Inhibition of low-density lipoprotein oxidation by carnosine histidine

Carnosine is a beta-alanylhistidine dipeptide found in skeletal muscle and nervous tissue that has been reported to possess antioxidant activity. Carnosine is a potential dietary antioxidant because it is absorbed into plasma intact. This research investigated the ability of carnosine to inhibit the oxidation of low-density lipoprotein (LDL) in comparison to its constituent amino acid, histidine. Carnosine (3 microM) inhibited Cu2+-promoted LDL (20 of protein/mL) oxidation at carnosine/copper ratios as low as 1:1, as determined by loss of tryptophan fluorescence and formation of conjugated dienes. Carnosine (6 microM) lost its ability to inhibit conjugated diene formation and tryptophan oxidation after 2 and 4 h of incubation, respectively, of LDL with 3 microM Cu2+. Compared to controls, histidine (3 microM) inhibited tryptophan oxidation and conjugated diene formation 36 and 58%, respectively, compared to 21 and 0% for carnosine (3 microM) after 3 h of oxidation. Histidine was more effective at inhibiting copper-promoted formation of carbonyls on bovine serum albumin than carnosine, but carnosine was more effective at inhibiting copper-induced ascorbic acid oxidation than histidine. Neither carnosine nor histidine was a strong inhibitor of 2,2'-azobis(2-amidinopropane) dihydrochloride-promoted oxidation of LDL, indicating that their main antioxidant mechanism is through copper chelation.     

Antioxidant activities of abietane diterpenoids isolated from Torreya nucifera leaves

Investigation on antioxidant compounds from the ethanolic extracts of Torreya nucifera leaves resulted in the isolation of abietane diterpenoids, a known 18-methylesterferruginol (1) and a new 18-dimethoxyferruginol (2). The structures of compounds 1 and 2 were elucidated on the basis of their spectroscopic analyses. Compounds 1 and 2 inhibited the Cu2+-mediated, 2,2'-azobis(2-amidinopropane)hydrochloride-mediated and 3-morpholinosydnonimine-1-mediated low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substances assay as well as the macrophage-mediated LDL oxidation. Compounds 1 and 2 exhibited the potent antioxidant activities in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Compound 1 also suppressed nitric oxide production and inducible nitric oxide synthase expression in lipopolysaccharide-stimulated RAW264.7 cells.     

Antioxidant capacity of oat (Avena sativa L.) extracts. 1. Inhibition of low-density lipoprotein oxidation and oxygen radical absorbance capacity

Milled oat groat pearlings, trichomes, flour, and bran were extracted with methanol and the fractions tested in vitro for antioxidant capacity against low-density lipoprotein (LDL) oxidation and R-phycoerythrin protein oxidation in the oxygen radical absorbance capacity (ORAC) assay. The oxidative reactions were generated by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or Cu(2+) in the LDL assay and by AAPH or Cu(2+) + H(2)O(2) in the ORAC assay and calibrated against a Trolox standard to calculate Trolox equivalents (1 Trolox equivalent = 1 TE = activity of 1 micromol of Trolox). The antioxidant capacity of the oat fractions was generally consistent with a potency rank of pearlings (2.89-8.58 TE/g) > flour (1.00-3.54 TE/g) > trichome (1.74 TE/g) = bran (1.02-1.62 TE/g) in both LDL and ORAC assays regardless of the free radical generator employed. A portion of the oat antioxidant constituents may be heat labile as the greatest activity was found among non-steam-treated pearlings. The contribution of oat tocols from the fractions accounted for <5% of the measured antioxidant capacity. AAPH-initiated oxidation of LDL was inhibited by the oat fractions in a dose-dependent manner, although complete suppression was not achieved with the highest doses tested. In contrast, Cu(2+)-initiated oxidation of LDL stimulated peroxide formation with low oat concentrations but completely inhibited oxidation with higher doses. Thus, oats possess antioxidant capacity most of which is likely derived from polar phenolic compounds in the aleurone.     

