FOX Assay Research Articles

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions. This study aimed to enhance the suitability of the ferrous-oxidized xylenol orange (FOX) assay for screening LOX activity across a wide pH range with different PUFAs. The narrow linear detection range of the standard FOX assay restricts its utility in screening LOX activity. To address this, the concentration of perchloric acid in the xylenol orange reagent was adjusted. The modified assay exhibited a fivefold expansion in the linear detection range for hydroperoxides and accommodated samples with pH values ranging from 3 to 10. The assay could quantify various hydroperoxide species, indicating its applicability in assessing LOX substrate preferences. Due to sensitivity to pH, buffer types, and hydroperoxide species, the assay required calibration using the respective standard compound diluted in the same buffer as the measured sample. The use of correction factors is suggested when financial constraints limit the use of FAHP standard compounds in routine LOX substrate preference analysis. FAHP quantification by the modified FOX assay aligned well with results obtained using the commonly used conjugated diene method, while offering a quicker and broader sample pH range assessment. Thus, the modified FOX assay can be used as a reliable high-throughput screening method for determining LOX activity. KEY POINTS: • Modifying perchloric acid level in FOX reagent expands its linear detection range • The modified FOX assay is applicable for screening LOX activity in a wide pH range • The modified FOX assay effectively assesses substrate specificity of LOX.

ROS Detection and Quantification in Plant-Nematode Interactions.

Reactive oxygen species (ROS) accumulation is one of the earliest hallmarks upon successful pathogen recognition in plants. H2O2 is considered the most important ROS in plant defense considering its relatively high stability and capacity to cross long distances in the plant. However, ROS also play roles in cell development and could hence facilitate nematode feeding site development. Several methods to analyze the cellular redox state exist, among which ROS detection and quantification and the evaluation of ROS scavenging enzyme activity (peroxidase activity, catalase activity, etc.). Here, we describe DAB staining, which is used to detect and localize ROS in planta upon an external trigger. Furthermore, ROS quantification using the FOX assay is described. Both methods have been used extensively in research and yield repeatable results in various plants.

Metformin Reverses the Effects of Angiotensin 2 in Human Mammary Arteries by Modulating the Expression of Nitric Oxide Synthases

Abstract Angiotensin 2 impairs vascular function by activation of reactive oxygen species (ROS) production and development of endothelial dysfunction. Metformin, the first-line therapeutic agent for type 2 diabetes mellitus, has vascular protective properties, beyond its glucose lowering effects. The aim of the present study was to investigate the interaction between metformin and angiotensin 2 in human internal mammary arteries harvested from patients with coronary heart disease undergoing revascularization procedure, by evaluation of vascular function, reactive oxygen species (ROS) production and the gene expression of nitric oxide (NO) synthases (endothelial – eNOS, neuronal – nNOS and inducible – iNOS). To this aim, vascular samples were incubated with angiotensin 2 (Ang2, 12 h) with/without metformin (Metf, 10 μM) and used for ROS measurement (FOX assay), vascular reactivity in organ bath (contractility to phenylephrine, relaxation to acetylcholine, contractility to NG-nitro-L-arginine methyl ester/L-NAME) and RTPCT studies. Acute incubation of the vascular rings with Ang2 impaired vascular reactivity (increase contractility, decrease relaxation), increased ROS production, supressed eNOS/nNOS and increased iNOS mRNA expression. Ex vivo incubation with metformin at a clinically relevant concentration reversed all these effects. These data suggest that Metformin might be useful in alleviating endothelial dysfunction by improving the endothelial-dependent relaxation and mitigating oxidative stress in clinical setting associated with cardiovascular disease regardless the presence of impaired glucose metabolism.

