SPECTROPHOTOMETRIC DETERMINATION OF OXIDIZED AND REDUCED PYRIDINE-NUCLEOTIDES IN ERYTHROCYTES USING A SINGLE EXTRACTION PROCEDURE
SPECTROPHOTOMETRIC DETERMINATION OF OXIDIZED AND REDUCED PYRIDINE-NUCLEOTIDES IN ERYTHROCYTES USING A SINGLE EXTRACTION PROCEDURE
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DOI：
10.1016/0003-2697(87)90506-9
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发表时间：
1987-08-01
影响因子：
2.9
通讯作者：
TANAKA, KR
中科院分区：
文献类型：
--
作者：
ZEREZ, CR;LEE, SJ;TANAKA, KR
有几种方法可用于提取和定量红细胞中氧化和还原的吡啶核苷酸。然而，酶法因血红蛋白的存在而变得复杂，在提取过程中会导致NADH和NADPH的氧化。虽然添加还原剂可以防止血红蛋白介导的氧化，但这会干扰对这些核苷酸的分光光度循环分析。因此，我们开发了一种用单一提取物测定人红细胞中氧化和还原的NAD和NADP的方法。我们的提取方法不需要还原剂，因此允许使用分光光度循环分析。使用这种方法，我们获得了所有添加的核苷酸与正常和网织红细胞丰富的红细胞的完全回收。本方法适用于正常人红细胞和网织红细胞比例较高的溶血性贫血患者红细胞中NAD+、NADH、NADP+和NADPH的测定。
Several methods are available for the extraction and quantitation of oxidized and reduced pyridine nucleotides in erythrocytes. Enzymatic methods, however, are complicated by the presence of hemoglobin, which causes oxidation of NADH and NADPH during extraction. Although hemoglobin-mediated oxidation can be prevented by the addition of reducing agents, these interfere with spectrophotometric cycling assays for these nucleotides. Therefore, we have developed a method for determining oxidized and reduced NAD and NADP in human erythrocytes using a single extract. Our extraction method eliminates the need for reducing agents and thus allows the use of a spectrophotometic cycling assay. Using this method, we obtained full recovery of all added nucleotides with both normal and reticulocyte-enriched red blood cells. Our method is suitable for the determination of NAD+, NADH, NADP+, and NADPH in normal human erythrocytes and in red cells from patients with hemolytic anemia with a higher proportion of reticulocytes.