Ferrous Oxidation-xylenol Orange Assay Research Articles

Modulation of M1/M2 Cytokines and Inflammatory Enzymes by Persicaria Species Leaf Extracts in Lipopolysaccharide-stimulated RAW 264.7 Cell Macrophages.

Investigating the impacts of plant-based substances on the regulation of pro-inflammatory M1 and anti-inflammatory M2 cytokines could have significant implications for immune-related health conditions. Seven Persicaria plant species from sub-Saharan Africa were specifically selected for analysis, based on their traditional use in treating inflammation. To investigate the inhibitory effects of methanol leaf extracts from selected plants on enzymes involved in chronic inflammation. The inhibition of nitric oxide production, acetylcholinesterase activity, and 15-lipoxygenase activity was assessed using the Griess reagent method, Ellman's colorimetric method, and the ferrous oxidation-xylenol orange assay. The quantity of M1/M2 cytokines released was quantified using a flow cytometer. At a concentration of 50 µg/mL, the methanol extracts of P. limbata exhibited the highest NO inhibition (97.67%), followed by P. nepalensis (93.06%) and P. setosula (92.78%). The NO inhibition caused by the plant extracts was correlated directly with the decrease in NO release by the LPS-stimulated macrophages. Furthermore, the pro-inflammatory enzyme assays indicated that the methanol extracts of P. setosula exhibited the highest enzyme inhibitory activity (LOX 89.59%, AChE 72.12 %). This was followed by P. limbata (with 92.76% for LOX and 56.93% for AChE) and P. nepalensis (with 88.16% for LOX and 47.17% for AChE). Cytokine assays revealed that the extracts of P. limbata had significant dose-dependent suppressive effects on IFN-γ and TNF-α expression while promoting the secretion of IL-2, IL-4, IL-6, and IL-10. Extracts of P. limbata contain immunomodulatory compounds that could be further explored as potential remedies to target the molecular drivers of chronic inflammation.

Naturally occurring benzophenones and xanthones from Garcinia smeathmannii (Planch. & Triana) Oliv. displayed anti-inflammatory effects by modulating the activities of inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

Introduction: There is a growing interest in studying natural products for the identification of novel lead compounds for drug development for treating inflammatory diseases. Although some studies have focused anti-inflammatory activity of benzophenones and xanthones, exploring additional targets such as enzymes and cytokines, involved in their inflammatory response could provide more comprehensive understanding of the compounds' anti-inflammatory effects. In this study, four xanthones ananixanthone (1), smeathxanthone A (2), smeathxanthone B (3), and 1,3,5,8-tetrahydroxy-2-(3-methybut-2-enyl)-4-(3,7-dimethyloct-2,6-dienyl) xanthone (4); and three benzophenones guttiferone O (5), guttiferone M (6), and aristophenone A (7) from Garcinia smeathmannii (Planch. & Triana) Oliv. were investigated for their effect on nitric oxide production, cyclooxygenase, lipoxygenase inhibition, and Th1/Th2 cytokines production in activated RAW 264.7 macrophages. Methods: The Griess reagent method and the ferrous oxidation-xylenol orange assay were used to evaluate the inhibition of NO production and the 15-lipoxygenase activity respectively. Cyclooxygenase activity was assessed using the fluorometric COX activity assay kit and measurement of Th1/Th2 cytokines was performed using a flow cytometer. Results: All the tested compounds exhibited a dose-dependent inhibition of NO production with varying degrees of inhibitory effects on 15-LOX activity. Compound (6), displays the best inhibitory effect on COX-1/COX-2 activity. A general trend of the tested compounds on cytokines profiles revealed that compound (5) showed a pronounced enhancement of anti-inflammatory cytokines (IL-4 and IL-10). Conclusion: This observation supports future exploration of ananixanthone (1), guttiferone O (5), and guttiferone (6) as potential candidates for the development of anti-inflammatory drugs.

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.

