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 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.
Powell
S R
SR
Department of Surgery, North Shore University Hospital, Manhasset, NY 11030.
Wapnir
R A
RA
D000818:Animals; D001205:Ascorbic Acid; D002021:Buffers; D002614:Chelating Agents; D003300:Copper / Q000032:analysis* / Q000737:chemistry; D004340:Drug Contamination; D005609:Free Radicals; D006321:Heart / Q000187:drug effects* / Q000502:physiology; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000592:standards; D007501:Iron / Q000032:analysis* / Q000737:chemistry; D008297:Male; D010084:Oxidation-Reduction; D010477:Perfusion / Q000379:methods* / Q000592:standards; D051381:Rats; D017207:Rats, Sprague-Dawley
D002021:Buffers; D002614:Chelating Agents; D005609:Free Radicals; D007202:Indicators and Reagents; D003300:Copper; D007501:Iron; D001205:Ascorbic Acid