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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概要
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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 the reduced form following oxidation, we developed an improved method for measurement of NADPH in human RBCs using high-performance liquid chromatography (HPLC). The present method with fluorescent detection 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 μM 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 pre-treatment with a glutathione reductase inhibitor. This method may be useful for the measurement of small amounts of NADPH from various biological sources.
- 日本薬学会の論文
著者
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Ishii Kazuyuki
Department of Cardiology, Toho University Ohashi Medical center
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Ogasawara Yuki
Department of Hygienic Chemistry, Meiji Pharmaceutical University
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Funakoshi Masayo
Department of Hygienic Chemistry, Meiji Pharmaceutical University
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Ogasawara Yuki
Department Of Environmental Biology Meiji Pharmaceutical University
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Ishii Kazuyuki
Dep. Of Hygienic Chemistry Meiji Pharmaceutical Univ.
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Ogasawara Yuki
Department of Environmental Biology, Meiji Pharmaceutical University
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Funakoshi Masayo
Department of Environmental Biology, Meiji Pharmaceutical University
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