Promoting physical activity and improving dietary quality of Singaporean adolescents: effectiveness of a school-based fitness and wellness program.

Limited data are available on the effectiveness of the school-based structured fitness and wellness program to influence dietary quality and physical activity levels in Singaporean adolescents. The study examined if a 20-h (over 10 weeks) school-based structured fitness and wellness module affects the diet quality indices, energy intakes, physical activity levels and the associated energy expenditures in a group of healthy, male adolescents with low diet quality and physical activity levels. Participant demography, anthropometry, dietary intake and daily physical activity were obtained at the beginning, mid-point and end of the 10-week program. Physical activity levels were assessed accelerometrically over a 1-weekday period. Dietary intake were taken using a structured 7-day food diary, and diet quality assessed using the Diet Quality Index-International (DQI-I). The 31 enrolled participants (age 19.8 ± 0.6 years) with body mass index (BMI) (19.8 ± 0.6 kg/m2) followed diets of low diet quality scores (48.3 ± 9.6 out of 100) and engaged in 3.87 ± 2.00 h of physical activity daily before the start of the intervention. Their dietary quality and physical activity levels did not change significantly throughout the intervention period. They scored poorly in the moderation and overall balance components of the diet quality assessment. The physical activity duration correlated inversely to the diet quality scores. Our results suggest that the prescribed school-based fitness and wellness module was ineffective in influencing the diet quality and physical activity levels of Singaporean male adolescents with low diet quality and physical activity levels.

Occurrence of azo food dyes and their effects on cellular inflammatory responses.

OBJECTIVES: The aim of this study was to examine the occurrence of five azo food dyes-tartrazine, sunset yellow, carmoisine, allura red, and ponceau 4 R-in the food supply chain of Singapore and their effects on the in vitro synthesis of leukotriene B4 (LTB4) and F2-isoprostanes. METHODS: Trained personnel recorded the names of foods and beverages sold in a local supermarket that contained at least one of the five azo dyes. The occurrence of the azo dyes in the local food supply was computed. The synthesis of LTB4 and F2-isoprostanes from freshly isolated blood neutrophils were measured using gas chromatography-mass spectrometry. RESULTS: Of the 1681 processed food items, 194 (11.54%) contained at least one of the five azo dyes. Tartrazine was most prevalent in food and beverage products sold in Singapore, followed by allura red, sunset yellow, ponceau 4 R, and carmoisine. The five azo dyes augmented the in vitro synthesis of LTB4 and F2-isoprostanes from blood neutrophils. Tartrazine was significantly more potent in increasing LTB4 synthesis than the other dyes, which exhibited similar potencies. The five food dyes increased the formation of F2-isoprostanes from blood neutrophils at all tested concentrations. CONCLUSION: The high prevalence of azo dyes in the food supply of Singapore and their ability to elicit proinflammatory responses in vitro suggest a potential health risk to the local population.

Common food antimicrobials: effects on cellular inflammation and oxidative damage and their estimated occurrence in Singapore.

BACKGROUND AND OBJECTIVES: The prevalence and potential health effects of common food antimicrobials in processed foods and beverages are relatively unknown in Singapore. The occurrence of chemical antimicrobials in processed foods and beverages and their effects on inflammation and oxidative stress in vitro were examined. METHODS AND STUDY DESIGN: The occurrence of antimicrobials in 1605 processed food and 359 beverage items were examined by surveying the ingredients on the product labels. Human neutrophils were exposed to physiologically relevant concentrations of common antimicrobials. Established markers of inflammation, l Leukotriene B4 and oxidative stress, F2-isoprostanes were measured using stable-isotope dilution gas chromatography-mass spectrometry. RESULTS: Antimicrobials were added to 23.2% of the processed foods and beverages. Sorbic, benzoic, lactic, propionic and acetic acids accounted for 84.8% of the added antimicrobials in the processed foods and beverages. 92.5% of the bread contained propionic acid. Lactic acid was the most common antimicrobial (44.4%) in cheeses. Sorbic acid was added to 63.2% of the margarines selected. Sauces (31.5%), energy drinks (50.0%), soft drinks (70.7%) and fruit cordials (66.6%) contained added benzoic acid. Benzoic and propionic acids at physiologically relevant concentrations augmented leukotriene B4formation (benzoicacid, EC50 = ~100 µg L-1and propionic acid, >200 µg L-1). Lactic and sorbic acids dose-dependently inhibited the F2-isoprostanes production (IC50 values ~100 µg L-1) and myeloperoxidase activity (IC50values ~100 µg L-1). CONCLUSIONS: Our results demonstrate that Singapore consumers are significantly exposed to food antimicrobials, and these molecules, in physiologically relevant concentrations, exert significant and differential effects in vitro.

