Green coffee polyphenols do not attenuate features of the metabolic syndrome and improve endothelial function in mice fed a high fat diet.

We have investigated the effects of the major polyphenol in coffee, chlorogenic acid (CGA), on obesity, glucose intolerance, insulin resistance, systemic oxidative stress and endothelial dysfunction in a mouse model of the metabolic syndrome. Thirty C57BL6 mice were randomly divided into (n=10/group) (i) normal diet (ND), (ii) high fat diet (HFD), or (iii) high fat diet supplemented with 0.5% w/w green coffee bean extract (GCE) rich in chlorogenic acid (HFD+GCE). The high fat diet consisted of 28% fat and all animals were maintained on their diets for 12 weeks. The mice fed a HFD and HFD+GCE displayed symptoms of the metabolic syndrome compared to their normal fed counterparts, although no endothelial dysfunction was detected in the abdominal aortas after 12 weeks. GCE did not attenuate HFD-induced obesity, glucose intolerance, insulin resistance or systemic oxidative stress. Furthermore, GCE did not protect against ex vivo oxidant (hypochlorous acid)-induced endothelial dysfunction.

Minimizing artifactual elevation of lipid peroxidation products (F2-isoprostanes) in plasma during collection and storage.

F2-isoprostanes (F2-IsoP's) are reliable measures of in vivo lipid oxidation, but care is required to prevent artifactual elevation. We examined the effects of blood collection and storage on plasma F2-IsoP's. Blood was collected into EDTA/butylated hydroxytoluene/reduced glutathione (EDTA/BHT/GSH) or EDTA, at 4 °C or room temperature. Plasma was stored at -20 or -80 °C for 1 or 6 months before F2-IsoP's were assayed by GC-MS. The temperature of blood collection did not affect F2-IsoP's. However, storage at -20 °C or collection into EDTA resulted in significant increases in F2-IsoP's. Blood collection into EDTA/BHT/GSH and storage at -80 °C minimizes artifactual elevation of plasma F2-IsoP's.

20-Hydroxyeicosatetraenoic acid synthesis is increased in human neutrophils and platelets by angiotensin II and endothelin-1.

The cytochrome P-450 arachidonic acid metabolite 20-HETE is central to the regulation of vascular tone, renal function, and blood pressure and is synthesized in the rat kidney in response to angiotensin II (ANG II) and endothelin-1 (ET-1). There are very few studies examining the cellular synthesis of 20-HETE in humans. We aimed to measure human neutrophil and platelet 20-HETE levels under basal conditions and after ANG II, ET-1, and calcium ionophore (CaI). 20-HETE was measured in human platelets and neutrophils after saline (control), CaI (2.5 µg/ml), and ANG II or ET-1 (10 nmol/l-1 µmol/l) incubations. The effect of cells, which were preincubated with the ω-hydroxylase inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl) (HET0016, 10 nM), ANG II types 1 or 2 (AT(1) or AT(2)) receptor inhibition with irbesartan (1 µmol/l) or PD-123319 (1 µmol/l), or endothelin receptor subtypes A or B (ET(A) or ET(B)) receptor inhibition with BQ-123 or BQ-778 (100 nmol/l), was studied. Neutrophil and platelet content and release of 20-HETE was significantly increased by CaI and blocked by the ω-hydroxylase inhibitor HET0016. ANG II and ET-1 significantly increased neutrophil and platelet content and release of 20-HETE. ANG II increased 20-HETE via the AT(2) receptor. ET-1 increased 20-HETE through the ET(B) receptor in platelets and both the ET(A) and ET(B) receptors in neutrophils. These studies show that human platelets and neutrophils synthesize 20-HETE in response to ANG II and ET-1. 20-HETE synthesis in both cell types was predominantly mediated via the AT(2) and ET(B) receptors. Stimulation via these receptor pathways has generally been thought to be cardioprotective and requires further studies in clinical situations associated with low-grade inflammation or where ANG II and ET-1 are elevated to clarify the role of 20-HETE.

