Werner A, Siems W, Grune T, Schreiter C
Institute of Biochemistry, Medical Faculty (Charité), Humboldt University, Berlin, G.D.R.
J Chromatogr. 1990 May 16;507:311-9. doi: 10.1016/s0021-9673(01)84208-4.
The mechanism by which hypoxia leads to irreversible cellular damage is poorly understood. A decrease in purine nucleotides is common to all ischaemic tissues, yielding hypoxanthine as the substrate of the xanthine oxidase reaction. Excessive production of radicals via xanthine oxidase induces peroxidation of unsaturated fatty acids, accompanied with the formation of aldehydes. The nucleotides and aldehydes were determined by high-performance liquid chromatography (HPLC) of red blood cell extracts. Nucleotides and their derivatives were determined by HPLC on an ODS column and elution with 10 mM phosphate buffer containing 2 mM tert.-butylammonium phosphate. The aldehyde production in glucose deprived red blood cells was stimulated by addition of xanthine oxidase and by inhibition of different haemotype enzymes with sodium azide. Aldehydes were analysed by derivatization to dinitrophenylhydrazones, followed by thin-layer chromatographic and HPLC separation with aqueous methanol on an ODS column. The HPLC methods presented are appropriate for the determination of nucleotides, nucleosides and nucleobases, in addition to alkenals and hydroxyalkenals in extracts of oxidatively stressed red blood cells.
缺氧导致不可逆细胞损伤的机制目前尚不清楚。嘌呤核苷酸减少是所有缺血组织共有的现象，次黄嘌呤成为黄嘌呤氧化酶反应的底物。通过黄嘌呤氧化酶过度产生自由基会诱导不饱和脂肪酸过氧化，并伴有醛类的形成。通过对红细胞提取物进行高效液相色谱（HPLC）测定核苷酸和醛类。核苷酸及其衍生物通过在ODS柱上进行HPLC测定，并用含有2 mM叔丁基磷酸铵的10 mM磷酸盐缓冲液洗脱。通过添加黄嘌呤氧化酶以及用叠氮化钠抑制不同血型的酶来刺激葡萄糖缺乏的红细胞中的醛生成。醛类通过衍生化为二硝基苯腙进行分析，然后在ODS柱上用水性甲醇进行薄层色谱和HPLC分离。所介绍的HPLC方法适用于测定氧化应激红细胞提取物中的核苷酸、核苷和核碱基，以及烯醛和羟基烯醛。