Natural Compounds Isolated from African Mistletoes (Loranthaceae) Exert Anti-Inflammatory and Acetylcholinesterase Inhibitory Potentials: In Vitro and In Silico Studies

Despite the medicinal uses of Phragmanthera capitata, Globimetula dinklagei and Tapinanthus bangwensis against memory loss, convulsions and pain, their efficacy against brain-dysfunction diseases and inflammation models has not yet been studied. Therefore, this study aims to investigate the anti-inflammatory and acetylcholinesterase (AChE) inhibitory potentials of their crude extracts and isolated natural compounds by combining in vitro and in silico experiments. Crude extracts and isolated compounds were tested in vitro for their AChE inhibitory activity by using Ellman’s method. Additionally, their anti-inflammatory activity was determined by evaluating the nitric oxide (NO) production inhibitory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells and the 15-lipoxygenase (15-LOX) inhibitory activity by using the ferrous oxidation xylenol orange assay. Furthermore, the in silico efficacy of natural compounds was investigated against ten putative target enzymes relevant in Alzheimer’s disease (AD) pathogenesis and inflammation. It was found that the crude extracts had weak to moderate inhibitory potential against AChE, with the extract of T. bangwensis being the most active (50% inhibitory concentration (IC50) = 48.97 µg/mL). Six natural compounds, namely, 3-O-β-D-glucopyranosyl-28-hydroxy-α-amyrin (2), apigenin- 8-C-β-D-glucopyranoside (3), globimetulin B (5), globimetulin C (6), bangwaursene B (8) and 3β-acetoxy-11,12-epoxytaraxerol (9), were identified for the first time as having anti-AChE potential, among which (9) had the highest efficacy with an IC50 of 13.89 µM. Among the anti-AChE compounds, (5) was also efficient against NO production and 15-LOX, and the data are in agreement with the docking score. In summary, compounds (5) and (9) are the most prominent lead compounds that should be further tested experimentally against molecular targets of AD and inflammation.

Moxonidine Increases Uptake of Oxidised Low-Density Lipoprotein in Cultured Vascular Smooth Muscle Cells and Inhibits Atherosclerosis in Apolipoprotein E-Deficient Mice.

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE-/- mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE-/- mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper.

Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.

Formation of \u03b1-tocopherol hydroperoxide and \u03b1-tocopheroxyl radical: relevance for photooxidative stress in Arabidopsis

Tocopherols, lipid-soluble antioxidants play a crucial role in the antioxidant defense system in higher plants. The antioxidant function of α-tocopherol has been widely studied; however, experimental data on the formation of its oxidation products is missing. In this study, we attempt to provide spectroscopic evidence on the detection of oxidation products of α-tocopherol formed by its interaction with singlet oxygen and lipid peroxyl radical. Singlet oxygen was formed using photosensitizer rose bengal and thylakoid membranes isolated from Arabidopsis thaliana. Singlet oxygen reacts with polyunsaturated fatty acid forming lipid hydroperoxide which is oxidized by ferric iron to lipid peroxyl radical. The addition of singlet oxygen to double bond carbon on the chromanol head of α-tocopherol forms α-tocopherol hydroperoxide detected using fluorescent probe swallow-tailed perylene derivative. The decomposition of α-tocopherol hydroperoxide forms α-tocopherol quinone. The hydrogen abstraction from α-tocopherol by lipid peroxyl radical forms α-tocopheroxyl radical detected by electron paramagnetic resonance. Quantification of lipid and protein hydroperoxide from the wild type and tocopherol deficient (vte1) mutant Arabidopsis leaves using a colorimetric ferrous oxidation-xylenol orange assay reveals that α-tocopherol prevents formation of both lipid and protein hydroperoxides at high light. Identification of oxidation products of α-tocopherol might contribute to a better understanding of the protective role of α-tocopherol in the prevention of oxidative damage in higher plants at high light.

Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation

Polysorbates are stabilizers typically required in therapeutic protein formulations. On account of their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects polysorbate 20 (PS20) and polysorbate 80 (PS80) against oxidative degradation. PS20 and PS80 solutions containing BHA, BHT—or as control without an antioxidant—were stressed by exposure to air at 40°C for 7 weeks. The following assays were performed: ferrous oxidation-xylenol orange assay, liquid chromatography coupled to UV and mass spectrometry (MS), pH measurement, liquid chromatography fluorescence micelle assay, headspace–gas chromatography coupled with MS. PS20 and PS80 solutions containing an antioxidant were found to be more stable, as demonstrated by lower peroxide levels, lower free fatty acid contents, stable pH, intact polysorbate micelle structure/composition, and less volatile degradants. PS20 and PS80 solutions containing BHT or BHA are more stable against oxidative degradation compared to nonstabilized solutions. It might be beneficial to formulate bulk polysorbate with the antioxidant(s) to ensure stabilization during all process steps.

