Helander A, Cronholm T, Tottmar O
Department of Zoophysiology, Uppsala University, Sweden.
Biochem Pharmacol. 1993 Dec 14;46(12):2135-8. doi: 10.1016/0006-2952(93)90601-r.
The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low-Km ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low-Km and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD+ concentration was apparent. For the rat liver low-Km ALDH, a Ki of 8.4 +/- 2.8 microM (mean +/- SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.
研究了氧化还原染料亚甲蓝对NADH稳定性以及醛脱氢酶（ALDH；EC 1.2.1.3）活性的影响。采用高效液相色谱法（HPLC）结合荧光或分光光度检测法测定NADH。通过使用HPLC和电化学检测法，跟踪3,4 - 二羟基苯乙醛（DOPAL）生成3,4 - 二羟基苯乙酸（DOPAC）的过程来进行ALDH活性测定。在亚甲蓝存在下孵育NADH溶液会导致NADH随时间直接氧化。亚甲蓝以浓度依赖性方式抑制人红细胞和白细胞中的ALDH以及大鼠肝线粒体低Km ALDH。稀释可使失活作用逆转，动力学分析表明，亚甲蓝对大鼠肝线粒体低Km ALDH和人红细胞ALDH的抑制作用是相对于DOPAL的竞争性抑制，而NAD⁺浓度未表现出明显影响。对于大鼠肝低Km ALDH，计算得出的Ki为8.4±2.8 μM（平均值±标准差；N = 5）。ALDH受到抑制以及由此导致的与乙醇脱氢酶（EC 1.1.1.1）结合的NAD系统氧化还原作用降低，可能解释了亚甲蓝对乙醇代谢氧化还原作用的保护效应。