Red wine antioxidants bind to human lipoproteins and protect them from metal ion-dependent and -independent oxidation

Plant-derived polyphenols may exert beneficial effects on atherosclerosis and cardiovascular diseases, in part, because of their antioxidant properties. In this study we compared the effects of unbound (free) and lipoprotein-associated red wine components on in vitro antioxidant protection of human low-density lipoprotein (LDL). Preincubation of LDL (1 mg protein/mL) with 0-2.5% (v/v) red wine for 3 h at 37 degrees C followed by gel filtration to remove unbound red wine components resulted in a dose-dependent, up to 4-fold increase in LDL-associated antioxidant capacity (measured as Trolox equivalents). Similar results were obtained with high-density lipoprotein (HDL) and bovine serum albumin (BSA). Furthermore, LDL was subjected to oxidation by copper and aqueous peroxyl radicals (2,2'-azobis[2-amidinopropane] dihydrochloride, AAPH). Under both types of oxidative stress, LDL-associated and free red wine components significantly decreased oxidation of the lipoprotein's protein moiety (assessed by tryptophan fluorescence) and lipid moiety (assessed by thiobarbituric acid-reactive substances and conjugated dienes). Similar protective effects of red wine components were observed against HDL oxidation. In contrast, red wine exerted a pro-oxidant effect on copper-induced oxidation of BSA tryptophan residues, while protecting them from AAPH-induced oxidation. Ascorbate strongly enhanced the protective effect of red wine against copper-induced LDL oxidation, and had an additive effect against AAPH-induced oxidation. Our data indicate that red wine components bind to LDL and HDL and protect these lipoproteins from metal ion-dependent and -independent protein and lipid oxidation.     

New insights into controversies on the antioxidant potential of the olive oil antioxidant hydroxytyrosol

In the present study, the antioxidant profile of olive oil antioxidants was investigated. Hydroxytyrosol and oleuropein are potent scavengers of hydroxyl radicals (OH*), peroxynitrite (ONOOH), and superoxide radicals (O(2)*(-)). Homovanillic alcohol, one of the main metabolites of hydroxytyrosol, and tyrosol are less potent scavengers of these reactive species. None of the olive oil antioxidants are good hypochlorous acid (HOCl) or hydrogen peroxide (H(2)O(2)) scavengers. Hydroxytyrosol efficiently protects against LDL oxidation in vitro and in vivo. However, no protective effect of hydroxytyrosol is usually demonstrated ex vivo against the oxidation of LDL isolated from humans after hydroxytyrosol consumption. The present study shows that this controversy is due to the isolation of LDL, which greatly reduces the protective effect of hydroxytyrosol against LDL oxidation. Hydroxytyrosol is an efficient scavenger of several free radicals. The physiological relevance of the high intrinsic antioxidant activity of hydroxytyrosol is illustrated by its protection against LDL oxidation.     

Buckwheat honey increases serum antioxidant capacity in humans

Honey has been known to exert significant in vitro antioxidant activity, in part due to its phenolic content. However, conclusions that the antioxidants in honey are or are not efficacious in the human body cannot be reached if its antioxidant action is not assessed as part of a human study. In the present study, the acute effect of consumption of 500 mL of water, water with buckwheat honey, black tea, black tea with sugar, or black tea with buckwheat honey on serum oxidative reactions was examined in 25 healthy men. Antioxidant capacity of human serum samples was measured using different methods: the oxygen radical absorbance capacity (ORAC) assay, ex vivo susceptibility of serum lipoprotein to Cu(2+)-induced oxidation, and the thiobarbituric acid reactive substances (TBARS) assay. The results showed that the serum antioxidant capacity determined by ORAC increased significantly (p < 0.05) by 7% following consumption of buckwheat honey in water. No significant changes in serum antioxidant capacity could be established after the consumption of any of the other beverages. Ex vivo serum lipoprotein oxidation and TBARS values were not significantly altered after consumption of any of the five beverages. This study provides primary evidence of the in vivo antioxidant activity of buckwheat honey. However, long-term studies and epidemiological data are necessary to investigate whether honey consumption can exert overall antioxidant-related health benefits.     

Antioxidative activity and active components of longan (Dimocarpus longan Lour.) flower extracts

Three different solvent extracts (methanol, ethyl acetate, and n-hexane) of longan ( Dimocarpus longan Lour.) flowers were assayed with three different antioxidant capacity methods, namely, the DPPH free radical scavenging effect, the oxygen radical absorbance capacity (ORAC) assay, and the inhibition of Cu(2+)-induced oxidation of human low-density lipoprotein (LDL). It was revealed that the methanol extract has the best antioxidative activity, followed by ethyl acetate and n-hexane extracts. The methanol extract was separated by liquid-liquid partition into n-hexane, ethyl acetate, n-butanol, and water fractions. The ethyl acetate fraction was found to have the highest activity of delaying LDL oxidation. After silica gel column chromatography, the fraction having a superior activity was identified as containing two major compounds, (-)-epicatechin and proanthocyanidin A2.     

