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Showing 1861–1880 of 2058 publications.
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Suarna, Cacang; Dean, Roger T.; Southwell-Keeley, Peter T.; Moore, Douglas Edwin; Stocker, RolandIn previous work we demonstrated that up to 30% of cholesteryl linoleate in homogenates of advanced human plaque samples is present in oxidized forms. Here we show that the material from plaque hexane extracts which co-elutes with cholesteryl hydroxy linoleate on reversed phase HPLC, is composed of several isomers of cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. Enzymatic hydrolysis and measurement of liberated cholesterol and disappearance of the esters revealed that almost all of the material consisted of unoxidized cholesterol esterified to oxidized derivatives of octadecadienoate. Semi-preparative reversed-phase HPLC was used to obtain sufficient quantities of this co-eluting material to undertake normal phase HPLC separation of these components. The nature of such separated and isolated compounds was identified, by co-chromatography with authentic standards, UV spectroscopy and chemical ionization and electron impact mass spectrometry, as cholesteryl hydroxy- and cholesteryl oxo-octadecadienoate. These oxidized fatty acids have been observed previously in plaque, in agreement with our new unambiguous demonstration of their presence as cholesteryl esters. The application of the methods described for the separation of the various forms of oxidized cholesteryl octadecadienoate may aid mechanistic studies of in vitro and in vivo lipoprotein lipid oxidation.
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Ho, Win F.; Gilbert, Bruce C.; Davies, Michael J.Nucleobase, nucleoside, RNA and DNA hydroperoxides have been generated by exposure of the parent compounds to high energy electrons in the presence of oxygen; EPR spin-trapping experiments using 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-4,5-dihydro-3H-pyrrole N-oxide (DMPO) have been employed to study the reactions of alkoxyl radicals generated from their reaction with Fe2+. Alkoxyl radicals generated from the pyrimidine hydroperoxides (nucleobases and nucleosides) are shown to be capable of reacting with a variety of substrates, which include the pyrimidine nucleobases and nucleosides themselves and histone proteins. Attack on the parent pyrimidine compounds involves addition to the C(5) and C(6) atoms of the pyrimidine ring; reaction with the histone proteins, amino acids and peptides gives carbon-centred species, providing direct evidence for transfer of damage via hydrogen-atom abstraction. Rapid reactions with antioxidants are also demonstrated.
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Ho, Win F.; Gilbert, Bruce C.; Davies, Michael J.EPR spin-trapping experiments using MNP (2-methyl-2-nitrosopropane) have been employed to study radicals formed by reaction of HO (generated from reaction of H<inf>2</inf>O<inf>2</inf> with Fe2+) with uridine as a model nucleoside; studies with [5-2H]uridine, [5,6-2H<inf>2</inf>]uridine and [1,3-15N<inf>2</inf>] have allowed us to obtain a more detailed analysis of the spectra following HO attack on uridine, for which EPR spectra cannot at present be unambiguously assigned. These studies provide detailed information concerning the analysis of hyperfine splittings and hence the quantification of the different amounts of the C(5)- and C(6)-hydroxyl-radical adducts formed following HO attack, which are comparable to results obtained from pulse radiolysis studies. Studies have also been extended to investigate the reactions of both SO<inf>4</inf>- and ButO with uridine.
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Suarna, Cacang; Dean, Roger T.; Southwell-Keely, Peter T.A range of ?-tocopherol analogues of varying side-chain length and structure has been prepared by the Wittig reaction of alkyltriphenylphosphonium bromides with either 6-benzyloxy-2,5,7,8-tetramethylchroman-2-carbaldehyde (8) or 6-acetoxy-2,5,7,8,-tetramethylchroman-2-carbaldehyde (14). These analogues include 2-hexyl-2,5,7,8-tetramethylchroman-6-ol (11), 2-heptyl-2,5,7,8-tetramethylchroman-6-ol (12) and 2,5,7,8-tetramethyl-2-(pent-l-enyl)chroman-6-ol (15). Methoxycarbonylmethyl 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (2) was formed by reaction of the triethylammonium salt of trolox (1) with methyl bromoacetate. Reaction of methoxycarbonylmethyltriphenylphosphonium bromide (16) with (8) did not produce the expected methyl 3-(6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl)prop-2-enoate (17), but rather 4-(6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl)but-3-en-2-one (22). A proposed mechanism for this unusual reaction is discussed.