Sesame supplementation does not improve cardiovascular disease risk markers in overweight men and women.

BACKGROUND AND AIMS: Pre-clinical studies suggest that sesame and its lignans induce beneficial changes in risk factors related to cardiovascular disease and increase the bioavailability of mammalian lignans. However, only very few intervention trials have investigated the potential bioactivities of sesame in humans. We aimed to investigate the effects of sesame supplementation in humans on blood lipids, blood pressure, systemic oxidative stress, inflammatory biomarkers and mammalian lignan metabolism. METHODS AND RESULTS: We conducted a randomized, placebo-controlled cross-over intervention trial at a university research centre. Overweight or obese men and women (n=33) consumed 25g/d of sesame ( approximately 50mg/d of sesame lignan) and an iso-caloric placebo matched for macronutrient composition for 5 wks each. Each intervention period was preceded by a 4-wk washout period. Blood lipid profiles, day time ambulatory blood pressure, oxidative stress and inflammatory biomarkers and urinary mammalian lignans were measured before and after each intervention. Results are presented as the effect of sesame supplementation relative to placebo. Urinary excretion of the mammalian lignans, enterolactone and enterodiol, increased by approximately 8-fold (P<0.001). Blood lipids and blood pressure were not altered. In addition, markers of systemic inflammation (C-reactive protein, interleukin-6, tumor necrosis factor-alpha) and lipid peroxidation (F(2)-isoprostanes) were not affected. CONCLUSION: Supplementation with 25g/d of sesame can significantly increase the exposure to mammalian lignans. However, this did not cause any improvement in markers of cardiovascular disease risk in overweight or obese men and women.

Effect of weight loss on neutrophil resolvins in the metabolic syndrome.

BACKGROUND: Non-resolving inflammation associates with obesity and insulin resistance, and may be dependent on the balance of inflammatory substances and specialised pro-resolving mediators of inflammation (SPM) that act to halt the inflammatory response. This controlled trial examined the effect of weight loss on neutrophil synthesis of SPM in volunteers with the metabolic syndrome (MetS). METHODS: Volunteers with MetS (n = 42) were matched for age and gender and randomly assigned to a 12-wk weight loss program followed by 4-wk weight stabilization or a 16-wk weight maintenance program. At baseline and 16 weeks, isolated neutrophils were stimulated with calcium ionophore and the released SPM were measured by LC-MS/MS. RESULTS: At baseline the SPM resolvin (Rv) E1, 18R-RvE3, RvD2 and Maresin-1 (MaR-1) were detected from stimulated neutrophils. The concentration of released RvE1 was at least 6-fold that of other detected SPM. Weight loss of 4.7 ±â€¯0.8 kg, led to a 2-fold increase in RvE1, P = 0.013, relative to the weight maintenance group. The increase in RvE1 after weight loss was related to, but independent of leukotriene B4. CONCLUSION: Following weight loss, human neutrophils from individuals with the metabolic syndrome are capable of releasing larger amounts of RvE1 upon stimulation.

Long-Term Dietary Nitrate Supplementation Does Not Prevent Development of the Metabolic Syndrome in Mice Fed a High-Fat Diet.