Antioxidative and antiproliferative properties of selected barley (Hordeum vulgarae L.) cultivars and their potential for inhibition of low-density lipoprotein (LDL) cholesterol oxidation

Aqueous methanolic extracts of whole kernels from six different barley cultivars, namely, Falcon, AC Metcalfe, Tyto, Tercel, Phoenix, and Peregrine, were examined for their total phenolic content (TPC), oxygen radical scavenging capacity (ORACFL), hydroxyl radical scavenging capacity (HORACFL), potency in prevention of lipid oxidation using the Rancimat method, efficacy in inhibition of Cu(II)-induced human LDL cholesterol oxidation, and antiproliferative activities using Caco-2 colorectal adenocarcinoma cell line. Total phenolic content as measured by Folin-Ciocalteu's method ranged from 0.68 to 1.19 mg of ferulic acid equiv/g of defatted material, whereas ORACFL and HORACFL values were 11.28-19.10 and 9.06-12.99 micromol of Trolox equiv/g of defatted material, respectively. Protection factor (PF), a measure of the effect of extracts on the prevention of oxidation of stripped corn oil as measured by Rancimat, ranged from 0.97 to 1.59. Furthermore, barley extracts showed 19.64-33.93% inhibition against Cu(II)-induced human LDL cholesterol oxidation at a final concentration of 0.02 mg/mL. The proliferation of Caco-2 colon cancer cells was significantly (p < 0.05) inhibited in a dose-dependent fashion in the presence of all barley extracts tested at the end of the day 4 of incubation. At the end of day 4, barley extracts rendered 29.3-51.2 and 9.3-15.9% inhibition of cell proliferation at 0.5 and 0.05 mg/mL, respectively. Phenolic extracts from whole barley kernel tested possessed high antioxidant, antiradical, and antiproliferative potentials. Therefore, inclusion of whole barley into the daily diet may render beneficial health benefits.     

Inhibition of low-density lipoprotein oxidation and oxidative burst in polymorphonuclear neutrophils by caffeic acid and hispidin derivatives isolated from sword brake fern (Pteris ensiformis Burm.)

Several antioxidant compounds have been previously identified from sword brake fern (Pteris ensiformis Burm.) by DPPH bleaching and Trolox equivalent antioxidant capacity (TEAC) analyses. Among the isolates, 7-O-caffeoylhydroxymaltol 3-O-beta-D-glucopyranoside and hispidin 4-O-beta- D-glucopyranoside [6-(3,4-dihydroxystyryl)-4-O-beta-D-glucopyranoside-2-pyrone] were two new compounds. The aim of this study is to elucidate the possible effect of the aqueous extract of sword brake fern (SBF) and these two compounds in preventing atherosclerosis. The results demonstrated that SBF and these two compounds strongly inhibited Cu2+-mediated low-density lipoprotein (LDL) oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene production, and relative electrophoretic mobility. The commercial antioxidant dl-alpha-tocopherol showed lower antioxidant activity than these two compounds at the same molecular concentration. SBF and these two compounds also suppressed N-formylmethionyl-leucylphenylalanine (fMLP)-stimulated reactive oxygen species (ROS) production in human polymorphonuclear neutrophils (PMN). These findings indicate that sword brake fern may prevent atherosclerosis via inhibition of both LDL oxidation and ROS production.     