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Neuil, Ji? Thomas, Shane R.; Stocker, Roland?-Tocopherol (?-TOH), generally regarded as the most important lipid- soluble, chain-breaking antioxidant in human plasma, can also be a pro- oxidant in isolated low-density lipoprotein (LDL) (Bowry V. W.; Stocker R. J. Am. Chem. Soc. 115:6029-6044; 1993). Here we examined whether this pro- oxidant activity of ?-TOH is of more general relevance. We compared the oxidizability of lipid hydroperoxide-free, in vivo or in vitro ?-TOH- depleted LDL and high-density lipoprotein (HDL), as well as plasma reconstituted with ?-TOH depleted lipoproteins, with that of the corresponding native and ?-TOH-supplemented samples, using water- and lipid- soluble peroxyl radicals (ROO.), hydroxyl radicals (OH), Cu2+, the transition metal-containing Ham's F-10 medium, soybean 15-lipoxygenase, and horseradish peroxidase as oxidants. Lipoprotein and plasma oxidizability was assessed by the loss of cholesteryl esters and ?-TOH and the accumulation of hydroperoxides of cholesteryl esters and phospholipids. Compared to native LDL, HDL, and plasma, the in vivo and in vitro ?-TOH depleted counterparts were highly resistant to peroxidation initiation by all oxidants when used at mild radical flux conditions. Wherever tested, the oxidizability of isolated LDL decreased proportionally with decreasing ?-TOH content. Initiation of LDL lipid oxidation by lipoxygenase and Cu2+ (even up to Cu2+:LDL ratio of 20:1) had an absolute requirement for ?-TOH. Oxidation of reconstituted plasma with ROO showed that in the absence of the vitamin, plasma lipids were largely resistant to oxidation, whereas bilirubin and urate oxidized more rapidly. Replenishing the in vitro depleted LDL with ?-TOH, but not with ?-tocopherol acetate, fully restored its original content of vitamin E and its oxidizability. Similarly, dietary supplementation with ?-TOH restored the vitamin content and oxidizability of the in vivo ?-TOH-depleted lipoproteins and plasma obtained from a patient with familial isolated vitamin E deficiency. Under high fluxes of ROO and OH, the activity of ?-TOH in LDL switched from pro- to anti-oxidant, with the switching point for OH observed at a lower radical flux than that for ROO. Together, our results show that ?-TOH generally makes lipoproteins more reactive towards radical oxidants; this can result in a pro-oxidant activity depending on the specific oxidation conditions.
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Silvester, Julie A.; Wei, X. D.; Davies, Michael J.; Timmins, Graham S.Photo-oxidation of bovine serum albumin (BSA) by porphyrins produces protein-centred radicals that can be spin trapped by 3,5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS) and 5,5-dimethyl-1 -pyrroline-N-oxide (DMPO). In the case of DMPO, a thiyl radical from the Cys-34 residue is trapped, whereas with DBNBS signals from both this thiyl and tertiary carbon-centred species are observed. However, specific chemical modification of the Cys-34 residue, in combination with dual-isotope spin-trapping techniques, shows that the signal assigned to the Cys-34 thiyl adduct with DBNBS is a nitroxide artefact resulting from sequential (non-radical) nucleophilic addition and oxidation. In contrast, both the Cys-34 thiyl DMPO adduct and the tertiary carbon-centred DBNBS adducts result from genuine spin-trapping. This study shows that such artefacts can be detected - even with anisotropic EPR spectra - through the use of appropriately substituted spin-traps, and that nitroso spin-traps need to be employed with great care in systems containing free thiol groups.