BACKGROUND: Nitric oxide (NO) is an important vascular signaling molecule that plays a role in vascular homeostasis. A reduction in NO bioavailability is thought to contribute to endothelial dysfunction, an early risk factor for both cardiovascular disease and type 2 diabetes. Dietary nitrate, through the nitrate-nitrite-NO pathway, may provide an alternate source of NO when the endogenous eNOS system is compromised. In addition to a role in the vascular system, NO may also play a role in the metabolic syndrome including obesity and glucose tolerance. AIM: To investigate the effect of long-term dietary nitrate supplementation on development of the metabolic syndrome in mice fed a high-fat diet. METHODS: Following 1 week of acclimatisation, male (6-8 weeks) C57BL6 mice were randomly assigned to the following groups (10/group) for 12 weeks: (i) normal chow + NaCl (1 mmol/kg/day), (ii) normal chow + NaNO3 (1 mmol/kg/day), (iii) high-fat diet + NaCl (1 mmol/kg/day), and (iv) high-fat diet + NaNO3 (1 mmol/kg/day). Body weight and food consumption were monitored weekly. A subset of mice (5/group) underwent running wheel assessment. At the end of the treatment period, all mice underwent fasting glucose tolerance testing. Caecum contents, serum, and tissues (liver, skeletal muscle, white and brown adipose, and kidney) were collected, frozen, and stored at -80°C until analysis. RESULTS: Consumption of the high-fat diet resulted in significantly greater weight gain that was not affected by dietary nitrate. Mice on the high-fat diet also had impaired glucose tolerance that was not affected by dietary nitrate. There was no difference in adipose tissue expression of thermogenic proteins or energy expenditure as assessed by the running wheel activity. Mice on the high-fat diet and those receiving dietary nitrate had reduced caecum concentrations of both butyrate and propionate. CONCLUSIONS: Dietary nitrate does not prevent development of the metabolic syndrome in mice fed a high-fat diet. This may be due, in part due, to reductions in the concentration of important short-chain fatty acids.

F2-isoprostanes affect macrophage migration and CSF-1 signalling.

F2-isoprostanes (F2-IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2-IsoP have been detected in atherosclerotic plaque. Colony stimulating factor-1 (CSF-1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2-IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F2t-isoprostane (15-F2t-IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F2t-IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF-1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages. We showed that 15-F2t-IsoP dose-dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF-1, with maximum effect at 10 µM. Analysis of CSF-1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15-F2t-IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15-F2t-IsoP signalling. Pyk2 silencing inhibited 15-F2t-IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F2t-IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F2t-IsoP-stimulated macrophage migration. These results demonstrate that 15-F2t-IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis.

Impaired ex vivo leukotriene B4 production characterizes the metabolic syndrome and is improved after weight reduction.

CONTEXT: Neutrophil (polymorphonuclear neutrophil) production of leukotriene B4 (LTB4) may be associated with alterations in immune and inflammatory function that characterize the metabolic syndrome (MetS). OBJECTIVE: We investigated whether polymorphonuclear neutrophil production of LTB(4) and its metabolites 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxyl-LTB4 were altered in subjects with features of the MetS before and after weight reduction. DESIGN, SETTING, PATIENTS, AND INTERVENTION: In a case-controlled comparison, men and postmenopausal women with features of the MetS were matched with controls. Subjects with MetS were then matched and randomly assigned to either a 12-wk weight reduction study followed by 4-wk weight stabilization or 16-wk weight maintenance. MAIN OUTCOME MEASURES: Measurements were performed at baseline and at the end of the 16-wk period. Stimulated neutrophil LTB4 and its metabolites were measured by HPLC. RESULTS: In the case-controlled study, body mass index, waist circumference, blood pressure, fasting triglycerides, and glucose were all significantly increased in subjects with features of the MetS (P < 0.05). Production of LTB4 and 20-OH-LTB4 was significantly lower compared with controls (P < 0.005). The weight loss intervention resulted in a 4.6-kg reduction in body weight and 6.6-cm decrease in waist circumference relative to controls and a significant increase in LTB4 and 20-OH-LTB4. CONCLUSIONS: Subjects with features of the MetS have lower stimulated LTB4, which is not due to increased metabolism of LTB4. Weight reduction restored the production of neutrophil LTB4, suggesting that in addition to modifying cardiovascular risk, weight loss may also help with the management of perturbed inflammatory responses in overweight subjects.

Vitamin E metabolism.

