Inhibition Of LDL Oxidation Research Articles

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: Flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H 2O 2/Cl − system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H 2O 2/Cl − system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC 50 = 1.1 ± 0.3 μM) is the most efficient inhibitor of the MPO/H 2O 2/Cl − system and nimesulide (IC 50 = 2.1 ± 0.3 μM) is more active than the other NSAIDs of the oxicam family (IC 50 = 8–12 μM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H 2O 2/Cl − system. Flufenamic acid has the best inhibiting activity towards the MPO/H 2O 2/Cl − system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.

Ex vivo inhibitory effect on tilapia LDL oxidation and hypolipidemia properties of Glycine tomentella root extract

The ethanolic extract of I-Tiao Gung (GT-E) (Glycine tomentella root extract) was found to reduce the oxidative rate and prolonged lag phase of LDL in human (Homo sapiens) and tilapia (Oreochromis mossambicus). The in vivo effect of GT-E was determined using tilapia as a model. Hyperlipidemia and hypercholesterolemia were induced in fish by feeding commercial feed daily at 2% body mass for 8 weeks, or at 1% body mass for 12 weeks. Thirty two adult male tilapia were randomly divided into two groups and fed with feed containing 1% (w/w) GT-E or control diet for 12 weeks. Specific growth rate was similar between the GT-E group and the control group. Total triacylglycerol, total cholesterol and low-density lipoprotein cholesterol (LDL-C) in plasma of the GT-E group were significantly lower, while plasma total antioxidant status was significantly higher than those of the control group. GT-E fed fish had longer lag phase of Cu2+-induced LDL oxidation and retained more alpha-tocopherol in LDL particles than the control fish. LDL from the GT-E group had more monounsaturated fatty acids and less polyunsaturated fatty acids than the control group indicative of its effect on fatty acids metabolism. GT-E demonstrated hypolipidemic and hypocholesterolemic effects and inhibiting LDL oxidation in tilapia similar to the effects in mammals, thus tilapia can serve as a surrogate animal model for prescreening anti-atherosclerosis effect of natural products.

Continuous intake of polyphenolic compounds containing cocoa powder reduces LDL oxidative susceptibility and has beneficial effects on plasma HDL-cholesterol concentrations in humans

Background:Cocoa powder is rich in polyphenols such as catechins and procyanidins and has been shown in various models to inhibit LDL oxidation and atherogenesis.Objective:We examined whether long-term intake of cocoa powder alters plasma lipid profiles in normocholesterolemic and mildly hypercholesterolemic human subjects.Design:Twenty-five subjects were randomly assigned to ingest either 12 g sugar/d (control group) or 26 g cocoa powder and 12 g sugar/d (cocoa group) for 12 wk. Blood samples were collected before the study and 12 wk after intake of the test drinks. Plasma lipids, LDL oxidative susceptibility, and urinary oxidative stress markers were measured.Results:At 12 wk, we measured a 9% prolongation from baseline levels in the lag time of LDL oxidation in the cocoa group. This prolongation in the cocoa group was significantly greater than the reduction measured in the control group (−13%). A significantly greater increase in plasma HDL cholesterol (24%) was observed in the cocoa group than in the control group (5%). A negative correlation was observed between plasma concentrations of HDL cholesterol and oxidized LDL. At 12 wk, there was a 24% reduction in dityrosine from baseline concentrations in the cocoa group. This reduction in the cocoa group was significantly greater than the reduction in the control group (−1%).Conclusion:It is possible that increases in HDL-cholesterol concentrations may contribute to the suppression of LDL oxidation and that polyphenolic substances derived from cocoa powder may contribute to an elevation in HDL cholesterol.

Antioxidant activity of cleomiscosins A and C isolated fromAcer okamotoanum

Phytochemical investigation of Acer okamotoanum leaf and twig led to the isolation of two coumarinolignans, cleomiscosin A (1) and cleomiscosin C (2). Here, we found that 2 dose-dependently inhibits LDL oxidation mediated by either catalytic copper ions (Cu2+) or free radicals generated with the azo compound 2,2'-azobis-(2-amidinopropane)dihydro-chloride (AAPH) with IC50s of 29.5 and 11.9 microM, respectively. By electrophoretic analysis, we also observed that 2 protects apolipoprotein B-100 (apoB-100) against Cu2+-induced fragmentation (65.3% inhibition at 5 microM). Furthermore, fluorescence analyses clearly indicated that both 1 and 2 protect against the oxidative modification of apoB-100 induced by either Cu2+ or HOCl (1, IC50s of 13.4 and 8.1 microM, respectively; 2, IC50s of 23.6 and 3.9 microM, respectively). These findings suggest that 1 and 2 could be beneficial in preventing LDL oxidation in atherosclerotic lesions.

