Metz E N, Balcerzak P, Sagone A L
J Clin Invest. 1976 Oct;58(4):797-802. doi: 10.1172/JCI108531.
The response of the hexose monophosphate shunt in erythrocytes was studied with the ionization chamber-electrometer apparatus to measure continuously 14CO2 derived from 14C-labeled substrates. The effect of methylene blue at high (0.1 mM) and low (1 muM) concentrations was evaluated under different gas mixtures; air, carbon monoxide, and 6% carbon monoxide in air. The latter gas mixture results in nearly 100% carboxyhemoglobin but provides a physiologic partial pressure of oxygen. The extent to which pentose is recycled through the shunt in response to methylene blue stimulation was examined with radioactive glucose substrates labeled on the first, second, and third carbon positions. Generation of hydrogen peroxide after stimulation of erythrocytes with methylene blue was evaluated by the catalase-aminotriazole trapping technique, [14C]formate oxidation, and oxidation of reduced glutatione. Stimulation of the shunt with 1 muM methylene blue was markedly impaired in the absence of oxyhemoglobin, but stimulation with 0.1 mM methylene blue was only slightly impaired under the carbon monoxide-air mixture. The higher concentration of methylene blue produced evidence of hydrogen peroxide generation of all three techniques. Despite the evidence for the involvement of oxygen, oxyhemoglobin, and hydrogen peroxide in the response to methylene blue, cells containing methemoglobin induced by sodium nitrite or from a patient with congenital methemoglobinemia responded normally to methylene blue in the absence of oxygen. These experiments indicate that the reactions induced by methylene blue in erythrocytes are more complex than generally thought and that high concentrations are associated with production of peroxide.
用电离室 - 静电计装置研究红细胞中磷酸己糖旁路的反应，以连续测量源自(^{14}C)标记底物的(^{14}CO_2)。在不同气体混合物（空气、一氧化碳以及空气中含(6%)一氧化碳）下评估高浓度（(0.1 mM)）和低浓度（(1 μM)）亚甲蓝的作用。后一种气体混合物可使羧基血红蛋白含量接近(100%)，但能提供生理氧分压。使用在第一、第二和第三碳原子位置标记的放射性葡萄糖底物，检测戊糖在亚甲蓝刺激下通过旁路循环的程度。通过过氧化氢酶 - 氨基三唑捕获技术、([^{14}C])甲酸氧化以及还原型谷胱甘肽氧化，评估亚甲蓝刺激红细胞后过氧化氢的生成情况。在没有氧合血红蛋白的情况下，(1 μM)亚甲蓝对旁路的刺激明显受损，但在一氧化碳 - 空气混合物中，(0.1 mM)亚甲蓝的刺激仅略有受损。较高浓度的亚甲蓝在所有三种技术中均显示有过氧化氢生成的证据。尽管有证据表明氧、氧合血红蛋白和过氧化氢参与了对亚甲蓝的反应，但由亚硝酸钠诱导产生高铁血红蛋白的细胞或先天性高铁血红蛋白血症患者的细胞，在无氧情况下对亚甲蓝仍有正常反应。这些实验表明，亚甲蓝在红细胞中诱导的反应比一般认为的更为复杂，且高浓度与过氧化物的产生有关。