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Mathieu, Christel; Swaraj, Kumari; Davies, Michael J.; Trinchant, Jean Charles; Puppo, Alain[No abstract available]
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Samman, Samir; Brown, Andrew J.; Beltran, C.; Singh, S.Objective: The aims of this study were to investigate the effect of ascorbic acid (AA) supplementation on the oxidisability of low density lipoprotein (LDL) in vitro and on plasma lipoproteins under controlled dietary conditions. Design: Randomised single-blind cross-over trial. Setting: Free living subjects. Subjects: Eight male smokers (age: 25 2.4 y, BMI: 20.7 0.5, cigarettes per day: 19.1 2.4; means s.e.). Interventions: Dietary intake was determined and all subjects were advised to achieve an intake as close as possible to the recommended dietary intake of AA (40 mg). After two weeks on the baseline diet, subjects were asked to consume 1 g AA per day for two weeks followed by two weeks of placebo supplementation, or vice versa. In view of the carry-over effects of plasma AA, a wash-out period was incorporated between treatments. Duplicate venous blood samples were collected before and after supplementation and the plasma concentrations of AA, lipids and lipoproteins were determined. The in vitro copper-induced oxidisability of LDL was assessed by monitoring of the absorbance of 234 nm. Results: No changes in the plasma lipids or the oxidisability of LDL were found after AA supplementation compared to placebo. Plasma AA concentrations doubled on average after supplementation indicating that the lack of effect was not a result of poor compliance. Conclusions: AA supplementation at this dose did not alter plasma lipids of LDL oxidisability in male smokers.
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Brown, Andrew J.; Leong, Sulin; Dean, Roger T.; Jessup, Wendy K.7-Hydroperoxycholesterols (7OOHs) are intermediates in cholesterol oxidation and potential cytotoxins. A normal-phase HPLC method with UV (205 nm) detection was developed that could resolve 7?OOH, 7?OOH, 7- ketocholesterol (7K), and the epimeric 7-hydroxycholesterols (7OHs). 7OOH formation was investigated when LDL was exposed to four different oxidizing systems: Cu2+; Ham's F-10; mouse peritoneal macrophages in Ham's F-10; and a metal-independent peroxyl-radical generating system (AAPH). With all four oxidizing systems, 7OOH (both free and esterified, mostly as the ?-isomer) was the major oxysterol formed at early times, with 7K dominating at later stages (?24 h) in Cu-oxLDL. When LDL was oxidized in the presence of cells there was transfer of free oxysterols from LDL to the cells. Negligible 7OOH, but significant amounts of 7OH, accumulated in the cells suggesting efficient cellular reduction of 7OOH. Lipid extracts from eight plaque samples obtained from patients undergoing carotid endarterectomy were analyzed. Only trace amounts of 7OOH (<0.02% of total cholesterol) could be detected using this normal-phase HPLC method with UV detection or with a more sensitive reverse- phase method utilizing chemiluminescence detection. 7K was the major 7- oxygenated sterol detected, at least 20-fold in excess of that calculated for 7OOH, followed by 7?OH and 7?OH. The trace concentrations of 7OOH in plaque indicate its lability in biological/cellular systems and may signify the ability of cells in the artery wall to metabolize it further.
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Cleary, Janelle A.; Mohr, Detlef; Adams, Mark R.; Celermajer, David S.; Stocker, Roland[No abstract available]
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Inhibition of LDL oxidation by ubiquinol-10. A protective mechanism for coenzyme Q in atherogenesis?Thomas, Shane R.; Neuil, Ji? Stocker, RolandThe oxidation of low density lipoprotein (LDL) is now commonly regarded as an important early event in atherogenesis. As such there is considerable interest in the ability of antioxidant supplementation to attenuate LDL oxidation and hence atherosclerosis. A majority of studies on LDL antioxidation have focused on ?-tocopherol (?-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to ?-TOH, circulating LDL also contains low levels of ubiquinol-10 (CoQ<inf>10</inf>H<inf>2</inf>; the reduced form of coenzyme Q). Recent studies have shown that in intact, isolated LDL, ?-TOH can act as either an anti- or pro-oxidant for the lipoprotein's lipids. This article reviews the molecular action of ?-TOH in LDL undergoing radical-initiated oxidation, and how the presence of CoQ<inf>10</inf>H<inf>2</inf> supresses the pro-oxidant or complements the antioxidant activity of the vitamin. We also comment on the plasma and intimal levels of ?-TOH and CoQ<inf>10</inf>H<inf>2</inf> in patients suffering from coronary artery disease and discuss the potential implications of these results for atherogenesis.