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St John's wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St John's wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.

LDL are oxidatively modified by plateletsviaGP91phoxand accumulate in human monocytes

Oxidative stress-mediated LDL modification has a key role in initiation of the atherosclerotic process. Platelets produce reactive oxidant species (ROS) upon stimulation with agonist, but it is uncertain whether they are able to oxidatively modify LDL. Human platelets taken from healthy subjects were incubated with LDL, then stimulated with collagen. Compared with unstimulated platelets, collagen-stimulated platelets induced LDL modification as shown by enhanced conjugated dienes and lysophosphatidylcholine formation, electrophoretic mobility, Apo B-100 degradation, and monocyte LDL uptake. Activated platelets also induced a marked reduction of vitamin E contained in LDL. A significant inhibition of LDL oxidation was observed in platelets treated with arachidonyl trifluomethyl ketone (AACOCF3), an inhibitor of phospholipase A2. The experiments reported above were also conducted in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, and in patients with hypercholesterolemia. Platelets from gp91 phox-deficient patients produced a small amount of ROS and weakly modified LDL. Conversely, platelets from hypercholesterolemic patients showed enhanced ROS formation and oxidized LDL more than platelets from healthy subjects. This study provides evidence that platelets modify LDL via NADPH oxidase-mediated oxidative stress, a phenomenon that could be dependent on arachidonic acid activation. This finding suggests a role for platelets in favoring LDL accumulation within atherosclerotic plaque.

Role of Scavenger Receptor Class B Type I and Sphingosine 1-Phosphate Receptors in High Density Lipoprotein-induced Inhibition of Adhesion Molecule Expression in Endothelial Cells

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) alpha in endothelial cells. HDL inhibited S1P-induced nuclear factor kappaB activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNFalpha was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P(1), in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P(3) receptors severely suppressed the stimulatory actions of S1P. Although G(i/o) proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P(3) receptors to G(12/13) proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P(3) receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNFalpha-induced actions through S1P receptors, especially S1P(1).

Isolation of ellagic acid from the aqueous extract of the roots of Decalepis hamiltonii: Antioxidant activity and cytoprotective effect

Roots of Decalepis hamiltonii are consumed as pickles and as a beverage and are believed to possess health-promoting properties. Roots of D. hamiltonii exhibit antioxidant properties. We have isolated an antioxidant compound from the aqueous extract of the roots of D. hamiltonii and identified it as ellagic acid (EA), based on NMR and MS. EA exhibited free radical-scavenging activity, inhibited LDL oxidation and showed cytoprotective effect against xenobiotic-induced oxidative stress in Ehrlich Ascites tumor cells. EA is a new addition to the list of antioxidant compounds in the roots of D. hamiltonii.

Marrubium vulgare extract inhibits human-LDL oxidation and enhances HDL-mediated cholesterol efflux in THP-1 macrophage

The objective of the present study was to elucidate the beneficial properties of aqueous extracts of Marrubium vulgare (AEM) towards cardiovascular disease by protecting human-LDL against lipid peroxidation and promoting HDL-mediated cholesterol efflux. Human-LDL were oxidised by incubation with CuSO 4 in the presence of increased concentrations of AEM (0–100 μg/ml). LDL lipid peroxidation was evaluated by conjugated diene formation, vitamin E disappearance as well as LDL-electrophoretic mobility. HDL-mediated cholesterol efflux assay was carried out in human THP-1 macrophages. Incubation of LDL with AEM significantly prolonged the lag phase ( P = 0.014), lowered the progression rate of lipid peroxidation ( P = 0.004), reduced the disappearance of vitamin E and the electrophoretic mobility in a dose-dependent manner. Also, incubation of HDL with AEM significantly increased HDL-mediated cholesterol efflux from THP-1 macrophages implicating an independent ATP binding cassette A1 (ABCA1) pathways. Our findings suggest that M. vulgare provides a source of natural antioxidants, which inhibit LDL oxidation and enhance reverse cholesterol transport and thus can prevent cardiovascular diseases development. These antioxidant properties increase the anti-atherogenic potential of HDL.

Salvianolic acid B attenuates MMP‐2 and MMP‐9 expression in vivo in apolipoprotein‐E‐deficient mouse aorta and in vitro in LPS‐treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.

Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activities

Our ex vivo study revealed that BRE had significantly stronger ability to inhibit LDL oxidation than white rice extract (WRE). The purpose of this study was to investigate whether black rice extract (BRE) supplementation might ameliorate oxidative stress and enhance antioxidant enzyme activities in HepG2 cells and in C57BL/6 mice. In the cellular study, superoxide anions (O2*-) and reactive oxygen species (ROS) in the BRE group were significantly suppressed. The BRE group also showed significant increases in superoxide dismutase (SOD) and catalase (CAT) activities by 161.6% and 73.4%, respectively. The major components responsible for the free-radical-scavenging and antioxidative properties might be cyanidin-3-O-glucoside chloride and peonidin-3-O-glucuside chloride. In the animal study, male C57BL/6 mice were divided into three groups (control, BRE, and WRE). Plasma HDL-cholesterol was significantly higher, and thiobarbituric, acid-reactive substances were significantly lower in the BRE group, whereas plasma levels of total cholesterol and triglyceride were not affected by BRE supplementation. Increased hepatic SOD and CAT activities were observed in BRE-treated mice as compared to the control mice. However, no changes were detected for the protein expression of antioxidant enzymes by Western blot analysis. Our data suggest that antioxidative effects exerted by BRE are mediated through decreases in free-radical generation as well as increases in SOD and CAT activities both in vitro and in vivo.

Antioxidant properties of extracts from juemingzi ( Cassia tora L.) evaluated in vitro

Methanol extract from juemingzi ( Cassia tora L.) was fractionated by liquid–liquid partition using ethyl acetate, n-butanol and water, respectively. The antioxidant activity of three different fractions was evaluated using different antioxidant tests, including inhibition of peroxidation of linoleic acid, reducing power, free-radical scavenging and ferrous ions chelating activity. In the above four assays, all the fractions showed antioxidant potential to varying degrees. Among these fractions, ethyl acetate fraction exhibited more antioxidant potency than other fractions. Furthermore, ethyl acetate fraction was found to be more effective in protecting LDL against oxidation in a concentration-dependent manner. The data suggest that juemingzi especially ethyl acetate-soluble fraction may have a preventive effect against atherosclerosis by inhibiting LDL oxidation.

<b>PHLOROGLUCINOL DERIVATIVES AND FLAVONES FROM <i>HELICHRYSUM PARONYCHIOIDES</i></b>

Investigation of Helichrysum paronychioides afforded a total of nine compounds: 4 phloroglucinol derivatives, 2 of which are novel natural products, and 5 flavone derivatives. Structures were established by various spectroscopic techniques (NMR, MS, UV, IR, CD) and by comparison with literature data for the known compounds. The four phloroglucinols, trans -(2 R ,3 R )-5,7-dihydroxy-2,3-dimethyl-4-chromanone ( 1 ), 2-butanoyl-4-prenyl-1-methoxy phloroglucinol ( 2 ), 2-(2-methylpropanoyl)-4-prenylphloroglucinol ( 3 ) and 2-(2-methyl- butanoyl)-4-prenylphloroglucinol ( 4 ) were screened for antioxidant activity against Cu-induced LDL oxidation. Compound 4 was found to be the most active inhibiting LDL oxidation at all concentrations (0.5-10 μM) while the other three showed moderate to no activity. KEY WORDS : Helichrysum paronychioides , Asteraceae, Phloroglucinol derivatives, CD spectroscopy, Synthesis Bull. Chem. Soc. Ethiop. 2006 , 20(1), 61-68.

The pleiotropic effect of statins in haemodialysis patients is not the consequence of an inhibition of LDL oxidation by myeloperoxidase

The pleiotropic effect of statins in haemodialysis patients is not the consequence of an inhibition of LDL oxidation by myeloperoxidase

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE −/−) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE −/− mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS −/−/apoE −/− mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS +/+/apoE −/− mice (30,993 ± 4746 vs. 26,648 ± 6815 μm 2/section; P = 0.608). After being fed the Western diet for 12 weeks, iNOS −/−/apoE −/− mice developed significantly smaller advanced lesions than iNOS +/+/apoE −/− mice (458,734 ± 14,942 vs. 519,570 ± 22,098 μm 2/section; P = 0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-α, and then incubated with native LDL in the absence or presence of N-Ω-nitro- l-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS +/+/apoE −/− mice ( P = 0.048), but it had no effect on LDL oxidation by cells from iNOS −/−/apoE −/− mice. iNOS −/−/apoE −/− mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74 ± 0.23 vs. 3.89 ± 0.41 μM MDA; P = 0.021) but not on chow diet (1.02 ± 0.07 vs. 1.51 ± 0.29 μM MDA; P = 0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS −/−/apoE −/− mice.