The aim of this paper is to provide an overview of vitamin E metabolism. The topics covered include: major classes of vitamin E metabolites; their production pathways and route of excretion; possible biological activities of vitamin E metabolites; and use of vitamin E metabolites as markers of oxidant generation. Recent investigations into vitamin E metabolism have also highlighted important new areas of research, such as the potential for high dose vitamin E supplementation to interfere with drug metabolism, as well as alternative methods to alter vitamin E bioavailability in vivo. These issues will also be discussed in the review.

Retinol supplementation in murine Plasmodium berghei malaria: effects on tissue levels, parasitaemia and lipid peroxidation.

Reduced plasma retinol concentrations occur in human malaria but the benefits of supplementation remain uncertain. We assessed the in vivo efficacy of retinol administration, and its effect on lipid peroxidation, in a Plasmodium berghei murine model. Animals received vehicle (n=17) or retinol (i) before P. berghei inoculation (four doses), (ii) at parasitaemia 10-15% (three to four doses) or (iii) before and after inoculation (six to seven doses; n=15 in each group), with euthanasia on day 8 post-inoculation or when the parasitaemia exceeded 50%. Multiple-dose pre-inoculation retinol reduced endpoint parasitaemia by 24% (P=0.001 versus controls). A reduction of 18% (P=0.042) was observed when retinol was given to parasitaemic animals. Retinol was ineffective when given both before and after infection (11% reduction; P=0.47). Although retinol supplementation did not change plasma retinol concentrations, liver retinol content increased and correlated inversely with endpoint parasitaemia (r=-0.45, P=0.001). Malaria infection augmented concentrations of the free radical lipid peroxidation end-product F(2)-isoprostanes in plasma, erythrocytes and liver by 1.8-, 2.8- and 4.9-fold, respectively, but retinol supplementation had no effect on these increases. Consistent with some human malaria studies, prophylactic retinol reduces P. berghei parasitaemia. This effect relates to augmentation of tissue retinol stores rather than to retinol-associated changes in oxidant status.

Structural requirements of flavonoids to induce heme oxygenase-1 expression.

Population studies suggest cardiovascular health benefits of consuming fruits and vegetables rich in polyphenolic compounds such as flavonoids. We reported previously that the flavonoid quercetin protects arteries from oxidant-induced endothelial dysfunction and attenuates atherosclerosis in apolipoprotein E gene knockout mice, with induction of heme oxygenase-1 (Hmox1) playing a critical role. The present study investigated the structural requirements of flavonoids to induce Hmox1 in human aortic endothelial cells (HAEC). We identified ortho-dihydroxyl groups and an α,ß-unsaturated system attached to a catechol as the key structural requirements for Hmox1 induction. Active but not inactive flavonoids had a low oxidation potential and prevented ascorbate autoxidation, suggesting that Hmox1 inducers readily undergo oxidation and that oxidized, rather than reduced, flavonoids may be the biological inducer of Hmox1. To test this hypothesis, we synthesized stable derivatives of caffeic acid (3-(3,4-dihyroxyphenyl)-2-propenoic acid) containing either ortho-dihydroxy or ortho-dioxo groups. Compared with the dihydroxy compound, the quinone analog induced Hmox1 more potently in HAEC and also provided enhanced protection to arteries of wild type animals against oxidant-induced endothelial dysfunction. In contrast, the quinone analog failed to provide protection against oxidant-induced endothelial dysfunction in arteries of Hmox1-/- mice, establishing a key role for Hmox1 in vascular protection. These results suggest that oxidized forms of dietary polyphenols are the likely inducers of Hmox1 and may explain in part the protective cardiovascular effects of diets rich in these compounds.

Low dose dietary nitrate improves endothelial dysfunction and plaque stability in the ApoE-/- mouse fed a high fat diet.