Dietary Plant Sterols Supplementation Increases In Vivo Nitrite and Nitrate Production in Healthy Adults: A Randomized, Controlled Study.

A randomized, double-blinded, placebo-controlled and crossover study was conducted to simultaneously measure the effects, 3 h after consumption and after 4-wk daily exposure to plant sterols-enriched food product, on in vivo nitrite and nitrate production in healthy adults. Eighteen healthy participants (67% female, 35.3 [mean] ± 9.5 [SD] years, mean body mass index 22.8 kg/m2 ) received 2 soy milk (20 g) treatments daily: placebo and one containing 2.0 g free plant sterols equivalent of their palmityl esters (ß-sitosterol, 55%; campesterol, 29%; and stigmasterol, 23%). Nitrite and nitrate concentrations were measured in the blood plasma and urine, using stable isotope-labeled gas chromatography-mass spectrometry. L-arginine and asymmetric dimethylarginine concentrations in blood serum were measured using commercially available enzyme immunoassays. Nitrite and nitrate concentrations in blood plasma (nitrite 5.83 ± 0.50 vs. 4.52 ± 0.27; nitrate 15.78 ± 0.96 vs. 13.43 ± 0.81 µmol/L) and urine (nitrite 1.12 ± 0.22 vs. 0.92 ± 0.36, nitrate 12.23 ± 1.15 vs. 9.71 ± 2.04 µmol/L) were significantly elevated after 4-wk plant sterols supplementation Placebo and 3-h treatments did not affect the blood plasma and urinary concentrations of nitrite and nitrate. Circulating levels of L-arginine and asymmetric dimethylarginine were unchanged in the placebo and treatment arms. Total plant sterols, ß-Sitosterol, campesterol, and stigmasterol concentrations were significantly elevated after 4-wk treatments compared to the placebo and 3-h treatments. Blood plasma nitrite and nitrate concentrations correlated significantly with the plasma total and specific plant sterol concentrations. Our results suggest that dietary plant sterols, in the combination used, can upregulate nitrite, and nitrate production in vivo.

Plant sterol-enriched soy milk consumption modulates 5-lipoxygenase, 12-lipoxygenase, and myeloperoxidase activities in healthy adults - a randomized-controlled trial.

A randomized, double blind, placebo-controlled and crossover study was conducted to simultaneously measure the effects, after 3-h and 4-week daily exposure to plant sterols-enriched food product, on inflammation, oxidative status, 5-lipoxygenase, 12-lipoxygenase, and myeloperoxidase activities in healthy adults. Eighteen healthy participants (67% female, 35.3 (mean) ± 9.5 (SD) years, mean body mass index 22.8 kg m-2) received two soy milk (20g) treatments daily: placebo and one containing 2.0 g free plant sterols equivalent of their palmitates (ß-sitosterol, 55%; campesterol, 29%; stigmasterol, 23%). F2-isoprostanes, leukotriene B4, sitosterol, campesterol, and stigmasterol concentrations were measured in the blood plasma and urine, using stable isotope-labeled gas chromatography-mass spectrometry. High-sensitivity c-reactive protein, tumor necrosis factor-α, and lipoxin A4 concentrations in blood serum were measured using commercially available enzyme immunoassays. Myeloperoxidase activity, serum lipid hydroperoxides, plasma and urinary F2-isoprostanes, plasma and urinary leukotriene B4, and plasma high-sensitivity c-reactive protein concentrations were significantly reduced, while circulating lipoxin A4 concentrations were significantly elevated after 4-week plant sterols treatment. Plant sterols treatment decreased plasma leukotriene B4 and increased plasma lipoxin A4 concentrations acutely. Total plant sterols, ß-sitosterol, campesterol, and stigmasterol concentrations were significantly elevated after 4-week treatments compared with the pre-treatment concentrations. Our results suggest that dietary plant sterols, in the combination used, can alleviate lipid peroxidation and inflammatory events in vivo. These effects are possibly exerted via the modulation of myeloperoxidase, 5-lipoxygenase, and 12-lipoxygenase activities.

