Measurement of Lipid Hydroperoxides by the Ferric-Xylenol Orange Method (2) Application to Lipoxygenase Assay

Abstract

SummaryThe ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe 3 ϩ ) 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/Fe 3 ϩ -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.

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“…However, the absorbance spectrum of NiO in combination with R6G molecules displayed a slight blue shift for the peaks at 346 and 485 nm. This is indicative of charge transfer from NiO to R6G molecules. − The charge-transfer phenomena were also investigated by acquiring the Raman spectra of R6G molecules at 532 and 785 nm. The multiwavelength spectra are shown in Supplementary Figure S1B.…”

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“…Glutathione (GSH) was measured according to the method of Fukuzawa and Tokumura . Next, 200 µl of supernatant was added to 1.1 ml of 0.25 M sodium phosphate buffer (pH 7.4) followed by the addition of 130 µl DTNB 0.04%.…”

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“…Xylenol orange or 3,39‐Bis[ N,N ‐bis‐(carboxymethyl)‐aminomethyl]‐o‐cresolsulfonephthalein is a molecule that can be employed for the spectrophotometric assay of hydroperoxides, both lipid and water soluble (Fukuzawa et al., ; Gay & Gebicki, , ; Reis et al., ; Takci et al., ), as well as for the assessment of total redox status in biological samples (Fukuzawa et al., ). The FOX method is based on the generation of a xylenol orange–Fe 3+ complex, as the reagent's Fe 2+ is oxidized upon interacting with the sample‐contained oxidants (Fukuzawa et al., ; Nourooz‐Zadeh, ; Reis et al., ). This is a sensitive and accurate method, with good stability to light/oxygen and with high reproducibility (Gay & Gebicki, ; Mehta, Darji, & Aparnathi, ; Orefice et al., ; Yu, Wang, Zhou, Zhao, & Qiu, ).…”