Mitigation of Monoamine Oxidase-Related Oxidative stress by Metformin and Empagliflozin in human atrial tissue

Both old and novel antidiabetic drugs have been reported to elicit cardioprotective, partially characterized pleiotropic effects. The present pilot study was aimed at assessing the effects of metformin (a biguanide) and empagliflozin (a SGLT2 inhibitor) on monoamine oxidase (MAO) A and B-related reactive oxygen species (ROS) production and vascular reactivity in atrial tissue isolated from patients subjected to elective cardiac surgery. Samples of atrial tissue were harvested during the surgical procedures, transferred to the laboratory and used for the: reactive oxygen species (ROS) measurements (immune-fluorescence, spectrophotometry–FOX assay) and assessment of MAO expression (qRT-PCR and immune-fluorescence) after incubation with metformin (10 μM, 12 h) and/or empagliflozin (10 μM, 12 h) in the presence vs absence of angiotensin 2 (Ang2, 100 nM), lipopolysaccharide (LPS, 1 μg/ml) and glucose (400 mg/dL). MAO-A and B are expressed in the atrial samples and acute incubation with Ang2, LPS and high glucose increased MAO expression and ROS production. All effects were alleviated in the presence of either antidiabetic drug. No cumulative effect was obtained when the drugs were added together. MAO-A and B are expressed in the human atrial tissue, up-regulated after ex-vivo stimulation with angiotensin 2, lipopolysaccharide and glucose and responsible for increased oxidative stress that was mitigated in the presence of either metformin or empagliflozin.

Formulations of selected Energy beverages promote pro-oxidant effects of ascorbic acid and long-term stability of hydrogen peroxide

Recently, autoxidation mediated by ascorbic acid (AA) and other ingredients, has been implicated in generation of hydrogen peroxide (H2O2) in so-called Energy beverages. Here, we report the use of cyclic voltammetry and the FOX assay to monitor at short and long incubation times, respectively, the production and stability of H2O2 generated by AA and redox-active ingredients. Levels of H2O2 in Energy drinks (36.5 ± 4.0 µM at 4 °C and 64.2 ± 7.6 µM at 20 °C) were found to be stable or increased (p < 0.05) upon vessel opening. A predictive model for the production of H2O2 as a function of AA concentration, temperature and incubation time, and depending on ingredients present, indicated that H2O2 peaked at 91–726 µM after 1 day and declined to ∼ 42–60 µM (4 °C) or zero after ∼10 days. The research supports that levels of H2O2 in beverages containing anti-oxidant mixtures and dissolved oxygen should be monitored and formulations modified to avoid AA autoxidation.

Relationship between oxidative stress and three-dimensional echocardiographic changes in myxomatous mitral valves with severe regurgitation - a pilot study

Abstract Funding Acknowledgements Type of funding sources: None. Background. Reactive oxygen species (ROS) have been reported to contribute to the molecular pathogenesis of severe mitral regurgitation induced by myxomatous degeneration. Data about oxidative stress and the possible relationship with the extent of prolapsing and/or flail elements is scarce in the literature. The pathogenic mechanism may prompt the development of therapeutic approaches aimed at delaying extensive valvular alteration. Purpose. The aim of the study is to investigate the possible correlation between ROS expression in myxomatous mitral valves with severe regurgitation and the extent of valvular alteration. Material and methods. Samples from myxomatous mitral valves with ruptured chordae and severe regurgitation were harvested during surgical intervention for valve repair/replacement from 9 patients. Besides transthoracic echocardiography, all patients were evaluated before surgery by transesophageal echocardiography (TEE) with three-dimensional (3D) reconstruction of the mitral valve, using 3D zoom mode acquisition with qualitative and quantitative analysis through the 4D Auto MVQ technology. Tissue from the valvular samples was further analysed for the assessment of ROS by 2 methods: spectrophotometry (ferrous oxidation xylenol orange - FOX assay) and confocal microscopy (dihydroethidium staining). Results. Patients had a mean age of 51.88 ± 13.54 years, with a left ventricular ejection fraction of 62.55 ± 7.33%, left ventricular end-diastolic diameter of 5.4 ± 0.72cm and end-diastolic volume of 168.55 ± 62.57ml, a mitral annulus area of 17.91 ± 5.74 cm2 and a number of prolapsing/flail scallops varying between 1 and 5 (average of 2 ± 1 scallops). All patients had diffuse thickening of the mitral leaflets with at least one primary and one secondary ruptured chordae and flail of the P2 scallop. Two patients had significant prolapse of at least 2 anterior mitral leaflet scallops and three patients had supplemental significant prolapse and/or flail of at least 1 posterior scallop, besides flail of P2. The mean value for ROS in the valvular tissue was 9.46 ± 2.03nM H2O2/mg tissue/h at FOX assay. A significant positive correlation between the ROS values and the extent of prolapsing/flail segments was found (R = 0.67, p = 0.04). Conclusion. Besides the diagnosis and accurate preoperatory valve description, 3D TEE along with molecular investigations contribute to the understanding of myxomatous mitral valves’ pathogenesis. Oxidative stress increased in the mitral valves with advanced myxomatous degeneration. Whether this occurs as a cause or a consequence contributing to the disease progression as well as the sources of ROS are worth further investigation.