Aged Garlic Supplement Protects Against Lipid Peroxidation in Hypercholesterolemic Individuals.

The health benefits of raw garlic intake has been extensively studied, but little is known about the biological effects of aged garlic consumption. A randomized, placebo-controlled, parallel-arm, double-blinded trial involving 41 hypercholesterolemic individuals was conducted to simultaneously examine and compare the blood lipid lowering and antioxidant effects after acute and extended exposures to aged and raw garlic supplements (1080 mg daily). Aged and raw garlic did not affect blood lipid concentrations in these hypercholesterolemic participants after acute and 13-week supplementation. The plasma and urinary F2-isoprostanes concentrations were significantly decreased after 13 weeks of aged garlic treatment. Aged garlic supplementation over 13 weeks also significantly decreased serum lipid hydroperoxide concentration and myeloperoxidase activity. Raw garlic treatments did not affect the F2-isoprostanes concentrations in blood plasma and urine, and lipid hydroperoxides in blood sera. Acute effects on the measured parameters were absent for both garlic treatments. In separate in vitro experiments, aqueous methanolic extract of aged garlic inhibited F2-isoprostanes formation and myeloperoxidase activity in freshly isolated human neutrophils to a greater extent than the raw garlic extract and S-allylcysteine at equivalent dosing concentrations. The aged garlic preparation was found to contain significantly higher total phenolic and S-allylcysteine contents than the raw garlic precursor. Our data showed that supplementation with aged garlic, not its raw garlic precursor, reduced oxidative stress and alleviated lipid peroxidation, possibly via the inhibition of myeloperoxidase. The differential antioxidant actions of the aged and raw garlic may be related to their different total phenolic contents and, to a lesser extent, their S-allylcysteine contents.

Relative Inhibitions of 5-Lipoxygenase and Myeloperoxidase and Free-Radical Scavenging Activities of Daidzein, Dihydrodaidzein, and Equol.

The effects of bioavailability and metabolic transformation on the biological activities of daidzein are relatively unknown. The effects of daidzein, dihydrodaidzein, and equol at physiologically relevant concentrations on the production of leukotriene B4 and F2-isoprostanes, and myeloperoxidase enzyme activity in freshly isolated human neutrophils were examined. Equol, at physiological concentrations, inhibited leukotriene B4 production (IC50-200 nmol/L) in human neutrophils significantly more than daidzein and dihydrodaidzein (IC50 values >1000 nmol/L). Daidzein, dihydrodaidzein, and equol did not affect the enzymatic hydrolysis of leukotriene A4 to leukotriene B4, suggesting that they exerted their inhibitory effects on the 5-lipoxygenase activity. Daidzein (IC50 = 600 nmol/L) protected against free radical peroxidation of arachidonic acid significantly more than did equol and dihydrodaidzein (IC50 values >1000 nmol/L). Equol also showed significantly greater inhibition of myeloperoxidase activity (IC50 = 450 nmol/L) when compared to daidzein and dihydrodaidzein. Equol accumulated within the human neutrophils at significantly higher concentrations than daidzein and dihydrodaidzein after incubation with the three compounds at physiologically relevant concentrations. Neutrophils were able to accumulate intracellular daidzein, dihydrodaidzein, and equol up to a concentration of ∼600 nmol/L. Our results provide in vitro evidence that the biological activities of daidzein are profoundly influenced by bioavailability and metabolic transformation.

Does influenza A infection increase oxidative damage?