Characterization of antioxidants present in bitter tea (Ligustrum pedunculare)

The present study examined the antioxidants present in bitter tea (Ligustrum pedunculare). It was found that the crude glycoside fraction strongly protected human low-density lipoprotein (LDL) from oxidation. Further column chromatography led to purification of eight phenylethanoid or monoterpene glycosides: lipedoside A-I, lipedoside A-II, lipedoside B-I, lipedoside B-III, lipedoside B-V, lipedoside B-VI, osmanthuside B, and anatolioside. It was found that lipedoside A-I, lipedoside A-II, lipedoside B-V, and lipedoside B-VI were protective, whereas the other four compounds did not protect human LDL from Cu(2+)-medicated oxidation. Lipedoside A-I, lipedoside A-II, lipedoside B-V, and lipedoside B-VI also had a scavenging effect on 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH), comparable to that of alpha-tocopherol. The inhibitory effect of these four phenylethanoid or monoterpene glycosides on oxidation of human LDL and alpha-tocopherol was dose-dependent at concentrations of 5-40 microM. The present results demonstrate that bitter tea as a beverage contains effective antioxidants that may have benefits similar to those of green tea in terms of antioxidant activity.     

Antioxidative activities of phenylethanoid glycosides from Ligustrum purpurascens.

Tea and kudingcha (bitter tea) are the two most popular beverages consumed in China. Tea derived from the leaves of Camellia sinensis has been well studied for its various health benefits, but there are very limited data on the biological activities of bitter tea derived from the leaves of Ligustrum purpurascens (LP). The present study was carried out to characterize the antioxidants present in the bitter tea brewed from the leaves of LP. It was found that the crude glycoside fraction possessed strong protection against oxidation of human low-density lipoprotein (LDL). The column chromatographic separation led to the isolatation of five phenylethanoid glycosides, namely, acteoside, ligupurpuroside A, cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B. When acteoside was heated in the boiling water, it was isomerized to form isoacteoside. Acteoside, isoacteoside, and ligupurpuroside A purified from LP were protective, whereas cis-ligupurpuroside B, trans-ligupurpuroside B, and osmanthuside B exhibited no protection to human LDL from Cu(2+)-medicated oxidation. Acteoside, isoacteoside, and ligupurpuroside A were also effective in preventing the peroxyl free radical-induced oxidation of alpha-tocopherol in human LDL. The antioxidant activities of acteoside, isoacteoside, and ligupurpuroside A were comparable to that observed for a green tea antioxidant, (-)-epicatechin gallate. The inhibitory effect of these three phenylethanoid glycosides on oxidation of human LDL and alpha-tocopherol was dose-dependent at concentrations of 5-40 microM. The present results suggest that the bitter tea beverage derived from LP contains effective antioxidants that may have an equal benefit as a green tea beverage.     

Comparison of protective effects between cultured Cordyceps militaris and natural Cordyceps sinensis against oxidative damage

The Chinese herb DongChong-XiaCao originating from Cordyceps sinensis is widely used as a traditional medicine in China for treatment of a wide variety of diseases. The extracts of Cordyceps sinensis (CSE) and Cordyceps militaris (CME) are well-known for their biological effects. In the present study, the antioxidant efficiency of CME and CSE in protecting lipid, protein, and low-density lipoprotein (LDL) against oxidative damage was investigated. CME and CSE showed weakly inhibitory effect on liposome oxidation, that of CME being superior to that of CSE. As for the protein oxidation model system, the inhibitory effect of CME on protein oxidation was inferior to that of CSE. CME and CSE at 1.0 mg/mL showed 50.5 and 67.1% inhibition of LDL oxidation, respectively. The contents of bioactive ingredients cordycepin and adenosine in CME are higher than those of CSE; however, both cordycepin and adenosine showed no significant antioxidant activity as determined by the Trolox equivalent antioxidant capacity method. Polyphenolic and flavonoid contents are 60.2 and 0.598 microg/mL in CME and 31.8 and 0.616 microg/mL in CSE, respectively, which may in part be responsible for their antioxidant activities. In addition, a polysaccharide present in CME and CSE displayed antioxidant activity, which suggested that the activity might be derived partly from polysaccharides of CME and CSE. The tendency to scavenge the ABTS(*)(+) free radical and the reducing ability of CME and CSE display concentration-dependent manners, suggesting that CME and CSE may be potent hydrogen donators. On the basis of the results obtained, the protective effects of CME and CSE against oxidative damage of biomolecules are a result of their free radical scavenging abilities.     

