Biaglow J E, Koch C J, Tuttle S W, Manevich Y, Ayene I S, Bernhard E J, McKenna W G, Kachur A V
Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Int J Radiat Oncol Biol Phys. 1998 Nov 1;42(4):769-73. doi: 10.1016/s0360-3016(98)00313-7.
Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions.
We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose.
Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption.
Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.
亚甲蓝（MB）可作为细胞内电子受体。本研究的目的是证明MB在测定细胞悬液总生物还原能力方面的实用性。
我们用Clark电极测量氧气消耗，并通过1-14C-葡萄糖释放14CO2来测定戊糖循环活性。
亚甲蓝催化细胞内还原剂烟酰胺腺嘌呤二核苷酸磷酸（NADPH）、烟酰胺腺嘌呤二核苷酸（NADH）和还原型谷胱甘肽（GSH）与氧气的反应，产生过氧化氢。反应速率与分子的负电荷相关（NADPH(-4) > NADH(-2) > GSH(-1)），这表明与带正电荷的氧化型MB反应是该反应的限速步骤。在细胞系统中，MB导致电子从细胞内源性底物流向氧气。由于多种过程导致NADP+/NADPH比值破坏，从而激活了MB。这些过程包括NADPH和GSH的直接氧化、GSH过氧化物酶催化GSH与H2O2的反应，随后氧化型谷胱甘肽（GSSG）将NADPH氧化。这导致细胞耗氧量增加，并在MB存在的情况下刺激戊糖循环（PC）的氧化分支。MB的细胞效应与其他电子接受药物不同。二酰胺和叔丁基过氧化氢作为直接氧化剂，而MB是氧气的电子载体。因此，MB对PC激活和氧气消耗的影响最大。
我们的结果表明，MB可用于通过耗氧量和PC激活来测定细胞的总生物还原能力。