Considerable data implicate oxidative damage in influenza pathogenesis. We examined temporal changes in oxidative damage using accurate biomarkers in an adult cohort with acute influenza infection and their relationships with clinical parameters. Clinical information and blood samples were collected during their acute illness and 3 months later. A fatigue questionnaire was administered 3 months following influenza infection. Thirty-five patients (mean age, 34 years) with polymerase chain reaction-confirmed influenza A infection were included; all patients returned for follow-up assessments. Adjusted levels of plasma F2-isoprostanes, total hydroxyeicosatetraenoic products (HETEs), 7ß-hydroxycholesterol and 7-ketocholesterol, serum gamma-glutamyltransferase, and high-sensitivity C-reactive protein (hsCRP) were increased during the acute illness compared with age-matched controls. Despite clinical recovery, levels of these biomarkers remained higher at month 3 compared with controls. A proportion of patients had persistent symptoms such as fatigue (23%), myalgia (14%), and arthralgia (11%) at month 3. Patients with significant fatigue had higher baseline levels of plasma F2-isoprostanes, F4-neuroprostanes, and total HETEs compared to those without fatigue. By contrast, patients with persistent arthralgia and myalgia had higher baseline levels of serum hsCRP compared to those without these symptoms. Our observations lead to the hypothesis that oxidative damage participates in the pathogenesis of influenza infection and postinfectious fatigue.

Cyclooxygenase-1 mediated platelet reactivity in young male smokers.

BACKGROUND: The purpose of this study is to investigate the acute and chronic effects of cigarette smoking on cyclooxygenase- 1(COX-1)-mediated platelet reactivity among cigarette smokers. METHODS: The levels of collagen-induced platelet aggregation, platelet COX-1 activity, and expressions were compared between smokers and age-matched nonsmokers. In smokers, the acute effects of cigarette smoking were assessed by repeating these measurements an hour after smoking. RESULTS: Twenty-five smokers and age-matched nonsmokers (all men; mean age, 29 years) were studied. Collagen-induced platelet aggregation and plasma/urinary thromboxane B2 (TXB2) and 11-dehydroxythromboxane B2 levels were higher in cigarette smokers compared to nonsmokers. Greater expression of platelet COX-1 was observed in smokers than in nonsmokers. Among smokers, collagen-induced platelet aggregation correlated positively with platelet volume and circulating nicotine and cotinine concentrations. The levels of plasma/urinary TXB2 were significantly increased an hour after cigarette smoking. CONCLUSION: Cigarette smoking aggravates COX-1-mediated platelet reactivity in young, otherwise healthy, smoking men.

Augmentation of 5-lipoxygenase activity and expression during dengue serotype-2 infection.

BACKGROUND: Leukotriene B4, a 5-lipoxygenase product of arachidonic acid with potent chemotactic effects on neutrophils, has not been assessed in dengue patients. In this study, plasma leukotriene B4 and serum high-sensitivity C-reactive protein levels were determined in adult patients during the febrile, convalescent and defervescent stages of dengue serotype-2 (DENV-2) infection, and compared with those of age-matched healthy and non-dengue febrile subjects. In vitro studies were performed to examine the effects of live and heat-inactivated DENV-2 on the activities and expression of 5-lipoxygenase in human neutrophils. RESULTS: Plasma leukotriene B4 was elevated during the febrile stages of dengue infection compared to levels during convalescence and in study controls. Plasma leukotriene B4 also correlated with serum high-sensitivity C-reactive protein in dengue patients (febrile, r = 0.91, p < 0.001; defervescence, r = 0.87, p < 0.001; convalescence, r = 0.87, p < 0.001). Exposure of human neutrophils to DENV-2 resulted in a significant rise in leukotriene B4; the extent of increase, however, did not differ between exposure to live and heat-inactivated DENV-2. Pre-incubation of either live or heat-inactivated DENV-2 resulted in reduced leukotriene B4 release by neutrophils, indicating that contact with dengue antigens (and not replication) triggers the neutrophil response. Production of leukotriene B4 was associated with an increase in 5-lipoxygenase expression in human neutrophils; addition of MK886 (a 5-lipoxygenase activating protein inhibitor) attenuated further increase in leukotriene B4 production. CONCLUSION: These findings provide important clinical and mechanistic data on the involvement of 5-lipoxygenase and its metabolites in dengue infection. Further studies are needed to elucidate the therapeutic implications of these findings.

Acute effects of cigarette smoking on insulin resistance and arterial stiffness in young adults.