Antioxidant activity of Sicilian pistachio (Pistacia vera L. var. Bronte) nut extract and its bioactive components

Pistacia vera L. is the only species of Pistacia genus producing edible nuts. This paper investigates the antioxidant potential of a Sicilian variety of pistachio nut by chemical as well as biological assays and measured antioxidant vitamins and a number of antioxidant polyphenols in either the hydrophilic and/or the lipophilic nut extract. In accordance with the majority of foods, the total antioxidant activity, measured as a TAA test, was much higher (50-fold) in the hydrophilic than in the lipophilic extract. Substantial amounts of total phenols were measured. The hydrophilic extract inhibited dose-dependently both the metal-dependent and -independent lipid oxidation of bovine liver microsomes, and the Cu+2-induced oxidation of human low-density lipoprotein (LDL). Peroxyl radical-scavenging as well as chelating activity of nut components may be suggested to explain the observed inhibition patterns. Among tocopherols, gamma-tocopherol was the only vitamin E isomer found in the lipophilic extract that did not contain any carotenoid. Vitamin C was found only in a modest amount. The hydrophilic extract was a source of polyphenol compounds among which trans-resveratrol, proanthocyanidins, and a remarkable amount of the isoflavones daidzein and genistein, 3.68 and 3.40 mg per 100 g of edible nut, respectively, were evaluated. With the exception of isoflavones that appeared unmodified, the amounts of other bioactive molecules were remarkably reduced in the pistachio nut after roasting, and the total antioxidant activity decreased by about 60%. Collectively, our findings provide evidence that the Sicilian pistachio nut may be considered for its bioactive components and can effectively contribute to a healthy status.     

Antioxidative activity of green tea polyphenol in cholesterol-fed rats

This study investigated the effects of green tea polyphenol on the serum antioxidative activity and cholesterol levels of cholesterol-fed rats and compared them with those of probucol, an antioxidant hypocholesterolemic agent. To evaluate the antioxidative activity, the susceptibility to oxidative modification of low-density lipoprotein (LDL) isolated from the serum of cholesterol-fed rats was measured, as was the serum antioxidative activity using the spontaneous autoxidation system of brain homogenate. Administration of green tea polyphenol effectively inhibited LDL oxidation and elevated serum antioxidative activity to the same degree as probucol. However, higher amounts of polyphenol than probucol needed to be administered to reduce the total, free, and LDL cholesterol levels. Furthermore, green tea polyphenol increased the levels of high-density lipoprotein (HDL) cholesterol, leading to dose-dependent improvement of the atherogenic index, an effect that was not seen with probucol. Thus, green tea polyphenol may exert an antiatherosclerotic action by virtue of its antioxidant properties and by increasing HDL cholesterol levels.     

Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid dehydrodimers on human low-density lipoproteins

Dietary antioxidants that protect low-density lipoprotein (LDL) from oxidation may help to prevent atherosclerosis and coronary heart disease. The antioxidant activities of purified monomeric and dimeric hydroxycinnamates and of phenolic extracts from rye (whole grain, bran, and flour) were investigated using an in vitro copper-catalyzed human LDL oxidation assay. The most abundant ferulic acid dehydrodimer (diFA) found in rye, 8-O-4-diFA, was a slightly better antioxidant than ferulic acid and p-coumaric acid. The antioxidant activity of the 8-5-diFA was comparable to that of ferulic acid, but neither 5-5-diFA nor 8-5-benzofuran-diFA inhibited LDL oxidation when added at 10-40 microM. The antioxidant activity of the monomeric hydroxycinnamates decreased in the following order: caffeic acid > sinapic acid > ferulic acid > p-coumaric acid. The antioxidant activity of rye extracts was significantly correlated with their total content of monomeric and dimeric hydroxycinnamates, and the rye bran extract was the most potent. The data suggest that especially rye bran provides a source of dietary phenolic antioxidants that may have potential health effects.     

Identification and in vitro biological activities of hop proanthocyanidins: inhibition of nNOS activity and scavenging of reactive nitrogen species

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.     