Evaluation of free radical scavenging and anti-lipoxygenase activity in various fractions of ayurvedic polyherbal decoction, Punarnavadi kashayam

Punarnavadi kashayam (PK), a classical ayurvedic polyherbal decoction prescribed for abating various inflammatory conditions, was analysed for its total phytochemical content, free radical scavenging, and anti-inflammatory activity in order to validate its traditional usage. PK was sequentially fractionated using solvents of different polarity and the preliminary phytochemical screenings were performed. Free radical scavenging activity was determined using various biochemical assays while the anti-inflammatory potential was evaluated by determining the anti-lipoxygenase activity using FOX assay. Ethyl acetate fraction showed better free radical scavenging activity as compared to the corresponding positive controls. The same fraction exhibited the better IC 50 for 5- and 12-lipoxygenase inhibition assay than the positive control, showing its anti-inflammatory property. The phytochemical analysis revealed a higher content of phytochemicals in ethyl acetate, dichloromethane and methanol fractions which displayed the correlation between phenolic content and the bioactivity. Overall, PK can effectively scavenge free radicals and inhibit lipoxygenase enzymes, thereby decreasing the production of free radicals and pro-inflammatory leukotrienes.

DETECTION OF HYDROPEROXIDES IN SOLUTIONS OF PHOTOOXIDIZED PSORALEN

Photooxidized psoralen solutions possess a variety of biological effects, which implementation mechanism may presumably involve hydroperoxides. Here, the hydroperoxide content in photooxidized psoralen solutions was assessed using photometric FOX assay (from Ferrous Oxidation + Xylenol Orange). FOX reagent with 10× content of Xylenol Orange, modified for quantitative analysis of up to 50 μM of hydroperoxides in aqueous phase was used in experiments. During photooxidation of 0.1 mM psoralen in phosphate buffer solution, hydroperoxide production increases with dose of UVA irradiation (~2.5 μM eq. of H2O2 for dose of 252 kJ/m2 and ~11 μM eq. of H2O2 for dose of 1512 kJ/m2) and reaches ~16.5 μM eq. of H2O2 at the highest dose investigated (3024 kJ/m2). A comparison of kinetics of psoralen photolysis and hydroperoxide generation allows us to suggest that generation of hydroperoxide results from the secondary photochemical processes involving psoralen photoproducts, presumably from photoinduced autooxidation of aldehydic photoproducts of psoralen.