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Celermajer, David S.Until recently, the endothelium was regarded as a relatively inert cell layer. However, over the past 20 years, research has revealed an extraordinary array of endothelial functions, including control over coagulation, fibrinolysis, arterial tone and vascular growth. Importantly, endothelial dysfunction has been implicated as a key event in the pathogenesis of atherosclerosis, coronary vasoconstriction and, probably, myocardial ischemia. The recent demonstration that endothelial dysfunction may be reversible raises the possibility of slowing the progression of atherosclerosis or modifying arterial function, or both, to decrease the risk of acute cardiovascular events.
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Jessup, Wendy K.; Squires, Brett; Kritharides, Leonard; Hume, David A.; Dean, Roger T.To assess whether human monocyte-specific colony-stimulating factor (CSF-1) might influence atherogenesis, CSF-1-induced macrophage responses that might contribute to enhanced clearance of low-density lipoprotein (LDL) or modified LDL were investigated. Careful account was made of cell preservation and increases in cell volume and protein (representing increased cell surface area, and thus endocytically active membrane) during culture with CSF-1. This permitted distinction between selective and nonspecific effects of CSF-1, the latter paralleling increases in cellular mass and volume, CSF-1 enhanced mouse peritoneal macrophage survival in vitro during exposure to lipoprotein-deficient serum with or without native LDL or acetylated LDL (Ac-LDL), as judged by maintenance of cellular DNA and cell numbers. In the presence of copper-oxidized LDL (Ox-LDL), such effects were very slight. In all conditions, CSF-1 increased cellular protein content. CSF-1 increased the uptake of both Ac-LDL and Ox-LDL calculated per culture, but this was entirely explicable by the increased cell protein, indicating that there was no selective enhancement of scavenger receptor or other routes for uptake of the modified LDLs. Similarly, CSF-1 also increased the accumulation of cholesterol and its esters nonspecifically. CSF-1 did have a marked and specific effect on the composition of cholesterol esters, decreasing the proportion of polyunsaturated esters relative to monounsaturated and saturated esters. Finally, cholesterol efflux induced by apolipoprotein A1 from Ac-LDL-loaded macrophages was not influenced by CSF- 1. Thus, the enhanced macrophage catabolism of modified LDLs by CSF-1 is part of a nonspecific action on the cells but could contribute to a reduction in circulating cholesterol, observed in some situations of CSF-1 presentation in humans.
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Brieger, David B.; Dawes, JoanLow molecular weight (LMW) heparins have prolonged circulating half-lives relative to unfractionated heparin, but the rates of plasma clearance differ between different LMW preparations. To determine the impact of method of production on their pharmacokinetic and ex vivo biological properties, two LMW heparins of similar molecular weight distribution, Logiparin and Fragmin, were radiolabelled with 125I, administered intravenously with 4 mg/kg of carrier drug into rabbits, and the circulating radiolabelled material and anti-Xa activity were analysed by size exclusion chromatography and affinity for antithrombin and Polybrene. Following administration of Logiparin, the anti-Xa amidolytic activity was eliminated-with the same half-life as the antithrombin-binding radiolabel and was not neutralised by antibody against tissue factor pathway inhibitor (TFPI). Larger molecules were cleared preferentially and were no longer detectable 8 h post injection. These findings resemble those we have previously described for Enoxaparin. After Fragmin administration the antithrombin binding radiolabel was cleared more rapidly than the anti-Xa activity, and at late times after injection a significant amount of this activity was neutralised by antibody against TFPI. Sulphated radiolabel was eliminated with a similar half-life to the anti-Xa activity and sulphated molecules > 6000 Da remained in the circulation 8 h after administration. Fragmin, unlike Logiparin and Enoxaparin, has no negatively charged sulphamino group at the reducing end of the molecule. We suggest that this minimises cellular interaction and protects the larger molecules from elimination. They remain in the circulation, contributing to anti-Xa activity by binding TFPI. Thus the method of production of LMW heparins may significantly influence their pharmacokinetic properties and circulating anticoagulant activities.