BACKGROUND: Nitric oxide (NO) is an important vascular signalling molecule. NO is synthesised endogenously by endothelial nitric oxide synthase (eNOS). An alternate pathway is exogenous dietary nitrate, which can be converted to nitrite and then stored or further converted to NO and used immediately. Atherosclerosis is associated with endothelial dysfunction and subsequent lesion formation. This is thought to arise due to a reduction in the bioavailability and/or bioactivity of endogenous NO. AIM: To determine if dietary nitrate can protect against endothelial dysfunction and lesion formation in the ApoE-/- mouse fed a high fat diet (HFD). METHODS AND RESULTS: ApoE-/- fed a HFD were randomized to receive (i) high nitrate (10mmol/kg/day, n=12), (ii) moderate nitrate (1mmol/kg/day, n=8), (iii) low nitrate (0.1mmol/kg/day, n=8), or (iv) sodium chloride supplemented drinking water (control, n=10) for 10 weeks. A group of C57BL6 mice (n=6) received regular water and served as a healthy reference group. At 10 weeks, ACh-induced vessel relaxation was significantly impaired in ApoE-/- mice versus C57BL6. Mice supplemented with low or moderate nitrate showed significant improvements in ACh-induced vessel relaxation compared to ApoE-/- mice given the high nitrate or sodium chloride. Plaque collagen expression was increased and lipid deposition reduced following supplementation with low or moderate nitrate compared to sodium chloride, reflecting increased plaque stability with nitrate supplementation. Plasma nitrate and nitrite levels were significantly increased in all three groups fed the nitrate-supplemented water. CONCLUSION: Low and moderate dose nitrate significantly improved endothelial function and atherosclerotic plaque composition in ApoE-/- mice fed a HFD.

Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation.

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.

Differential inhibition of thromboxane B2 and leukotriene B4 biosynthesis by two naturally occurring acetylenic fatty acids.

The seed oil of the plant Ixiolaena brevicompta is a rich source of crepenynic acid (octadec-cis-9-en-12-ynoic acid), which has been linked with extensive sheep mortalities in Western New South Wales and Queensland, Australia. A number of acetylenic fatty acids have been found to interfere with lipid and fatty acid metabolism and inhibit cyclooxygenase and lipoxygenase enzymes in a variety of tissues. We have investigated the effects of crepenynic acid and ximenynic acid (octadec-trans-11-en-9-ynoic acid) on leukotriene B4 and thromboxane B2 production in rat peritoneal leukocytes and compare them with non-acetylenic compounds linoleic and ricinoleic acids. In concentrations ranging from 10 to 100 microM linoleic acid and ricinoleic acid had only minimal effects on leukotriene B4 and thromboxane B2 production in ionophore-stimulated cells. Ximenynic acid gave dose-dependent inhibition of leukotriene B4, thromboxane B2 and 6-ketoprostaglandin F1 alpha production. Ximenynic acid appears to be a more effective inhibitor of leukotriene B4 than crepenynic acid with an IC50 of 60 microM compared to 85 microM. On the other hand, crepenynic acid is a much more effective inhibitor of the cyclooxygenase products, having an IC50 for thromboxane B2 of less than 10 microM. Both acetylenic fatty acids inhibited phospholipase activity in these cells by 40-50% at a concentration of 100 microM but had no inhibitory effect at 10 microM. These results indicate that crepenynic acid and ximenynic acid differentially inhibit the cyclooxygenase and lipoxygenase products of stimulated leukocytes, and that at high doses of these fatty acids the effect on these products may be partially due to inhibition of phospholipase A2.

Dietary modification of fatty acid and prostaglandin synthesis in the rat. Effect of variations in the level of dietary fat.

