Department of Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, Chiba 274-8510, Japan.
Biol Pharm Bull. 2010;33(3):364-9. doi: 10.1248/bpb.33.364.
Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.
抗坏血酸（AA）具有很强的抗氧化功能，表现在其体外清除超氧自由基的能力上。此外，AA 是胶原分子翻译后脯氨酸羟化所必需的成分。这些反应会生成脱氢抗坏血酸（DHA），AA 的氧化形式。在这项研究中，我们描述了一种改进的方法来评估生物样本中的 DHA。使用 35mM 三（2-羧乙基）膦盐酸盐（TCEP）作为还原剂，在 pH 1.5 的 5%偏磷酸溶液中加入 1mM 乙二胺四乙酸（EDTA），在冰上 2 小时后，可将 DHA 完全还原为 AA。该方法使我们能够测量 6 周龄小鼠的多种组织和血浆中的 DHA 含量。这些组织中的 DHA 占总 AA 的百分比差异很大，即 0.8%至 19.5%。肺、心脏、脾脏和血浆中的 DHA 含量占总 AA 含量的 10%以上，而大脑、小脑、肝脏、肾脏和小肠中的 DHA 含量不到总 AA 含量的 5%。DHA 含量的这种差异可能表明生理部位的氧化应激水平存在重要差异。因此，这种改进的方法为所有 DHA 测定提供了一个有用的标准。