Croton gratissimus leaf extracts inhibit cancer cell growth by inducing caspase 3/7 activation with additional anti-inflammatory and antioxidant activities

BackgroundCroton species (Euphorbiaceae) are distributed in different parts of the world, and are used in traditional medicine to treat various ailments including cancer, inflammation, parasitic infections and oxidative stress related diseases. The present study aimed to evaluate the antioxidant, anti-inflammatory and cytotoxic properties of different extracts from three Croton species.MethodsAcetone, ethanol and water leaf extracts from C. gratissimus, C. pseudopulchellus, and C. sylvaticus were tested for their free radical scavenging activity. Anti-inflammatory activity was determined via the nitric oxide (NO) inhibitory assay on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and the 15-lipoxygenase inhibitory assay using the ferrous oxidation-xylenol orange assay. The cytotoxicity of the extracts was determined on four cancerous cell lines (A549, Caco-2, HeLa, MCF-7), and a non-cancerous African green monkey (Vero) kidney cells using the tetrazolium-based colorimetric (MTT) assay. The potential mechanism of action of the active extracts was explored by quantifying the caspase-3/− 7 activity with the Caspase-Glo® 3/7 assay kit (Promega).ResultsThe acetone and ethanol leaf extracts of C. pseudopulchellus and C. sylvaticus were highly cytotoxic to the non-cancerous cells with LC50 varying between 7.86 and 48.19 μg/mL. In contrast, the acetone and ethanol extracts of C. gratissimus were less cytotoxic to non-cancerous cells and more selective with LC50 varying between 152.30 and 462.88 μg/mL, and selectivity index (SI) ranging between 1.56 and 11.64. Regarding the anti-inflammatory activity, the acetone leaf extract of C. pseudopulchellus had the highest NO inhibitory potency with an IC50 of 34.64 μg/mL, while the ethanol leaf extract of the same plant was very active against 15-lipoxygenase with an IC50 of 0.57 μg/mL. A linear correlation (r<0.5) was found between phytochemical contents, antioxidant, anti-inflammatory and cytotoxic activities of active extracts. These extracts induced differentially the activation of caspases − 3 and − 7 enzymes in all the four cancerous cells with the highest induction (1.83-fold change) obtained on HeLa cells with the acetone leaf extract of C. gratissimus.ConclusionBased on their selective toxicity, good antioxidant and anti-inflammatory activities, the acetone and ethanol leaf extracts of C. gratissimus represent promising alternative sources of compounds against cancer and other oxidative stress related diseases.

Contribution of monoamine oxidases to vascular oxidative stress in patients with end-stage renal disease requiring hemodialysis.

Arteriovenous fistula (AVF) is the "lifeline" for patients with end-stage renal disease (ESRD) undergoing hemodialysis. AVF maturation failure is a poorly understood process, one of the contributors being endothelial dysfunction due to oxidative stress. Monoamine oxidases (MAOs) A and B were recently identified as novel sources of vascular oxidative stress. The aim of the present study was to assess the contribution of MAOs to the endothelial dysfunction in patients with ESDR with indication of hemodialysis. Fragments of brachial artery collaterals were harvested from ESRD patients during the surgical procedure aimed at creating the vascular access in the cubital fossa. The effect of increasing concentrations (10, 30, 100 μmol/L) of the irreversible MAO-A inhibitor, clorgyline, and MAO-B inhibitor, selegiline, on endothelial-dependent relaxation (EDR) in response to cumulative doses of acetylcholine was studied in isolated phenylephrine-preconstricted vascular rings. Hydrogen peroxide (H2O2) production was assessed using ferrous oxidation xylenol orange assay. We showed that incubation of brachial rings with MAO inhibitors significantly improved EDR and attenuated H2O2 generation in patients with ESRD. MAO-related oxidative stress might contribute to the primary dysfunction/non-maturation of the AVF and MAO inhibitors could improve maturation and long-term patency of the vascular access in dialysis patients.

Inhibition of Nitric Oxide Production in LPS-Stimulated RAW 264.7 Macrophages and 15-LOX Activity by Anthraquinones from Pentas schimperi.