Dietary supplements with safflower oil, linseed oil, cod liver oil and hydrogenated coconut oil were given to rats at levels of 5, 20 and 40 energy % to simultaneously assess the effects of both type and level of dietary fat on tissue fatty acid composition and prostanoid synthesis. There was no significant change in weight gain or blood pressure between the dietary groups after the 4-week dietary regimen. The liver oil and linseed oil diets depressed the arachidonic acid content of kidney phospholipids at all levels of supplementation. The arachidonic acid content of plasma lipids was significantly elevated in animals on the safflower oil diet at 20 and 40 energy % while those on the same level of liver oil diet showed a marked reduction in arachidonic acid. Whole blood synthesis of thromboxane B2 varied significantly at all levels of fat supplementation, with the 20 energy % safflower oil fed group showing maximally enhanced thromboxane B2 production compared to the coconut oil group (P less than 0.001). Conversely, the liver oil groups showed depressed thromboxane B2 synthesis at 20 and 40 energy % (P less than 0.01) compared to the coconut oil group and at 5 energy % compared to the safflower oil group (P less than 0.05). Production of 6-ketoprostaglandin F1 alpha and prostaglandin E2 by incubated kidney homogenates only differed significantly between the dietary groups at 40 energy %. Urinary excretion of 6-ketoprostaglandin F1 alpha was increased by 45% and 55% in rats fed the safflower oil diet at 20 and 40 energy %, respectively compared to the saturated fat diet, while in the liver oil groups excretion was reduced by 20% and 32%. Dietary suppression of prostanoid synthesis is explained in part by changes in available arachidonic acid and competitive inhibition of cyclooxygenase by (n - 3) fatty acids. Thus, minor changes in dietary fat can readily alter tissue fatty acid composition, but both the extent and nature of changes in phospholipid and prostanoid metabolism vary markedly according to the tissue site.

Oxidation of low-density lipoproteins: effect of antioxidant content, fatty acid composition and intrinsic phospholipase activity on susceptibility to metal ion-induced oxidation.

The oxidative modification of low-density lipoprotein (LDL) may play an important role in atherogenesis. Our understanding of the mechanism of LDL oxidation and the factors that determine its susceptibility to oxidation is still incomplete. We have isolated LDL from 45 healthy individuals and studied the relationship between LDL fatty acid, vitamin E and beta-carotene composition, intrinsic phospholipase A2-like activity and parameters of LDL oxidation. LDL was exposed to a copper ion-dependent oxidising system and the kinetics of oxidation studied by monitoring formation of fatty acid conjugated dienes. The length of the lag phase of inhibited lipid peroxidation was measured as well as the rate of lipid peroxidation during the propagation phase. There was no significant correlation between LDL antioxidant vitamin or fatty acid composition and lag time to LDL oxidation. Oleic acid was negatively correlated with the rate of LDL oxidation (r = -0.41, P < 0.01) whilst linoleic acid was significantly correlated with the extent of LDL oxidation measured by the production of total dienes (r = 0.34, P < 0.05). Interestingly, LDL vitamin E content was positively correlated with both the rate (r = 0.28, P < 0.05) and extent of LDL oxidation (r = 0.43, P < 0.01). LDL isolated from this group of subjects showed significant intrinsic phospholipase-like activity. The phospholipase activity, whilst not correlated with lag time, was significantly correlated with both rate (r = 0.43, P < 0.01) and total diene production (r = 0.44, P < 0.01) of LDL oxidation. We conclude that antioxidant content, fatty acid composition and intrinsic phospholipase activity have little influence on the lag time of Cu-induced LDL oxidation. These components do however, significantly influence both the rate and extent of LDL oxidation, with increased vitamin E, linoleic acid content and phospholipase activity associated with faster and more extensive oxidation. The possible pro-oxidant effect of vitamin E has interesting implications for the postulated 'protective' effects of vitamin E on atherogenesis.

The effect of vitreous humour on prostaglandin production by cultured rabbit chorioretinal fibroblasts.