Modulation of proinflammatory cytokines and enzymes by polyherbal formulation Guggulutiktaka ghritam

BackgroundGuggulutiktaka ghritam is an ayurvedic medicine which has been traditionally used to treat various chronic inflammatory conditions. However, the mechanism of action of the Ayurvedic medication in control of inflammatory conditions has not been clearly evaluated. ObjectiveIn the current study, the effect of the Guggulutiktaka ghritam extract (GTG) on the lipoxygenase pathway and in the production of proinflammatory cytokines involved in the pathogenesis of chronic inflammation was studied. Materials and methodsThe effect of GTG in the production of leukotriene was determined by enzyme inhibition studies on 12- lipoxygenase. The assay was carried out by ferrous oxidation of xylenol orange (FOX assay) and was compared to a positive control nordihydroguaiaretic acid. The effect of GTG on the production of proinflammatory cytokines TNF-α and IL-1β in monocytes were studied. For this, the monocytes were pretreated with various concentrations of GTG and subsequently stimulated with lipopolysaccharide. The cytokines TNF-α and IL-1β produced were quantified by ELISA and the results were compared to positive controls Rolipram and Dexamethasone respectively. The gene expression studies were carried out using qRT-PCR. The IC50 values were calculated and evaluated statistically. ResultsThe result indicates that GTG in comparison to the positive control Nordihydroguaiaretic acid significantly reduced the activity of 12- lipoxygenase. Also, there was significant inhibition in the production of proinflammatory cytokines in LPS stimulated monocytes pretreated with GTG as compared to positive control Rolipram and Dexamethasone. There was significant downregulation of IL-1β gene in LPS stimulated monocytes pretreated with GTG as compared to control. These changes are further supported by Raman spectra obtained for GTG treated and untreated cells. ConclusionThe study revealed that GTG is a leukotriene and cytokine inhibitor. The inhibition in the production of cytokines may be due to the down-regulation of genes for TNF-α and IL-1β. The study provides a scientific validation on the possible anti-inflammatory mechanism of action of this traditionally used medicine. Identification of bioactive molecules would aid in developing newer therapeutics for control of chronic inflammation.

Age‐ and Muscle‐Specific Oxidative Stress Management Strategies in a Long‐Lived Diver, the Weddell Seal

The free radical theory of aging purports that the accumulation of cellular oxidative damage leads to aging. While this process is not expected to manifest in wild populations, which can suffer from early predation or injury‐induced mortality, physically active long‐lived mammals are a model in which cellular aging may occur. Air breathing divers such as seals experience repeated bouts of ischemia‐reperfusion, potentially exposing their tissues to oxidative stress and thus damage and accelerated aging. We have previously demonstrated that skeletal muscle cellular morphology “ages” in Weddell seals without a corresponding decrease in diving behavior. This suggests stress response may protect against oxidative damage and functional impairment, providing insight into mechanisms for managing oxidative stress. We studied whether muscular senescence might arise from oxidative damage by comparing muscle from old (age 17–27 years) and young (age 9–16 years) cohorts of adult Weddell seals. Muscle biopsies were collected from healthy, active, non‐reproductive seals and frozen in liquid nitrogen. Apoptosis was examined with a cell‐death ELISA, while markers of oxidative damage to lipids (FOX assay) and proteins (4‐HNE western blot) were contrasted with antioxidant profiles (kinetic activity assays or western blots). These variables were statistically compared between age cohorts with an ANOVA model that included animal sex and biopsy site as cofactors. Old seals exhibited a 43% increase (p = 0.002) in apoptosis over young animals. Lipid peroxidation and oxidative protein damage, however, did not differ with age, suggesting that apoptotic differences were not due to oxidative stress. We also assessed antioxidant profiles and found that glutathione peroxidase (GPx) activity was 22% higher in the old cohort (p = 0.026). GPx reduces lipid hydroperoxides; this enhanced antioxidant protection may limit lipid peroxidation and downstream protein modification in old seals. The locomotory (longissimus dorsi, LD) and maneuvering (pectoralis, P) muscles of Weddell seals are perfused differently from one another during a dive, thus we also considered whether oxidative damage and antioxidant patterns are distinct between muscles using the same ANOVA. Lipid peroxidation was highest in LD (24% increase, p = 0.001), though no difference in protein modification was detected. Antioxidant protein levels revealed increased superoxide dismutase (SOD2, p = 0.009), thioredoxin reductase (TRXR2, p = 0.027) and heat shock protein 70 (HSP70, p = 0.00001) in LD. On the other hand, P exhibited a 14% increase in glutathione S‐transferase (GST, p = 0.004), which is consistent with regionally lower levels of oxidized lipid. Together, these findings illustrate that long‐lived air breathing divers display enhanced antioxidant and stress responses, possibly utilizing the pre‐conditioning effect of repeated diving. Thus, the aging Weddell seal is a compelling model for studying the successful management of oxidative stress.Support or Funding InformationNational Science Foundation Office of Polar ProgramsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Ionic Functional Groups on the Oxidation State and Interfacial Binding Property of Catechol-Based Adhesive.

