Adventitious Redox-Active Metals in Krebs-Henseleit Buffer can Contribute to Langendorff Heart Experimental Results

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 from 0.32 to 0.17 μM. 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 nonchelexed 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-ischemia 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.