Stolze K, Nohl H
Institute of Pharmacology and Toxicology, Veterinary University of Vienna, Austria.
Free Radic Res Commun. 1992;16(3):159-66. doi: 10.3109/10715769209049168.
The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.
广泛使用的食品添加剂丁基羟基茴香醚（BHA）和丁基羟基甲苯（BHT）与氧合血红蛋白发生反应，从而形成高铁血红蛋白。使用可见光谱法测量反应速率，并确定了BHA的二级反应速率常数，并与对羟基茴香醚进行了比较。我们使用电子自旋共振（ESR）研究了自由基反应中间体的参与情况。BHA和BHT预期的单电子氧化产物苯氧自由基，仅在纯3-叔丁基-4-羟基茴香醚和氧合血红蛋白中才能检测到。对于2-和3-叔丁基-4-羟基茴香醚的商业混合物，观察到一种非常强的二级自由基物种的ESR信号，类似于之前在对羟基茴香醚中观察到的信号，并且依赖于游离巯基的存在，因此我们假定存在过氧亚铁物种，即高铁血红蛋白-H2O2加合物。在第二系列实验中，我们研究了这种假定中间体与BHA和BHT的反应活性，实验从与ESR光谱仪相连的停流装置中的纯高铁血红蛋白/H2O2-苯酚混合物开始，检测到来自BHA和对羟基茴香醚的预期苯氧自由基。由于BHT的低溶解度和反应活性降低，仅发现痕量的苯氧型自由基以及高浓度的未反应过氧亚铁物种。在存在巯基阻断物质N-乙基马来酰胺（NEM）的情况下，反应速率增加，这证明了BHA、BHT和对羟基茴香醚与游离巯基的反应活性。
