Measurement of lipid hydroperoxides by the ferric-xylenol orange method (2) application to lipoxygenase assay.
The ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe3+) product with a peak absorbance at 560 nm. We recently reported a modified FOX method, the sensitivity of which was increased in the presence of membranous phosphatidylcholine (PC) by forming a XO/Fe3+-PC complex with a peak absorbance at 610 nm. Lipoxygenases (LOXs) and their metabolites have been implicated in a wide range of disease states. We applied our newly developed FOX method to the assay of human 15-lipoxygenase-2 (15-LOX-2) and soybean lipoxygenase (SLOX) as typical animal and plant lipoxygenases, respectively. The amounts of 15-S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (15-HPETE) produced by 15-LOX-2 measured by UV-absorption at 237 nm attributed to the conjugated diene, coincided with the results of our FOX method measuring absorbance at 610 nm. The 15-HPETE production time courses measured by the two methods also correlated well. SLOX rapidly oxidized unesterified linoleic acids (LA) and slowly esterified fatty acids in egg yolk PC (EYPC). Availability of EYPC was increased if the membrane structure was moderately disturbed by MeOH and Triton X-100, but LA oxidation was readily decreased by them. These results indicate that our method is useful for lipoxygenase assay. Furthermore, our method was applicable to assaying the inhibitory effect of 5,8,11,14-eicosatetraynoic acid (ETYA) on SLOX activity using LA and EYPC as substrates. The inhibition dose-dependency of ETYA was almost the same in the LA and EYPC systems, although the enzyme concentrations differed by a factor of 1,000, suggesting that ETYA functioned as a competitive inhibitor. These results indicate that our method may be useful as a screen for the identification of novel inhibitors of lipoxygenases.
Faculty of Pharmacy, Yasuda Women's University, Hiroshima 731-0153, Japan. fukuzawa@yasuda-u.ac.jp
Kenji
K
Fukuzawa
Masumi
M
Sano
Kaori
K
Akai
Junichi
J
Morishige
Akira
A
Tokumura
Mitsuo
M
Jisaka
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000378:metabolism; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D004530:Egg Yolk; D005227:Fatty Acids / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008041:Linoleic Acids / Q000378:metabolism; D008054:Lipid Peroxides / Q000032:analysis* / Q000378:metabolism; D008084:Lipoxygenase / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D010084:Oxidation-Reduction; D010636:Phenols; D013454:Sulfoxides; D014992:Xylenes / Q000378:metabolism*
D005227:Fatty Acids; D005290:Ferric Compounds; D005456:Fluorescent Dyes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D010636:Phenols; D013454:Sulfoxides; D014992:Xylenes; D015117:5,8,11,14-Eicosatetraynoic Acid; C101084:lipoxygenase L-1; D008084:Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; C016833:xylenol orange