This study pursued whether singlet oxygen (O-1(2)) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The O-1(2) formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl- 4-piperidone (TMPD) by O-1(2) to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta -carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that O-1(2) is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the O-1(2) formation in living organisms following the advancement of peroxidation of constitutive lipids. (C) 2001 Elsevier Science Inc. All rights reserved.