liquid Chromatography with Fluorescence Detection: Ratio of the Reduced Form as a Biomarker of Oxidative Stress
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DOI：
10.1248/bpb.32.1819
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发表时间：
2009-11-01
影响因子：
2
通讯作者：
Ishii, Kazuyuki
中科院分区：
文献类型：
--
作者：
Ogasawara, Yuki;Funakoshi, Masayo;Ishii, Kazuyuki
还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）是许多具有生理重要性的过程中还原力的主要来源。我们研究了氧化应激对人红细胞（RBC）中NADPH相对含量的影响。为了确定氧化后以还原形式存在的NADPH的稳态，我们开发了一种使用高效液相色谱（HPLC）测量人RBC中NADPH的改进方法。与酶循环法和HPLC光谱检测法相比，本方法具有更好的重现性和选择性。校准曲线在0.1-5.0 μ M范围内呈线性，相关系数为0.999。人红细胞中总NADPH（NADPH+NADP（+））和NADPH浓度测定的运行内精密度分别为2.4%和8.6%（n=5）。将RBC悬液暴露于叔丁基过氧化氢（t-BHP）后，谷胱甘肽过氧化物酶（GPX）沿着NADPH消耗清除t-BHP，在还原型谷胱甘肽短暂降低和快速恢复后，观察到NADPH比值[（NADPH/（NADPH+NADP+）]显著降低。在不存在葡萄糖的情况下，观察到t-BHP暴露诱导的NADPH比率显著降低。然而，NADPH的比例并没有减少t-BHP暴露后，用谷胱甘肽还原酶抑制剂预处理。该方法可用于各种生物来源的少量NADPH的测定。
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the principal source of reducing power in numerous processes of physiological importance. We examined the influence of oxidative stress on the relative amounts of NADPH in human red blood cells (RBCs). To determine the homeostasis of the NADPH existing in (lie reduced form following oxidation, we developed an improved method for measurement of NADPH in human RBCs using high-performance liquid chromatography (H PLC). The present method with fluorescent defection is reproducible and selective than enzymatic cycling method and HPLC methods with spectrometric detection. The calibration curve is linear over the range of 0.1-5.0 mu M with a correlation coefficient of 0.999. The within-run precision of the assays for total NADPH (NADPH+NADP(+)) and NADPH concentrations in human RBC were 2.4% and 8.6%, respectively (n=5). After the RBC suspension was exposed to tert-butyl hydroperoxide (t-BHP), which is scavenged by glutathione peroxidase (GPX) along with NADPH consumption, a significant decrease in the NADPH ratio [(NADPH/(NADPH+NADP+)] was observed after a transient decrease and rapid recovery of the reduced form of glutathione. The marked decrease in the NADPH ratio induced by t-BHP exposure was observed in the absence of glucose. However, the NADPH ratio was not decreased by t-BHP exposure after pretreatment with a glutathione reductase inhibitor. This method may be useful for the measurement of small amounts of NADPH front various biological sources.