Adhesive hydrogels were prepared by copolymerizing dopamine methacrylamide with either acrylic acid (AAc) or N-(3-aminopropyl)methacrylamide hydrochloride (APMH). The effect of incorporating the anionic and cationic side chains on the oxidation state of catechol was characterized using the FOX assay to track the production of hydrogen peroxide byproduct generated during the autoxidation of catechol, and the interfacial binding property of the adhesive was determined by performing Johnson-Kendall-Roberts contact mechanics tests tested over a wide range of pH values (pH 3.0-9.0). The ionic species contributed to interfacial binding to surfaces with the opposite charge with measured work of adhesion values that were comparable to or in some cases higher than those of catechol. Addition of AAc minimized the oxidation of catechol even at a pH of 8.5 and correspondingly preserved the elevated adhesive property of catechol to both quartz and amine-functionalized surfaces. However, AAc lost its buffering capacity at pH 9.0, and catechol was oxidized at this pH. On the other hand, catechol formed a cohesive covalent bond with the network-bound amine side chain of APMH at basic pH, which interfered with the interfacial binding capability of APMH and the catechol.

Interference of single walled carbon nanotubes (SWCNT) in the measurement of lipid peroxidation in aquatic organisms through TBARS assay

Nanomaterials (NM) exhibit unique properties due their size and relative area, but the mechanisms and effects in the living organisms are yet to be unfold in their totality. Potential toxicity mechanisms concerning NM as carbon nanotubes include oxidative stress generation. Several fluorimetric and colorimetric methods have been systematically used to measure NM toxicity, and controversial results have been reported. One of the problems can be related to the interference effects induced by NM, leading to artifacts that can lead to misleading conclusions. In present study, it was performed in vitro assays with two aquatic species: the zebrafish Danio rerio and the polychaete Laeonereis acuta to evaluate the potential interference capacity of single-wall carbon nanotubes (SWCNT) in a fluorometric method (TBARS assay) to measure lipid peroxidation. Obtained results indicated that gills and brain of zebrafish presented a lowered fluorescence only at extremely high concentrations (50 and 500mg/L). Determinations in anterior, middle, and posterior body regions of L. acuta showed a quite different pattern: high fluorescence at low SWCNT concentrations (0.5mg/L) and lowering at the highest (500mg/L). To eliminate matrix effect of biological samples, tests employing the standard for TBARS assay, 1,3,3-tetramethoxipropane, were run and the results showed again higher fluorescence values at low concentrations (0.5–5mg SWCNT/L), a technique artifact that could lead to misleading conclusions since higher fluorescence values implicate higher TBARS concentration, implying oxidative stress. Using the colorimetric FOX assay with cumene hydroperoxide as standard presented remarkable better results since no artifacts were observed in the same SWCNT concentration range that employed with the TBARS technique.