Factors in vitreous humour which regulate prostaglandin production were investigated using cultured rabbit chorioretinal fibroblasts. These cells produced predominantly prostaglandin E2, 6-ketoprostaglandin F1 alpha, a compound likely to be a metabolite of prostaglandin E2 and 5-hydroxyeicosatetraenoic acid. The synthesis of 6-ketoprostaglandin F1 alpha was nearly completely inhibited by the cyclooxygenase inhibitor aspirin and partially inhibited by 10(-6) M dexamethasone (49%) and 10(-5) M forskolin (68%). Addition of 10% rabbit vitreous humour to subconfluent cells maintained in Dulbecco's modified Eagle's medium plus 1% fetal bovine serum resulted in stimulation of 6-ketoprostaglandin F1 alpha production by as much as 246% as measured by radioimmunoassay. Chorioretinal fibroblasts labelled by [3H]arachidonic acid incorporation into cellular phospholipids synthesised greater amounts of all labelled arachidonic acid metabolites in response to vitreous humour. It was concluded, therefore, that there are factors present in vitreous humour of molecular weight above 10 kDa which are capable of stimulating cellular cyclooxygenase activity. Confluent cells also responded to a factor(s) present in vitreous humour. The fraction of less than 10 kDa inhibited 6-ketoprostaglandin F1 alpha production by 50% when used at a concentration of 10%. Furthermore, 6-ketoprostaglandin F1 alpha production in confluent cells (but not subconfluent cells) was inhibited to 40% of control levels by vitamin C at a concentration of 1 mg/100 ml. The latter result points to an inhibitory role for vitamin C in vitreous humour. We conclude, therefore, that vitreous humour contains factors important for the regulation of prostaglandin metabolism in the eye.

Onset of changes in phospholipid fatty acid composition and prostaglandin synthesis following dietary manipulation with n-6 and n-3 fatty acids in the rat.

A synthetic diet preparation supplemented with 10% by weight of either safflower oil, hydrogenated coconut oil containing 3% safflower oil, or 'max EPA' fish oil was fed to rats over a 8-week period. Serial measurements of serum fatty acids, serum thromboxane B2 and urinary prostaglandin excretion were taken during the treatment period to assess the rate of change in fatty acid composition and prostaglandin synthesis following dietary manipulation. There was no significant change in weight gain between the dietary groups during the treatment period. Significant changes in serum fatty acids occurred within 48 h of treatment, with the 'max EPA' oil group having arachidonic acid levels reduced by 23% (P less than 0.01) compared to the coconut oil group. Conversely, rats fed safflower oil had an 18% enhancement of arachidonic acid during the same time period. Whole blood synthesis of thromboxane B2 was significantly depressed (P less than 0.01) after 48 h in rats fed 'max EPA' oil compared to the safflower oil or coconut oil groups. This suppression reached a maximum of 65% (P less than 0.001) after 7 days of dietary 'max EPA' oil treatment. The safflower oil and coconut oil-fed groups showed the same levels of serum thromboxane B2 production over the treatment period. Urinary excretion of both 6-ketoprostaglandin F1 alpha and prostaglandin E2 varied significantly (P less than 0.01) between the groups after 7 days of dietary treatment. Rats fed 'max EPA' oil had depressed urinary prostanoid excretion compared to the safflower and coconut oil groups which remained very similar to each other. After the 8-week treatment period rats were killed and the phospholipid fatty acid composition and prostaglandin-generating capacity of platelets, aorta and renal tissue was examined. Prostanoid production by kidney cortex and medulla and segments of aorta was consistently suppressed in rats fed 'max EPA' oil. These observations correlated well with changes in the phospholipid fatty acid profiles in these tissues. This study shows rapid changes in serum fatty acids and thromboxane B2 generation following dietary manipulation, while changes in urinary excretion or prostanoid metabolites occur only after a longer time period.

Dietary cosupplementation with vitamin E and coenzyme Q(10) inhibits atherosclerosis in apolipoprotein E gene knockout mice.

