Powell S R, Wapnir R A
Department of Surgery, North Shore University Hospital, Manhasset, NY 11030.
J Mol Cell Cardiol. 1994 Jun;26(6):769-78. doi: 10.1006/jmcc.1994.1091.
Adventitious redox-active metals in Krebs-Henseleit buffer exhibit a significant enhancement of damage to isolated rat hearts. Using atomic absorption spectroscopy, it was determined that Krebs-Henseleit buffer contains substantial amounts of contaminating iron and copper. Significant copper contamination was found in ACS Reagent grade sodium chloride and sodium bicarbonate; iron contamination in sodium chloride, potassium chloride, sodium bicarbonate, and calcium chloride. Chelating resin treatment of individual reagents was found to decrease copper content of Krebs-Henseleit buffer from 0.32 to 0.17 microM. Using salicylate as a probe for .OH formation, it was determined that considerable amounts of this radical are formed when 0.25 mM ascorbate is added to the buffer indicating significant metal-catalysed autoxidation. Isolated rat hearts, perfused with non-chelexed Krebs-Henseleit buffer plus 0.25 mM ascorbate for 60 min, sustained moderate injury with developed systolic pressure, +dP/dtmax and -dP/dtmax decreased by 30 to 35% by the end of experiment. Hearts perfused with chelating resin-treated Krebs-Henseleit buffer sustained no significant injury within the same time frame. Furthermore, it was observed that hearts perfused with non-chelexed Krebs-Henseleit buffer accumulate significant amounts of copper depending on the amount of contamination and length of perfusion. Significant effects on post-ischemic end diastolic pressure were observed in hearts perfused with a Krebs-Henseleit buffer subsequently found to be contaminated with high levels of copper. These results clearly demonstrate that adventitious redox-active transition metals may be a confounding factor in experimental results. Further, it is recommended that all perfusion media be routinely examined for adventitious metals and treated if deemed necessary.
克雷布斯 - 亨泽莱特缓冲液中存在的外来氧化还原活性金属会显著增强对离体大鼠心脏的损伤。通过原子吸收光谱法测定发现，克雷布斯 - 亨泽莱特缓冲液含有大量的铁和铜污染物。在ACS试剂级氯化钠和碳酸氢钠中发现了显著的铜污染；在氯化钠、氯化钾、碳酸氢钠和氯化钙中发现了铁污染。发现对各试剂进行螯合树脂处理可使克雷布斯 - 亨泽莱特缓冲液中的铜含量从0.32微摩尔降至0.17微摩尔。使用水杨酸盐作为·OH形成的探针，确定当向缓冲液中加入0.25毫摩尔抗坏血酸时会形成大量这种自由基，表明存在显著的金属催化自氧化。用未经过螯合处理的克雷布斯 - 亨泽莱特缓冲液加0.25毫摩尔抗坏血酸灌注离体大鼠心脏60分钟，心脏会受到中度损伤，实验结束时收缩压、+dP/dtmax和 -dP/dtmax降低30%至35%。在相同时间框架内，用经螯合树脂处理的克雷布斯 - 亨泽莱特缓冲液灌注的心脏未受到显著损伤。此外，观察到用未经过螯合处理的克雷布斯 - 亨泽莱特缓冲液灌注的心脏会根据污染量和灌注时间积累大量铜。在用随后发现被高水平铜污染的克雷布斯 - 亨泽莱特缓冲液灌注的心脏中，观察到对缺血后舒张末期压力有显著影响。这些结果清楚地表明，外来的氧化还原活性过渡金属可能是实验结果中的一个混杂因素。此外，建议对所有灌注介质常规检查是否存在外来金属，并在必要时进行处理。