The anti-inflammatory activity of a coumarin and nine anthraquinone derivatives, 3-hydroxy-1-methoxy-2-methylanthraquinone (1), 2-hydroxymethyl anthraquinone (2), schimperiquinone B (3), cleomiscosin A (4), damnacanthal (5), 1,2-dihydroxy anthraquinone (6), damnacanthol (7), 3-hydroxy-2-hydroxymethyl anthraquinone (8), 1-hydroxy-2-methoxyanthraquinone (9), and 2-hydroxymethyl-3-O-prenylanthraquinone (10), isolated from the roots of Pentas schimperi were determined. The anti-15-lipoxygenase activity and nitric oxide production inhibition on lipopolysaccharide-activated macrophages RAW 264.7 cells were determined as indicators of anti-inflammatory activity. The Griess assay was used to measure nitric oxide production and the ferrous oxidation-xylenol orange assay was used to determine the 15-lipoxygenase inhibitory activity. All the compounds significantly decreased nitrite + nitrate accumulation in lipopolysaccharide-stimulated RAW 264.7 cells in a concentration-dependent manner with 85.67 % to 119.75 % inhibition of nitrite + nitrate production at 20 µg/mL. Most of the compounds had a moderate inhibitory effect on 15-lipoxygenase activity. Compounds 8 and 10 were the most potent inhibitor both in nitrite + nitrate production with respective IC50 values of 1.56 µM and 6.80 µM. Compounds 2, 7, and 8 had good anti-15-lipoxygenase activity with respective IC50 values of 13.80 µM, 14.80 µM, and 15.80 µM compared to quercetin, which was used as a standard lipoxygenase inhibitor (IC50 of 16.80 µM). Our study revealed 3-hydroxy-2-hydroxymethyl anthraquinone and damnacanthol as potent inhibitors of both 15-lipoxygenase activity and nitric oxide production. Further studies are needed in order to envisage its possible future use as a therapeutic alternative against inflammatory diseases.

Anti-inflammatory activity of benzophenone and xanthone derivatives isolated from Garcinia (Clusiaceae) species

Species of the genus Garcinia have been the source of many benzophenone and xanthone derivatives. Recent data regarding potent biological properties of natural compounds in Garcinia species led us to investigate the in vitro anti-inflammatory effect of three known xanthones, lichexanthone (1), 1,3,6,7-tetrahydroxyxanthone (2), 1,3,5,6-tetrahydroxyxanthone (3), two new xanthones 1-hydroxy-3,6,7-tri-O,O,O-triprenylxanthone (4), 1,6-dihydroxy-3,7-di-O,O-diprenylxanthone (5) and two benzophenones isoxanthochymol (6), guttiferone E (7), isolated from Garcinia nobilis and Garcinia punctata. The Griess assay was used for the measurement of nitric oxide (NO) production in RAW264.7 macrophages and the ferrous oxidation-xylenol orange assay was used to determine the 15-lipoxygenase (15-LOX) inhibitory activity. All the compounds had inhibitory effect on 15-LOX activity to different extents. Compound (7) had the highest anti-LOX activity with an IC50 value of 43.05μg/mL. At the highest studied concentration (25μg/mL), compound (4) had the most potent inhibitory activity against NO release with a% of inhibition of 95.42% and less cytotoxic effect on RAW264.7 cells (% of cell viability of 81.40).The results presented here suggest that 1,3,5,6-tetrahydroxyxanthone (3) and guttiferone E (7) are promising inhibitors of NO production and 15-LOX activity. Further studies should be considered in order to elucidate the mechanism by which these compounds exert their inhibitory activities.

Advanced oxidation protein products are more related to metabolic syndrome components than biomarkers of lipid peroxidation

Although advanced oxidation protein products (AOPPs) have been reported as the most appropriate parameter for determination of oxidative stress in patients with metabolic syndrome (MetS), a direct comparison between protein and lipid peroxidation has not been performed yet. The aim of this study was to compare protein peroxidation with lipid peroxidation measured by 2 different methodologies (tert-butyl hydroperoxide-initiated chemiluminescence and ferrous oxidation-xylenol orange assay). The hypothesis of this study was that AOPPs would be more related to MetS than to oxidative markers of lipid peroxidation. This cross-sectional study evaluated 76 patients with MetS and 20 healthy subjects. Prooxidant-antioxidant index (PAI) assessed as AOPP/total radical-trapping antioxidant parameter ratio progressively increased (P < .05) according to the number of MetS components, whereas AOPPs and total radical-trapping antioxidant parameter increased (P < .05) when 5 components were compared with 3 components. Spearman test showed a positive correlation between AOPPs and waist circumference (r = 0.318, P < .01), fasting glucose (r = 0.250, P < .05), homeostasis model assessment insulin resistance (r = 0.043, P < .01), triacylglycerol (r = 0.713, P < .0001), highly sensitive C-reactive protein (r = 0.275, P < .05), and uric acid (r = 0.356, P < .01), whereas there was an inverse correlation with high-density lipoprotein cholesterol (r = −0.399, P < .001). Prooxidant-antioxidant index demonstrated a positive correlation with waist circumference (r = 0.386, P < .01), fasting glucose (r = 0.388, P < .01), fasting insulin (r = 0.344, P < .05), homeostasis model assessment insulin resistance (r = 0.519, P < .001), triacylglycerol (r = 0.687, P < .0001), highly sensitive C-reactive protein (r = 0.278, P < .05), and uric acid (r = 0.557, P < .0001), whereas there was an inverse correlation with high-density lipoprotein cholesterol (r = −0.480, P < .0001). In conclusion, protein peroxidation determined by AOPPs, and especially by PAI, is more related to MetS components than lipid peroxidation. In addition, PAI progressively increased with the number of MetS components.