Intimal oxidation of LDL is considered an important early event in atherogenesis, and certain antioxidants are antiatherogenic. Dietary coenrichment with vitamin E (VitE) plus ubiquinone-10 (CoQ(10), which is reduced during intestinal uptake to the antioxidant ubiquinol-10, CoQ(10)H(2)) protects, whereas enrichment with VitE alone can increase oxidizability of LDL lipid against ex vivo oxidation. In the present study, we tested whether VitE plus CoQ(10) cosupplementation is more antiatherogenic than either antioxidant alone, by use of apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet without (control) or with 0.2% (wt/wt) VitE, 0.5% CoQ(10), or 0.2% VitE plus 0.5% CoQ(10) (VitE+CoQ(10)) for 24 weeks. None of the supplements affected plasma cholesterol concentrations, whereas in the VitE and CoQ(10) groups, plasma level of the respective supplement increased. Compared with control, plasma from CoQ(10) or VitE+CoQ(10) but not VitE-supplemented animals was more resistant to ex vivo lipid peroxidation induced by peroxyl radicals. VitE supplementation increased VitE levels in aorta, heart, brain, and skeletal muscle, whereas CoQ(10) supplementation increased CoQ(10) only in plasma and aorta and lowered tissue VITE: All treatments significantly lowered aortic cholesterol compared with control, but only VitE+CoQ(10) supplementation significantly decreased tissue lipid hydroperoxides when expressed per parent lipid. In contrast, none of the treatments affected aortic ratios of 7-ketocholesterol to cholesterol. Compared with controls, VitE+CoQ(10) supplementation decreased atherosclerosis at the aortic root and arch and descending thoracic aorta to an extent that increased with increasing distance from the aortic root. CoQ(10) significantly inhibited atherosclerosis at aortic root and arch, whereas VitE decreased disease at aortic root only. Thus, in apoE-/- mice, VitE+CoQ(10) supplements are more antiatherogenic than CoQ(10) or VitE supplements alone and disease inhibition is associated with a decrease in aortic lipid hydroperoxides but not 7-ketocholesterol.

Regular consumption of black tea increases circulating kynurenine concentrations: A randomized controlled trial.

BACKGROUND: Circulating neopterin and the ratio of kynurenine to tryptophan (KYN/TRP) concentrations are biomarkers of immune activation that have been linked to cardiovascular and total mortality. Several in vitro studies indicated that tea flavonoids and other antioxidants can modulate tryptophan breakdown rates and neopterin production in immune cells. We aimed to assess the effects of regular black tea consumption on tryptophan and neopterin metabolisms in vivo. METHODS: Participants were healthy individuals, with no major illnesses and having normal to mildly elevated systolic blood pressure. They were randomly assigned to consume 3 cups/day of either powdered black tea solids (tea; n = 45) or a flavonoid-free caffeine-matched beverage (control; n = 49). Serum concentrations of tryptophan, kynurenine and neopterin were assessed at baseline and again at 3 and 6 months after daily ingestion of the respective beverage. RESULTS: Regular consumption of tea over 6 months, compared to control, did not significantly alter neopterin (p = 0.13) or tryptophan (p = 0.85) concentrations, but did result in significantly higher kynurenine (p = 0.016) and KYN/TRP (p = 0.012). Relative to the control group, in the tea group kynurenine and KYN/TRP increased during the treatment period by 0.28 µmol/L (95% CI: - 0.04, 0.60) and 3.2 µmol/mmol (95% CI: - 1.6, 8.0), respectively at 3 months, and by 0.48 µmol/L (95% CI: 0.16, 0.80) and 7.5 µmol/mmol (95% CI: 2.5, 12.5), respectively at 6 months. CONCLUSIONS: Increased circulation of kynurenine and KYN/TRP following regular black tea consumption may indicate enhanced tryptophan breakdown, possibly due to immune activation-induced tryptophan degrading enzyme indoleamine 2,3-dioxygenase. GENERAL SIGNIFICANCE: The influence of black tea consumption on biomarkers of immune system activation could relate to its general health benefits. Data suggests that the net effect strongly depends on the individual immune state, being stimulatory in healthy individuals, while acting more immune dampening in situations with an inflammatory background.