BACKGROUND: It is unclear whether changes in insulin sensitivity or arterial stiffness in cigarette smokers could explain the link between cigarette smoking and diabetes mellitus. The purpose of the study was to evaluate the acute effects of cigarette smoking on insulin resistance and arterial stiffness in a cohort of young healthy adults. METHODS: Metabolic risk components, hemodynamic parameters, plasma nitrite/nitrate and high-sensitivity C-reactive protein (hsCRP) levels, were compared between smokers and age- and gender-matched controls (non-smokers). In smokers, these levels were determined 8-h following cigarette abstinence and an hour after smoking. RESULTS: One hundred nineteen smokers and age-matched non-smokers (mean age, 32 years; 83% men) were included in this study. Compared with non-smokers, smokers had a significantly higher number of abnormal metabolic risk components, HOMA-IR index and total nitrite/nitrate levels. There were no differences in brachial/central blood pressure, augmentation index and hsCRP between smokers and non-smokers. An hour after smoking, smokers had significantly higher levels of HOMA-IR, total nitrite/nitrate, hsCRP and heart rate compared with baseline levels. By contrast, brachial/central blood pressure and augmentation index were unchanged after cigarette smoking. Baseline vascular and insulin resistance status predicted the extent of rise in the HOMA-IR and augmentation indices acutely after cigarette smoking (adjusted R(2) 0.358 and 0.124, p < 0.001 respectively). CONCLUSIONS: Individuals with more advanced vascular damage and insulin resistance are vulnerable to the acute effects of cigarette smoking.

Products of 5-lipoxygenase and myeloperoxidase activities are increased in young male cigarette smokers.

The significance of 5-lipoxygenase and myeloperoxidase activities has not been extensively studied among young male smokers. Leukotriene B(4), 20-hydroxy-leukotriene B(4), 20-carboxy-leukotriene B(4) and 3-chlorotyrosine were measured in plasma and urinary samples of young male smokers at 8 hours following cigarette abstinence and an hour after cigarette smoking. Leukotriene B(4) and 3-chlorotyrosine were determined in neutrophils isolated from these individuals. The levels of these markers were compared with those of age-matched controls. In vitro studies were performed to evaluate the production of leukotriene B(4) and 3-chlorotyrosine from human neutrophils following exposure to nicotine and cotinine. Thirty male smokers (mean age, 27.4 years) and 28 male non-smokers (mean age, 28.7 years) were studied. Plasma levels of leukotriene B(4), 20-carboxy-leukotriene B(4) and 3-chlorotyrosine were higher in smokers than in non-smokers; leukotriene B(4) and 20-carboxy-leukotriene B(4) levels increased further an hour after cigarette smoking. Peripheral neutrophils isolated from smokers showed greater expressions of myeloperoxidase and 5-lipoxygenase activities compared with non-smokers, while plasma leukotriene B(4) and 3-chlorotyrosine were correlated significantly with high-sensitivity C-reactive protein and plasma nicotine concentrations. Exposure of human neutrophils to nicotine and cotinine resulted in a higher production of leukotriene B(4) and 3-chlorotyrosine. To conclude, leukotriene B(4) and 3-chlorotyrosine levels are increased in young male cigarette smokers. These results suggest that cigarette smoking aggravates neutrophil-mediated inflammation by modulating the activities of myeloperoxidase and 5-lipoxygenase pathways.

Biomarkers of oxidative damage in cigarette smokers: which biomarkers might reflect acute versus chronic oxidative stress?

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F(2)-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F(4)-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F(2)-isoprostanes, HETEs, and 8-hydroxy-2'-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F(2)-isoprostanes, allantoin, and 7ß-hydroxycholesterol and urinary F(2)-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F(2)-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2'-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress.

Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction.

OBJECTIVE: Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (-)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)(-/-) gene-knockout mouse. METHODS AND RESULTS: Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE(-/-) control mice). Quercetin significantly reduced aortic F(2)-isoprostane, vascular superoxide, vascular leukotriene B(4), and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE(-/-) mice). Theaflavin showed similar, although less extensive, significant effects. Although (-)-epicatechin significantly reduced F(2)-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE(-/-) mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (-)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE(-/-) controls. CONCLUSIONS: Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE(-/-) gene-knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.

A metabolite profiling approach to identify biomarkers of flavonoid intake in humans.

