NEW REACTIVE OXIDIZING SPECIES CAUSES FORMATION OF CARBON-CENTERED RADICAL ADDUCTS IN ORGANIC EXTRACTS OF BLOOD FOLLOWING LIVER-TRANSPLANTATION

alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) radical adducts from Folch (chloroform:methanol) extraction of blood of transplanted Livers exhibited a large 6-line electron paramagnetic resonance (EPR) spectrum. Slow EPR sample preparation involving freezing and thawing prior to extraction over 15 min yielded a spectrum assigned as a lipid-derived free radical species, whereas rapid (< 1 min) extraction without a freeze-thaw cycle yielded a mixture of radicals, one with coupling constants similar to the alpha-hydroxymethyl-4-POBN adduct (4-POBN/(CH2OH)-C-.). Extraction with purified chloroform, however, yielded a much weaker, probably lipid-derived signal. Use of C-13-methanol in the Folch extracting solution yielded a 12-line EPR spectrum, indicating that a new, highly reactive oxidant species from blood following liver transplantation can convert organic solvents used in tissue extractions to free radicals. This hypothesis was supported by simulation of EPR spectra of free radicals extracted rapidly with Folch, which indicated that the spectrum contained two carbon-centered species, one with hyperfine coupling constants similar to the alpha-methylhydroxyl-4-POBN adduct, the other probably lipid-derived. Because the former originates from methanol in the Folch, extraction of samples with alcohol free organic solvent is most likely superior when the potential for formation of stable oxidant species exists, such as after liver transplantation.