Analysis of the addition products of alpha-tocopherol with phosphatidylcholine-peroxyl radicals by high-performance liquid chromatography with chemiluminescent detection.
A chemiluminescence-based high-performance liquid chromatographic method was developed for the analysis of the addition products of alpha-tocopherol with phosphatidylcholine-peroxyl radicals (TOO-PC). The TOO-PC eluted from a reversed-phase column was reacted with a chemiluminescent reagent consisting of a Cypridina luciferin analog and a lipid-soluble iron chelate in acidic methanol at 50 degrees C, and the generated chemiluminescence was monitored. The detection limit for TOO-PC by this method was about 1 pmol. This method was applied to the detection of TOO-PC in the peroxidized membranes prepared from rabbit erythrocyte ghosts. When the erythrocyte ghosts were peroxidized by the addition of a water-soluble free radical initiator, a peak corresponding to TOO-PC was detected on the chromatogram with chemiluminescent detection. The amount of TOO-PC in the erythrocyte membranes increased with the depletion of endogenous alpha-tocopherol. The results indicate that this method proved useful for the detection of the TOO-PC formed by the peroxyl-radical scavenging reactions of alpha-tocopherol in biological systems.
Yamauchi
R
R
Department of Bioprocessing, Faculty of Agriculture, Gifu University, Japan. yamautir@cc.gifu-u.ac.jp
Hara
Y
Y
Murase
H
H
Kato
K
K
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004910:Erythrocyte Membrane / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements; D008956:Models, Chemical; D010545:Peroxides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D011817:Rabbits; D012680:Sensitivity and Specificity; D013997:Time Factors; D014810:Vitamin E / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism
D010545:Peroxides; D010713:Phosphatidylcholines; D014810:Vitamin E; C049375:perhydroxyl radical