Flavonoids are phytochemicals that are widespread in the human diet. Despite limitations in their bioavailability, experimental and epidemiological data suggest health benefits of flavonoid consumption. Valid biomarkers of flavonoid intake may be useful for estimating exposure in a range of settings. However, to date, few useful flavonoid biomarkers have been identified. In this study, we used a metabolite profiling approach to examine the aromatic and phenolic profile of plasma and urine of healthy men after oral consumption of 200 mg of the pure flavonoids, quercetin, (-)-epicatechin, and epigallocatechin gallate, which represent major flavonoid constituents in the diet. Following enzymatic hydrolysis, 71 aromatic compounds were quantified in plasma and urine at 2 and 5 h, respectively, after flavonoid ingestion. Plasma concentrations of different aromatic compounds ranged widely, from 0.01 to 10 micromol/L, with variation among volunteers. None of the aromatic compounds was significantly elevated in plasma 2 h after consumption of either flavonoid compared with water placebo. This indicates that flavonoid-derived aromatic compounds are not responsible for the acute physiological effects reported within 2 h in previous human intervention studies involving flavonoids or flavonoid-rich food consumption. These effects are more likely due to absorption of the intact flavonoid. Our urine analysis suggested that urinary 4-ethylphenol, benzoic acid, and 4-ethylbenzoic acid may be potential biomarkers of quercetin intake and 1,3,5-trimethoxybenzene, 4-O-methylgallic acid, 3-O-methylgallic acid, and gallic acid may be potential markers of epigallocatechin gallate intake. Potential biomarkers of (-)-epicatechin were not identified. These urinary biomarkers may provide an accurate indication of flavonoid exposure.

HDL is the major lipoprotein carrier of plasma F2-isoprostanes.

Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and animal models. F(2)-isoprostanes are formed in vivo via free radical peroxidation of arachidonic acid, and their quantification has allowed assessment of oxidative stress in vivo. F(2)-isoprostanes associate with lipids, although their distribution in human plasma lipoproteins is unknown. Our aim was to determine the distribution and levels of F(2)-isoprostanes in lipoproteins isolated from human plasma by ultracentrifugation and fast protein liquid chromatography (FPLC). F(2)-isoprostanes were significantly higher in HDL compared with LDL or VLDL after isolation by ultracentrifugation or FPLC. Furthermore, HDL3 particles contained elevated levels of F(2)-isoprostanes compared with HDL2. Platelet activating factor acetylhydrolase (PAF-AH), which hydrolyses esterified F(2)-isoprostanes from phospholipids, was predominantly associated with LDL. Reduced F(2)-isoprostanes in LDL may be related to higher PAF-AH activity in LDL. Paraoxonase 1 (PON-1) activity was associated with HDL2 and may be a contributing factor to the lower F(2)-isoprostanes in HDL2 compared with HDL3. Further studies are required to establish the implications of these findings on HDL function.

Pure dietary flavonoids quercetin and (-)-epicatechin augment nitric oxide products and reduce endothelin-1 acutely in healthy men.

BACKGROUND: Dietary flavonoids may improve endothelial function and ultimately lead to beneficial cardiovascular effects. OBJECTIVE: The objective was to assess whether pure dietary flavonoids can modulate nitric oxide and endothelin-1 production and thereby improve endothelial function. DESIGN: A randomized, placebo-controlled, crossover trial in 12 healthy men was conducted to compare the acute effects of the oral administration of 200 mg quercetin, (-)-epicatechin, or epigallocatechin gallate on nitric oxide, endothelin-1, and oxidative stress after nitric oxide production was assessed via the measurement of plasma S-nitrosothiols and plasma and urinary nitrite and nitrate concentrations. The effects on oxidative stress were assessed by measuring plasma and urinary F(2)-isoprostanes. Plasma and urinary concentrations of quercetin, (-)-epicatechin, and epigallocatechin gallate were measured to establish the absorption of these flavonoids. RESULTS: Relative to water (control), quercetin and (-)-epicatechin resulted in a significant increase in plasma S-nitrosothiols, plasma nitrite, and urinary nitrate concentrations (P < 0.05), but not in plasma nitrate or urinary nitrite. Epigallocatechin gallate did not alter any of the measures of nitric oxide production. Quercetin and (-)-epicatechin resulted in a significant reduction in plasma endothelin-1 concentration (P < 0.05), but only quercetin significantly decreased the urinary endothelin-1 concentration. None of the 3 treatments significantly changed plasma or urinary F(2)-isoprostane concentrations. Significant increases in the circulating concentrations of the 3 flavonoids were observed (P < 0.05) after the corresponding treatment. CONCLUSIONS: Dietary flavonoids, such as quercetin and (-)-epicatechin, can augment nitric oxide status and reduce endothelin-1 concentrations and may thereby improve endothelial function.

