A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid.
An assay for the detection of hydroperoxy derivatives of linoleic acid formed by the action of 15-lipoxygenase is described. The assay developed is based on a method first reported by Ohishi et al. (1985) Biochem. Int. 10, 205-211) with some important modifications. The assay described herein takes advantage of the ability of (9Z,11E)-13-hydroperoxyoctadecadienoic acid (13-HPODE), the product of the action of 15-lipoxygenase on linoleic acid, to oxidize N-benzoyl leucomethylene blue to methylene blue in the presence of hemoglobin. The resultant blue color is stable to light and air and can be quantified spectrophometrically at 660 nm. The linear range of the assay is 1.6-32 nmol (0.5-10 micrograms) of 13-HPODE. The utility of the assay can be extended to detect other peroxides as well as inhibitors of 15-lipoxygenase. The assay is a rapid, reliable method for the detection of lipid hydroperoxide production.
Auerbach
B J
BJ
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48106-1047.
Kiely
J S
JS
Cornicelli
J A
JA
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000032:analysis / Q000378:metabolism*; D015227:Lipid Peroxidation; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors / Q000032:analysis; D008751:Methylene Blue; D011817:Rabbits; D013053:Spectrophotometry / Q000379:methods*
D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D019787:Linoleic Acid; D001093:Arachidonate 15-Lipoxygenase; D008751:Methylene Blue