Sensitive Photonic System to Measure Oxidative Potential of Airborne Nanoparticles and ROS Levels in Exhaled Air

A photonic system has been developed that enables sensitive quantitative determination of reactive oxygen species (ROS) – mainly hydrogen peroxide (H2O2) – in aerosol samples such as airborne nanoparticles and exhaled air from patients. The detection principle relies on the amplification of the absorbance under multiple scattering conditions due to optical path lengthening [1,2]. In this study, the presence of cellulose membrane that acts as random medium into the glass optical cell considerably improved the sensitivity of the detection based on colorimetric FOX assay (FeII/orange xylenol). Despite the loss of assay volume (cellulose occupies 75% of cell volume) the limit of detection is enhanced by one order of magnitude reaching the value of 9nM (H2O2 equivalents). Spectral analysis is performed automatically with a periodicity of 5 to 15 s, giving rise to real-time ROS measurements. Moreover, the elution of air sample into the collection chamber via a micro-diffuser (impinger) enables quantitative determination of ROS contained in or generated from airborne samples. As proof-of-concept the photonic ROS detection system was used in the determination of both ROS generated from traffic pollution and ROS contained in the exhaled breath as lung inflammation biomarkers.

Estradiol prevents olfactory dysfunction induced by A-β 25–35 injection in hippocampus

BackgroundSome neurodegenerative diseases, such as Alzheimer and Parkinson, present an olfactory impairment in early stages, and sometimes even before the clinical symptoms begin. In this study, we assess the role of CA1 hippocampus (structure highly affected in Alzheimer disease) subfield in the rats’ olfactory behavior, and the neuroprotective effect of 17 beta estradiol (E2) against the oxidative stress produced by the injection of amyloid beta 25–35.Results162 Wistar rats were ovariectomized and two weeks after injected with 2 μl of amyloid beta 25–35 (A-β25–35) in CA1 subfield. Olfactory behavior was evaluated with a social recognition test, odor discrimination, and search tests. Oxidative stress was evaluated with FOX assay and Western Blot against 4-HNE, Fluoro Jade staining was made to quantify degenerated neurons; all these evaluations were performed 24 h, 8 or 15 days after A-β25–35 injection. Three additional groups treated with 17 beta estradiol (E2) were also evaluated. The injection of A-β25–35 produced an olfactory impairment 24 h and 8 days after, whereas a partial recovery of the olfactory behavior was observed at 15 days. A complete prevention of the olfactory impairment was observed with the administration of E2 two weeks before the amyloid injection (A-β25–35 24 h + E2) and one or two weeks after (groups 8 A-β +E2 and 15 A-β +E2 days, respectively); a decrease of the oxidative stress and neurodegeneration were also observed.ConclusionsOur finding shows that CA1 hippocampus subfield plays an important role in the olfactory behavior of the rat. The oxidative stress generated by the administration of A-β25–35 is enough to produce an olfactory impairment. This can be prevented with the administration of E2 before and after amyloid injection. This suggests a possible therapeutic use of estradiol in Alzheimer’s disease.

A modified ferrous oxidation-xylenol orange assay for lipoxygenase activity in rice grains

Ferrous oxidation-xylenol orange assay reagent was reformulated by using spectral analysis of ferric–xylenol orange complex to detect low concentrations of lipoxygenase rice grain products. Reducing the levels of ferrous sulphate and xylenol orange in the FOX reagent enabled the detection of low concentrations of hydroperoxy fatty acid derived from lipoxygenase activity in the range of 0.1–1.5μM. Protein, substrate and time courses of the modified FOX assay were studied to determine lipoxygenase activity in rice grain. The assay was also applicable as a high throughput technique for comparisons of lipoxygenase activity from various rice varieties. This has important implications for rapid screening for low-lipoxygenase containing rice cultivars in rice breeding program and grain quality during storage.