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Adams, Mark R.; Jessup, Wendy K.; Hailstones, Deborah L.; Celermajer, David S.Background: Monocyte adhesion to endothelial cells is a key early event in atherogenesis. Because L-arginine has been shown to reduce atheroma and to decrease monocyte-endothelial cell adhesion in an animal model of atherosclerosis, we studied the effects of L-arginine on human monocyte adhesion to human endothelial cells and endothelial expression of cell adhesion molecules. Methods and Results: Human umbilical vein endothelial cells (HUVECs) were grown to confluence, then incubated for 24 hours with arginine-deficient media to which was added saline (control), 100 or 1000 ?mol/L L-arginine, 100 ?mol/L D-arginine, 100 ?mol/L N(G)-monomethyl-L- arginine (L-NMMA), or 100 ?mol/L L-arginine with 100 ?mol/L L-NMMA. Human monocytes obtained by elutriation were incubated for 1 hour with HUVECs and adhesion was measured by light microscopy. Compared with control, monocyte adhesion was reduced by L-arginine (5910%, P=.01) and increased by L-NMMA (12320%, P=.01). Surface expression of cell adhesion molecules by HUVECs was assessed by an ELISA under the above conditions with and without stimulation with interleukin-1?. Expression of ICAM-1 was reduced with both concentrations of L-arginine compared with control in both the basal (4312%, P<.01), and stimulated (4615%, P<.01) states, which correlated with decreased levels of mRNA. Expression of VCAM-1 was reduced only in the stimulated state and only in the presence of 1000 ?mol/L L-arginine (7224%, P=.02). Conclusions: L-Arginine reduces human monocyte adhesion to endothelial cells and decreases expression of certain endothelial cell adhesion molecules.
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Vassiliou, Gerard G.Suramin is a polysulfated drug used in the treatment of cancer and AIDS. High concentrations (1 mg/ml) of suramin did not affect the ability of native ?<inf>2</inf>-macroglobulin (?<inf>2</inf>M) to inhibit proteinases nor did it prevent conversion of native ?<inf>2</inf>M to the 'fast' receptor-binding form. Nevertheless, pharmacological concentrations (below 250 ?g/ml) of suramin prevented the interaction between methylamine-activated ?<inf>2</inf>M and its receptor, the low-density-lipoprotein-receptor-related protein. Inhibition was demonstrated at the molecular level and was not due to-calcium sequestration by the drug, irreversible denaturation of the receptor, or a non-specific polyanion effect (since heparin and dextran sulfate did not alter the binding of ?<inf>2</inf>M). The ability of suramin to accelerate the dissociation of pre-bound ?<inf>2</inf>M was consistent with a noncompetitive mechanism of inhibition although the possibility of a competitive component cannot be eliminated. I discuss how the inhibition of ?<inf>2</inf>M-binding by suramin may contribute to the antiproliferative properties of this drug.
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Witting, Paul Kenneth; Stocker, RolandThe reduction of nitroxide compounds in vivo has in most part been assigned to the reaction with reductants such as ascorbic acid, reduced thiols and quinols such as ubiquinol or by reaction with simple carbon-centred radicals. In this study a water-soluble nitroxide, 2,2,5,5-tetramethyl-4-phenylimidazolin-3-oxide-1-oxyl (TPI), was exposed to both native and partially oxidized human low-density lipoprotein (LDL<inf>n</inf> and LDL<inf>pox</inf>, respectively) and it was found that TPI decayed in each case to the corresponding hydroxylamine, 2,2,5,5-tetramethyl-4-phenyl-3-oxide-1-hydroxylimidazoline (TPHI). In particular, the reduction of TPI in the presence of LDL<inf>pox</inf> occurred via a complex mechanism involving the consumption of both ?-tocopherol (?-TOH) and cholesteryl linoleate hydroperoxides (Ch18: 2-OOH). The EPR signal of the nitroxide also diminished when TPI was exposed to the water-soluble vitamin E analogue, Trolox C, but neither Ch18:2-OOH nor linoleate hydroperoxides alone caused significant decay of the nitroxide signal. Together these results indicate that water-soluble nitroxides may be reduced in circulation by reaction with ?-TOH in LDL, thereby adding to the complexity of the reduction of nitroxide spin labels in vivo. 1997 by John Wiley & Sons, Ltd.