Quercetin and its in vivo metabolites inhibit neutrophil-mediated low-density lipoprotein oxidation.

This study examined the effects of metabolic transformation of the common dietary flavonoid, quercetin, on its ability to protect low-density lipoprotein (LDL) from neutrophil-mediated modification. Quercetin was shown to be effective in protecting LDL against neutrophil-mediated modification at physiological concentrations (1 microM) and appears to act by inhibiting myeloperoxidase (MPO)-catalyzed oxidation (IC(50) = 1.0 microM). Quercetin was also shown to protect against radical-induced [2,2'-azobis(2-methylpropionamidine)dihydrochloride] oxidation (IC(50) = 1.5 microM). Studies of structure-activity relationships showed that methylation at the 3'-position or glucuronidation at the 3-position did not significantly affect inhibition by quercetin of the MPO activity, but conjugations at both positions significantly reduce its activity. Our results suggest that the common dietary flavonoid, quercetin, and some of its major in vivo metabolites are potential inhibitors of MPO at physiological concentrations. Dietary flavonoids that could modify MPO activity could protect lipoproteins from damage in chronic inflammatory states such as cardiovascular disease.

Metabolic transformation has a profound effect on anti-inflammatory activity of flavonoids such as quercetin: lack of association between antioxidant and lipoxygenase inhibitory activity.

Dietary flavonoids are thought to have health benefits possibly due to antioxidant and anti-inflammatory properties. Many previous in vitro studies examining the bioactivity of flavonoids have failed to consider the effects of metabolic transformation on flavonoid activity. In this study we examined the effect of quercetin and its major metabolites on the production of pro-inflammatory eicosanoids by human leukocytes. Studies comparing free radical scavenging, antioxidant activity and eicosanoid production demonstrate that there are different structural requirements for antioxidant and anti-inflammatory activity. We also investigated the effect of metabolic transformation on flavonoid bioactivity by comparing the activity of quercetin and its major metabolites to inhibit inflammatory eicosanoid production from human leukocytes. Quercetin was a potent inhibitor of leukotriene B4 formation in leukocytes (IC50 approximately 2 microM), and its activity was dependent on specific structural features, particularly the 2,3-double bond of the C-ring. Functionalisation of the 3'-OH group with either methyl or sulfate reduced inhibitory activity up to 50% while a glucuronide substituent at the 3-OH effectively removed the LTB4 inhibitory activity. The major quercetin metabolite quercetin-3'-O-sulfate retained considerable lipoxygenase inhibitory activity (IC50 approximately 7 microM) while quercetin-3-O-glucuronide maintained antioxidant activity but had no lipoxygenase inhibitory activity at physiological concentrations. In conclusion, we have found that structural modification of quercetin due to metabolic transformation had a profound effect on bioactivity, and that the structural features required for antioxidant activity of quercetin and related flavonoids were unrelated to those required for inhibition of inflammatory eicosanoids.

Augmentation of monocyte intracellular ascorbate in vitro protects cells from oxidative damage and inflammatory responses.

Ascorbic acid is present as a primary antioxidant in plasma and within cells, protecting both cytosolic and membrane components of cells from oxidative damage. The effects of intracellular ascorbic acid on F(2)-isoprostanes (biomarkers of oxidative stress) and monocyte chemoattractant protein-1 (marker of inflammatory responses) production in monocytic THP-1 cells were investigated under conditions of 2,2'-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) induced oxidative stress. Cells cultured under normal conditions have extremely low ascorbate levels and the intracellular ascorbate can be augmented significantly by adding ascorbate to the culture medium. While AAPH treatment reduced cell viability, increased F(2)-isoprostanes and MCP-1 production, the presence of intracellular ascorbic acid maintained high cell viability and attenuated both F(2)-isoprostanes and MCP-1 production. Measurement of intracellular ascorbic acid and its oxidised products showed that intracellular ASC was oxidised to a significantly greater extent during AAPH treatment and may be utilised to protect the cells under conditions of oxidative stress. This study demonstrates the importance of intracellular ascorbate, which may be lacking under normal cell culture conditions, under conditions of increased oxidative stress.