Anti-inflammatory and anticholinesterase activity of six flavonoids isolated from Polygonum and Dorstenia species.

This study was aimed at investigating the anti-inflammatory and anticholinesterase activity of six naturally occurring flavonoids: (-) pinostrobin (1), 2',4'-dihydroxy-3',6'-dimethoxychalcone (2), 6-8-diprenyleriodictyol (3), isobavachalcone (4), 4-hydroxylonchocarpin (5) and 6-prenylapigenin (6). These compounds were isolated from Dorstenia and Polygonum species used traditionally to treat pain. The anti-inflammatory activity was determined by using the Griess assay and the 15-lipoxygenase inhibitory activity was determined with the ferrous oxidation-xylenol orange assay. Acetylcholinesterase inhibition was determined by the Ellman's method. At the lowest concentration tested (3.12µg/ml), compounds 2, 3 and 4 had significant NO inhibitory activity with 90.71, 84.65 and 79.57% inhibition respectively compared to the positive control quercetin (67.93%). At this concentration there was no significant cytotoxicity against macrophages with 91.67, 72.86 and 70.86% cell viability respectively, compared to 73.1% for quercetin. Compound 4 had the most potent lipoxygenase inhibitory activity (IC50 of 25.92µg/ml). With the exception of (-) pinostrobin (1), all the flavonoids had selective anticholinesterase activity with IC50 values ranging between 5.93 and 8.76µg/ml compared to the IC50 4.94µg/ml of eserine the positive control. These results indicate that the studied flavonoids especially isobavachalcone are potential anti-inflammatory natural products that may have the potential to be developed as therapeutic agents against inflammatory conditions and even 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.

Monoamine Oxidases Are Mediators of Endothelial Dysfunction in the Mouse Aorta

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease models.

Evaluation of Oxidative Stress in Overweight Subjects With or Without Metabolic Syndrome

Although oxidative stress is considered the underlying mechanism by which dysfunctional metabolism occurs in obese subjects, there are few studies on oxidative stress in overweight subjects. The objective of this study was to verify the influence of metabolic syndrome (MetS) on oxidative stress and antioxidant defense in overweight subjects. There were 123 subjects (50 in the control group and 73 in the overweight group) chosen to participate in this cross-sectional study. The control group included 50 healthy individuals with a BMI between 20 and 24.9 kg/m(2) and without MetS. The overweight group included 73 subjects with a BMI between 25 and 29.9 kg/m(2). Overweight subjects were divided into two groups: with MetS (29 subjects) and without MetS (44 subjects). Control group and overweight group subjects without MetS showed no differences in oxidative stress parameters and total antioxidant capacity (TRAP). Overweight subjects with MetS had higher hydroperoxide concentrations measured by chemiluminescence compared to the control group (P < 0.05), higher hydroperoxide and hydrogen peroxide concentrations determined by ferrous oxidation-xylenol orange assay compared to overweight subjects without MetS (P < 0.001), and higher advanced oxidation protein product (AOPP) concentrations (P < 0.001) compared to the other groups. AOPP was directly correlated with uric acid concentrations. Overweight subjects with MetS had lower TRAP concentrations compared to the control group (P < 0.001). In conclusion, this study showed that overweight subjects with MetS, in contrast to overweight subjects without MetS, have a redox imbalance characterized by increased plasma oxidation and reduced antioxidant capacity.