Salvianolic acid B attenuates cyclooxygenase‐2 expression in vitro in LPS‐treated human aortic smooth muscle cells and in vivo in the apolipoprotein‐E‐deficient mouse aorta

Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.

We-P14:394 Inhibition of LDL oxidation in red yeast rice

We-P14:394 Inhibition of LDL oxidation in red yeast rice

Polyphenols synergistically inhibit oxidative stress in subjects given red and white wine

Aim of this study was to analyse the relationship between the plasma levels of polyphenols and the antioxidant activity of red and white wine. Twenty healthy subjects (HS) were randomly allocated to drink 300 ml of red ( n = 10) or white n = 10 wine for 15 days. Ten HS who refrained from any alcohol beverage for 15 days were used as control. Urinary PGF-2α-III, a marker of oxidative stress and plasma levels of polyphenols were measured. Urinary PGF-2α-III significantly fell in subjects taking wine with a higher percentage decrease in subjects given red wine (−38.5 ± 6%, p < 0.001) than in those given white wine (−23.1 ± 6%). Subjects taking red wine had higher plasma polyphenols than those taking white wine (1.9 ± 0.6 μM versus 1.5 ± 0.33 μM, p < 0.001). Plasma polyphenols were inversely correlated with urinary PGF2α ( r = 0.77, p < 0.001). No changes of urinary isoprostanes were observed in subjects who refrained from wine intake. In vitro study demonstrated that only a mixture of polyphenols, all in a range corresponding to that found in human circulation, inhibited LDL oxidation and PKC-mediated NADPH oxidase activation. Such inhibitory effects were more marked using the concentrations of polyphenols detected in human circulation after red wine intake. This study shows that red wine is more antioxidant than white wine in virtue of its higher content of polyphenols, an effect that may be dependent upon a synergism among polyphenols.

Electron Paramagnetic Resonance Study of Cell-Free Hemoglobin in Sickle Cell Disease: Potential Antioxidant Role of Haptoglobin.

It has previously been shown that red blood cell-free oxyhemoglobin (oxyHb) can affect endothelial function in Sickle Cell Disease (SCD) by virtue of its NO scavenging properties (Reiter et al, Nat Med, 2002). This reaction may have down-stream consequence via formation of metHb which has been demonstrated to have pro-oxidant properties and can oxidize low-density lipoprotein in vitro. In this study we have examined the fate of metHb in SCD vs normal plasma by examining the characteristic single line EPR signature of metHb in the g = 6 region. We show that the EPR signature of metHb differs in SCD compared with normals and exhibits a split EPR line indicative of a more rhombic heme geometry (See Figure). [Display omitted] Addition of purified metHb to SCD plasma results in a conversion of the single line EPR spectrum to the rhombic form. By addition of hemin and hemoglobin to plasma constituents we have determined that the rhombic EPR spectrum is consistent with heme bound to serum albumin. In addition, immunoprecipitation of serum albumin from SCD plasma with EPR analysis of the IP fraction revealed the presence of albumin associated metHb. Interestingly, the transfer of heme from metHb to albumin is accelereated by the presence of low-density lipoprotein and inhibited by the addition of haptoglobin suggesting that plasma components can modulate heme release from metHb and that the lack of haptoglobin in SCD plasma allowed the transfer of heme from metHb to albumin. To confirm this we added metHb to SCD plasma in the presence and absence of haptoglobin and observed that the addition of haptoglobin prevented conversion of the metHb spectrum to the albumin associated split (rhombic) species. From these studies we conclude that plasma haptoglobin not only binds hemoglobin but also limits heme release from metHb. To examine potential oxidative consequences of this effect, we incubated LDL with metHb in the presence and absence of haptoglobin. Haptoglobin inhibited LDL oxidation in a dose-dependent manner. Interestingly, the 1-1 isoform of haptoglobin was much more effective than the 2-2 isoform, in agreement with published studies on the efficacy of hemoglobin binding to the two haptoglobin varieties. Additionally, we have measured levels of 8-isoprostanes in the plasma of SCD patients as an index of lipid oxidation and show that 8-isoprostane levels are significantly elevated in SCD patients (295 209 pg/ml, mean S.D., n= 13, vs. 69 37 pg/ml, mean S.D. n= 5 for normal controls). In conclusion, these studies provide evidence that the saturation of the haptoglobin clearance mechanisms in hemolytic disorders such as SCD can not only affect the NO-axis of endothelial function due to increases in cell-free hemoglobin, but can also lead to altered heme metabolism leading to lipid oxidation and formation of pro-inflammatory lipids which have the potential to alter both endothelial and red cell function.