Selective Cytotoxicity of a Grape Seed Proanthocyanidin Extract to Human Oral Carcinoma HSC-2 Cells

Grape seed extract (GSE), a nutraceutical rich in polyphenol proanthocyanidins, was evaluated for its toxicity to human oral cells. Using the neutral red cytotoxicity assay, human squamous carcinoma (HSC-2) cells were shown to be more sensitive to GSE than were gingival fibroblasts. Polyphenols undergo auto-oxidation reactions in cell culture medium, to generate reactive oxygen species, in particular, hydrogen peroxide. Using the FOX assay to quantify hydrogen peroxide, GSE, when added to cell culture medium, generated hydrogen peroxide, albeit at relatively low levels. No hydrogen peroxide was detected in the presence of catalase, which catalyzes the decomposition of hydrogen peroxide, and minor levels were detected in the presence of superoxide dismutase, which stabilizes polyphenols. Superoxide free radical, detected with nitroblue tetrazolium, was identified in GSE-amended medium. Focusing on carcinoma HSC-2 cells, the 24-hr toxicity of GSE was unaffected by the hydrogen peroxide scavengers, catalase and pyruvate, indicating that hydrogen peroxide played no role in toxicity. For HSC-2 cells cotreated with GSE and D,L-buthionine-[S,R]-sulfoximine, a depleter of intracellular glutathione, no potentiation of toxicity occurred. Over the 24 hr toxicity range, GSE did not affect the level of intracellular glutathione; however, some depletion occurred, but only at elevated GSE concentrations. GSE toxicity, apparently, was independent of oxidative stress. The 24-hr cytotoxicity of GSE to HSC-2 cells was potentiated in the presence of SOD, indicating that the proanthocyanidins per se, rather than their auto-oxidation products, accounted for toxicity. To confirm the instability of GSE in cell culture medium with a concomitant lowering of potency, studies compared medium freshly-amended with GSE to “spent” medium, i.e., GSE-amended medium left in the incubator for 24 hr prior to usage. The potency of GSE to HSC-2 cells was significantly lowered following incubated in “spent” medium for 24 hours.

Use of a Ferrous Oxidation-Xylenol Orange (FOX) Assay to Determine Lipoxygenase Activity in Barley and Malt

The objective of this research work was to develop a new high-throughput screening method for lipoxygenase (LOX) activity, which was based on the determination of lipid hydroperoxides by ferrous oxidation-xylenol orange (FOX) assay. The final method employed extraction of a finely ground sample at a grist/buffer ratio of 1:20 for 30 min at 4°C. Extracts were incubated with linoleic acid for 5 min at 25°C, and the hydroperoxides formed were determined with the FOX reagent. Up to 100 samples can be analyzed per day with this method. The new method showed good reproducibility (coefficient of variation = 1.4–6.5%) and accuracy, although there are some disadvantages and limitations compared with the traditional UV assay. The FOX assay principal may be useful to barley breeders for screening large numbers of progeny, and also would have value to maltsters and brewers when conducting research work on LOX or performing routine quality assurance.

Estimation of Lipid Hydroperoxide Levels in Tropical Marine Macroalgae.

The incipient levels of lipid hydroperoxides (LHPOs) were determined in selected green, brown, and red macroalgae by the FOX assay using hydroperoxy HPLC mix. The LHPOs contents varied between the investigated species and showed relatively low values in this study. Among the greens, it varied from 12±6.2μg·g(-1) (Codium sursum) to 31.5±2.8μg·g(-1) (Ulva lactuca), whereas in reds, from 5.7±1.6μg·g(-1) (Gracilaria corticata) to 46.2± 6μg·g(-1) (Sarconema filiforme), and in browns, from 4.6±4.4μg·g(-1) (Dictyota bartayresiana) to 79±5.0μg·g(-1) (Sargassum tenerrimum), on fresh weight basis. These hydroperoxides represented a minor fraction of total lipids and ranged from 0.04% (S.swartzii) to 1.1% (S.tenerrimum) despite being a rich source of highly unsaturated fatty acids. The susceptibility of peroxidation was assessed by specific lipid peroxidazibility (SLP) values for macroalgal tissues. The LHPO values were found to be independent of both the PUFAs contents and their degree of unsaturation (DBI), as evident from the PCA analysis. SLP values were positively correlated with the LHPOs and negatively with DBI. The FOX assay gave ≥20-fold higher values for LHPOs as compared to the TBARS method for all the samples investigated in this study. Furthermore, U.lactuca cultured in artificial seawater (ASW) enriched with nutrients (N, P, and NP) showed a sharp decline in LHPOs contents relative to those cultured in ASW alone P≤0.05. It is inferred from this study that the FOX assay is an efficient, rapid, sensitive, and inexpensive technique for detecting the incipient lipid peroxidation in macroalgal tissues.