Soy and alfalfa phytoestrogen extracts become potent low-density lipoprotein antioxidants in the presence of acerola cherry extract

Postmenopausal women have an increased risk of coronary heart disease. Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis, and the presence of modified LDL (LDL(-)) in plasma appears to represent LDL oxidation in vivo. Because previous studies have demonstrated a strong antiatherogenic effect of estrogen due to its antioxidant activity and similar antioxidant activity was found for specific isoflavones derived from soy extract, the antioxidant activity of a phytoestrogen extract derived from soy and alfalfa was studied. Copper-mediated LDL oxidation was inhibited in the presence of soy and alfalfa extracts, and this effect was further enhanced in the presence of acerola cherry extract, which is rich in ascorbic acid. Male rabbit aortic endothelial cells pretreated with soy extract were resistant to the toxic effects of high levels of LDL and LDL(-), and a lesser, but significant protection, was also afforded by alfalfa extract. Cell-mediated oxidation of LDL, measured by LDL(-) formation, was inhibited in the presence of soy extract but not alfalfa extract. However, in the presence of acerola cherry extract, both soy and alfalfa extracts potently inhibited the formation of LDL(-). These findings show that acerola cherry extract can enhance the antioxidant activity of soy and alfalfa extracts in a variety of LDL oxidation systems. The protective effect of these extracts is attributed to the presence of flavonoids in soy and alfalfa extracts and ascorbic acid in acerola cherry extract, which may act synergistically as antioxidants. It is postulated that this synergistic interaction among phytoestrogens, flavonoids, and ascorbic acid is due to the "peroxidolitic" action of ascorbic acid, which facilitates the copper-dependent decomposition of LDL peroxides to nonradical products; this synergy is complemented by a mechanism in which phytoestrogens stabilize the LDL structure and suppress the propagation of radical chain reactions. The combination of these extracts markedly lowers the concentrations of phytoestrogens required to achieve significant antioxidant activity toward LDL.     

Phenolic antioxidants and the protection of low density lipoprotein from peroxynitrite-mediated oxidations at physiologic CO2

Dietary phenolic antioxidants have been shown to prevent LDL modifications mediated by several physiologic oxidants including peroxynitrite. However, more recent data demonstrated that CO(2) affected the fate of peroxynitrite in biological fluids and significantly reduced peroxynitrite scavenging by polyphenols, raising doubts concerning their antioxidant activity. We found that the oxidation of LDL lipids mediated by peroxynitrite decreased in the presence of bicarbonate, while Trp oxidation and 3-nitroTyr formation increased, suggesting a redirection of peroxynitrite reactivity toward the protein moiety. We therefore evaluated the protective activity of some phenolic antioxidants (quercetin, oleuropein, resveratrol, (+)-catechin, (-)-epicatechin, tyrosol, alpha- and gamma-tocopherol, ascorbate) on peroxynitrite-mediated oxidation of LDL aromatic residues. Some of these phenols protected LDL Trp from oxidation better than ascorbate or alpha-tocopherol, although protection at 100 microM did not exceed 30-40%. However, the same phenolic antioxidants were more active in inhibiting 3-nitroTyr formation and those with a catechin structure provided significant protection (IC(50%) 40-50 microM). Red wine, a polyphenol-rich beverage, showed a protective effect comparable to that of the most active phenolic antioxidants. Direct EPR studies showed that bicarbonate significantly increased the peroxynitrite-dependent formation of O-semiquinone radicals in red wine, supporting the hypothesis that polyphenols are efficient scavengers of radicals formed by peroxynitrite/CO(2). Ascorbate was a poor inhibitor of peroxynitrite/CO(2)-induced LDL tyrosine nitration, but the simultaneous addition to the most active polyphenols halved their IC(50%). In conclusion, although cooperation with other antioxidants can further decrease the IC(50%) of polyphenolics, as demonstrated for ascorbate, their antioxidant activity appears to occur at concentrations at least 1 order of magnitude higher than their bioavailability.     

Modes of action of nitrification inhibitors

 In recent years, substantial progress has been made towards understanding the modes of action for the specific inhibition of autotrophic NH₃ oxidation. This has included demonstrating that NH₃ monooxygenase (AMO) has a broad substrate range for catalytic oxidation, and the inhibitory effects of many compounds are due to competition for the active site. Other compounds, such as acetylenes, are oxidized by the normal catalytic cycle of AMO to highly reactive products which covalently bind the enzyme causing irreversible inhibition. Substantial evidence has shown the important role of Cu in the activity of AMO, and indicated that a large class of compounds containing thiono-S inhibit AMO activity by binding with Cu within the active site. Heterocyclic N compounds form another important class of nitrification inhibitors with little known about their mode of action, although evidence suggests that their inhibitory influence is closely related to the presence of ring N. Received: 21 July 1998