No abstract

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.

Abstract-Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis.Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. Key Words: low density lipoproteins Ⅲ myeloperoxidase Ⅲ reactive nitrogen species Ⅲ vitamin C Ⅲ vitamin E T he hypothesis that oxidative stress plays an important role in the pathogenesis of atherosclerosis has gained considerable support. Although there are many determinants in the development of an atherosclerotic lesion, substantial in vitro evidence links LDL oxidation to potentially atherogenic processes at the molecular and cellular level. 1-3 However, the in vivo mechanism(s) of the initiation and progression of LDL oxidation is presently unclear and is a topic of much research. The most relevant information concerning this mechanism has come from immunohistochemical and biochemical analyses of animal and human atherosclerotic lesions and lipoproteins extracted from these lesions. Ceruloplasmin, 15-lipoxygenase, myeloperoxidase (MPO), and inducible (in addition to endothelial) nitric oxide synthase (NOS) have been found in animal and human lesions and can cause or contribute to LDL oxidation in vitro. 2,4 -10 With respect to a role for ceruloplasmin in in vivo LDL oxidation, specific markers of metal ion-catalyzed protein damage are not elevated in early or intermediate lesions, in contrast to advanced lesions. 9,11 Immunohistochemical detection of aldehyde-modified LDL in human and animal lesions 12,13 also has been used as evidence for metal ionmediated oxidation; however, the specificity of antibodies raised against aldehyde-modified or copper-oxidized LDL has been questioned, because these can cross-react with LDL modified by hypochlorous acid (HOCl). 14 Therefore, it appears unlikely that ceruloplasmin and other sources of redoxactive metal ions contribute significantly to LDL oxidation in vivo during the early stages of atherosclerotic lesion development.The role of 15-lipoxygenase in LDL oxidation and atherogenesis is controversial, because the mechanism by which the intracellular enzyme "seeds" extracellular LDL with hydroperoxides is unclear. 15,16 Furthermore, there is limited evidence for the presence of 15-lipoxygenase-modified lipids in lesions, because only small increases in the S/R enantiomeric ratio of lipid hydro(pero)xides were detected. 17,18 Nevertheless, immunohistochemical studies have demonstrated the presence of 15-lipoxygenase in macrophage-rich regions of human and rabbit atherosclerotic lesions, 19,20 and epitopes of oxidized LDL colocalize with 15-lipoxygenase. 20 Interestingly, cholesterol-fed transgenic rabbits overexpressing the human 15-lipoxygenase gene in macrophages develop significantly less atherosclerosis than do the...

Experimental evidence suggests that aldehydes generated as a consequence of lipid peroxidation may be involved in the pathogenesis of atherosclerosis. It is well documented that aldehydes modify LDL: however, less is known concerning the effects of aldehydes on other plasma and interstitial fluid components. In the present study, we investigated the effects of five physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal [HNE], and malondialdehyde [MDA]) on two key constituents of the antiatherogenic reverse cholesterol transport pathway, lecithin-cholesterol acyltransferase (LCAT) and HDL. Human plasma was incubated for 3 hours at 37 degrees C with each one of the five aldehydes at concentrations ranging from 0.16 to 84 mmol/L. Dose-dependent decreases in LCAT activity were observed. The short-chain (acrolein) and long-chain (HNE) alpha,beta-unsaturated aldehydes were the most effective LCAT inhibitors. Micromolar concentrations of these unsaturated aldehydes resulted in significant reductions in plasma LCAT activity. The short- and longer-chain saturated aldehydes acetaldehyde and hexanal and the dialdehyde MDA were considerably less effective at inhibiting LCAT than were acrolein and HNE. In addition to inhibiting LCAT, aldehydes increased HDL electrophoretic mobility and cross-linked HDL apolipoproteins. Cross-linking of apolipoproteins A-I and A-II required higher aldehyde concentrations than inhibition of LCAT. The alpha,beta-unsaturated aldehydes acrolein and HNE were fourfold to eightfold more effective cross-linkers of apolipoproteins A-I and A-II than the other aldehydes studied. These data suggest that products of lipid peroxidation, especially unsaturated aldehydes, may interfere with normal HDL cholesterol transport by inhibiting LCAT and modifying HDL apolipoproteins.

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or`foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoproteindeficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance sitedirected oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants. ß