Abstract 610: The role of oxidative stress in the growth inhibitory and proapoptotic effects of brown button mushrooms (Agaricus bisporus) in human prostate cancer cells

Abstract An increasing body of clinical and in vivo/in vitro studies suggests that some chemical components in specialty mushrooms like maitake (Grifola frondosa) may exert cancer preventive effects. Edible mushrooms, which have a similar content of polysaccharides, proteoglycans, steroids, etc., have been less well-studied for their anticancer and cancer preventive activity. For example, only one publication has reported the inhibition of human breast cancer cell growth by brown button mushrooms (Agaricus bisporus). The objectives of our research were to evaluate the anti-prostate cancer activity and mechanism(s) of action of brown button mushroom extracts. A hot water extract was prepared from freeze-dried brown button mushroom powder and fractioned with hexane and ethyl acetate. The IC50 of ethyl acetate fraction from brown button mushrooms (BBEA) was ∼0.1mg/ml. The inhibitory potency against LNCaP human prostate cancer cells correlated with the total phenol content, suggesting that these may be the active anticancer components. To further investigate the mechanisms of BBEA's anticancer effects, we examined the production of H2O2 by the extract using FOX assay and examined oxidative stress, proapoptotic, and cell cycle arrest markers by western blot. BBEA (0.1mg/ml) produced ∼50µM H2O2 under cell culture conditions after 6h. The expression of the oxidative stress marker, αH2A.X, in LNCaP cells was enhanced by BBEA after 12 and 24h, suggesting that pro-oxidative effects may be a possible anticancer mechanism. BBEA also increased caspase-3 and PARP cleavage and reduced the expression of Cyclin D1 and Cyclin E2, suggesting induction of apoptosis and cell cycle arrest by BBEA. In summary, this is the first report of the anticancer effects of phenolic compounds in brown button mushrooms and these results demonstrate that further study of BBEA is warranted. Microarray studies are currently under way to better characterize the anticancer activity of BBEA against human prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 610. doi:1538-7445.AM2012-610

Development of Estimation Methods of Skin Oxidation and Evaluation of Anti-Oxidative Effects of Genistein in Topical Formulations

The objective of the present study was to establish the method of measurement of hydrogen peroxide and to estimate the anti-oxidative effect of genistein in the skin. UVB induced skin oxidation and anti-oxidative effect of genistein formulations were evaluated by determining levels of hydrogen peroxide. The mechanism involved in the determination of hydrogen peroxide is based on a color reaction between ferric ion (Fe3+) and xylenol orange, often called FOX assay and subsequent monitoring of absorbance values of the reactant at 540 nm. The reaction was to some extent pH-dependent and detection sensitivity was greatest at pH 1.75. Genistein liposomal gel demonstrated better anti-oxidative effect with regard to lowering hydrogen peroxide levels elevated by UVB irradiation compared to genistein-suspended gel. A linear relationship has been observed between anti-oxidative effect of genistein and drug deposition in the skin tissue. Genistein liposomal gel resulting in the localization of the drug in the deeper skin led to improved anti-oxidative effect compared to genistein gel. The suggested method for evaluation of oxidation of the skin can be used as a tool to screen effective anti-oxidative agents and their delivery systems acting on the skin.