Intravenous anesthetic propofol suppresses leukotriene production in murine dendritic cells

Leukotrienes, divided into cysteinyl leukotrienes (CysLTs), which are important mediators of asthmatic responses, and leukotriene B4 (LTB4), a chemotactic and chemokinetic agent for leukocytes, are potent lipid mediators generated from arachidonic acid by 5-lipoxygenase (5-LO). Leukotrienes are also considered to have immunoregulatory and pro-inflammatory actions. Propofol is an intravenous anesthetic widely used for anesthesia and sedation that is alleged to possess anti-inflammatory properties. The present study examined the effect of propofol on leukotriene production by dendritic cells (DC). In murine bone marrow-derived DC, propofol significantly suppressed CysLT and LTB4 production after short-term stimulation with zymosan. The protein levels of cytosolic phospholipase A2 and 5-LO, or arachidonic acid release from plasma membranes, were not affected by the presence of propofol. Although zymosan treatment induced or enhanced the phosphorylation of ERK1/2, p-38 MAPK, and JNK, which presumably up-regulates the activity of 5-LO, the presence of propofol had no additional effect on the phosphorylation status of any of these MAPKs. Similarly, zymosan significantly increased the concentration of intracellular calcium, which is the most crucial activator of 5-LO, but no additional concentration changes were observed with the addition of propofol. Lastly, in an in-vitro cell-free ferrous oxidation-xylenol orange assay, propofol significantly inhibited the 5-LO activity of purified human recombinant 5-LO enzyme with an IC50 of ~7.5 µM. Thus, propofol’s inhibition of 5-LO is not likely restricted to the circumstances surrounding the production of leukotrienes from DC, but applicable to other types of immune and non-immune cells that produce leukotrienes. The 5-LO-inhibiting activity of propofol may, at least in part, contribute to the well-known anti-inflammatory activity of propofol.

Cellular adaptive response to chronic radiation exposure in interventional cardiologists

Aims Invasive cardiologists are the most exposed to ionizing radiation among health professionals and show an increased rate of somatic DNA damage. To evaluate the effects of chronic low-dose exposure to ionizing radiation on redox state and apoptotic activation. Methods and results We enrolled 10 healthy exposed professionals (all interventional cardiologists, Group II, exposed: age = 38 ± 5 years) and 10 age- and gender-matched unexposed controls (Group I, non-exposed). Exposed subjects had a median exposure of 4 mSv/year (range 1-8) by film badge dosimetry (below lead apron). We measured reduced glutathione (GSH, a marker of antioxidant response) in erythrocytes and plasma generation of hydrogen peroxide (a marker of oxyradical stress) by ferrous oxidation-xylenol orange assay in plasma. In both groups, lymphocytes were isolated and caspase-3 activity (a marker of apoptotic response) measured at baseline and following 2 Gy in vitro irradiation. Exposed subjects showed a three-fold increase in hydrogen peroxide (Group I = 2.21 ± 1.03 vs. II = 6.51 ± 1.55 μM H(2)O(2) equivalents) and a 1.7-fold increase in GSH (I = 12.37 ± 1.22 vs. II = 20.61 ± 2.16 mM). Exposed subjects also showed higher values of caspase-3 activity, both at baseline and-more strikingly-following high-dose radiation challenge. Conclusion In interventional cardiologists, chronic exposure to low-dose radiation is associated with an altered redox balance mirrored by an increase in hydrogen peroxide and with two possibly adaptive cellular responses: (i) an enhanced antioxidant defence (increase in GSH, counteracting increased oxyradical stress) and (ii) an increased susceptibility to apoptotic induction which might efficiently remove genetically damaged cells.

Measurement of Plasma Hydroperoxide Concentrations by the Ferrous Oxidation-Xylenol Orange Assay in Conjunction with Triphenylphosphine

We describe the application of the FOX2 (ferrous oxidation in xylenol orange, version 2) method to the measurement of hydroperoxides in plasma. Authentic plasma hydroperoxides can be determined by a strategy in which the hydroperoxide reductant, triphenylphosphine, is used to discriminate between the background signal generated by ferric ions present in plasma and that generated by hydroperoxide in plasma. The approach was validated by extraction of total lipids from plasma using ethyl acetate prior to assay with the FOX2 reagent. Plasma from 23 normal individuals contained hydroperoxide in the range of 0.22 to 7.8 μM with a mean of 3.02 μM and a population standard deviation of 1.85 μM. After partitioning with ethyl acetate, plasma hydroperoxide levels ranged from 0.22 to 6.22 μM, with a mean value of 2.52 μM and a population standard deviation of 1.65 μM.