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Thomas, Shane R.; Witting, Paul Kenneth; Stocker, Roland?-Tocopherol (?-TOH) can promote lipid peroxidation in human low density lipoprotein (LDL) unless co-anti-oxidants are present that eliminate the chain-carrying ?-tocopheroxyl radical (?-TO) (Bowry, V. W., Mohr, D., Cleary, J., and Stocker, R. (1995) J. Biol. Chem. 270, 5750-5763). Interferon-? inhibits human monocyte/macrophage-facilitated LDL lipid peroxidation via induction of cellular tryptophan degradation and production and release of 3-hydroxyanthranilic acid (3HAA) (Christen, S., Thomas, S. R., Garner, B., and Stocker, R. (1994) J. Clin. Invest. 93, 2149-2158). We now report on the mechanism of antioxidant action of 3HAA. 3HAA directly reduced ?-TO in UV-exposed micellar dispersions of ?-TOH or in LDL incubated with soybean 15-lipoxygenase (SLO), as assessed by electron paramagnetic resonance spectroscopy. 3HAA did not inhibit SLO enzyme activity. Anthranilic acid, which lacks the phenoxyl group, was incapable of reducing a-TO. 3HAA dose- dependently inhibited the peroxidation of surface phospholipids and core cholesteryl esters in LDL exposed to SLO, peroxyl radicals (ROO), or Cu2+; oxidants that convert ?-TOH to ?-TO. In all cases, sparing of LDL's ?-TOH, but not ubiquinol-10 (CoQ<inf>10</inf>H<inf>2</inf>), was observed until the majority of 3HAA was consumed. Addition of 3HAA or ascorbate prevented further consumption of ?-TOH and accumulation of lipid hydroperoxides when added to aqueous or lipophilic ROO-oxidizing LDL after complete and partial consumption of CoQ<inf>10</inf>H<inf>2</inf> and ?-TOH, respectively. In contrast, addition of urate, an efficient ROO scavenger incapable of scavenging ?-TO, did not efficiently inhibit ongoing lipid peroxidation. Oxidation of 3HAA- supplemented human plasma by aqueous ROO resulted in the successive consumption of ascorbate, CoQ<inf>10</inf>H<inf>2</inf>, 3HAA, bilirubin, ?-TOH, and urate. Lipid peroxidation was prevented as long as ascorbate, CoQ<inf>10</inf>H<inf>2</inf>, and 3HAA were present, but subsequently proceeded as a free-radical chain reaction concomitant with ?-TOH, bilirubin, and urate consumption. Addition of 3HAA to aqueous ROO-oxidizing plasma, after complete consumption of ascorbate and CoQ<inf>10</inf>H<inf>2</inf>, strongly inhibited ongoing lipid peroxidation and consumption of ?-TOH, bilirubin, and urate immediately and as efficiently as did ascorbate. These findings demonstrate that 3HAA is a highly efficient co- antioxidant for plasma lipid peroxidation by virtue of its ability to interact with ?-TO in lipoproteins. Since interferon-?, is the principal inducer of tryptophan degradation and release of 3HAA by monocytes/macrophages, this may represent a localized extracellular antioxidant defense against LDL oxidation in inflammation.
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Davies, Michael J.Previous studies have demonstrated that ?-irradiation of some free amino acids in the presence of oxygen gives high yields of side-chain hydroperoxides. It is shown in the present study that N-acetyl amino acids and peptides also give high levels of hydroperoxides on ?-irradiation, even when the free amino acid does not, and that hydroperoxides can be formed on both the backbone (at ?-carbon positions) and the side chain. Decomposition of ?-carbon hydroperoxides by Fe(II)-EDTA gives initially an alkoxyl radical via a pseudo-Fenton reaction; these radicals fragment rapidly with k estimated as ?107 s-1. With N-acetyl amino acids and dipeptides ?- scission of an alkoxyl radical at the C-terminal ?-carbon results in C- terminal decarboxylation, with release of CO<inf>2</inf>/(); the corresponding amides undergo deamidation with release of ()C(O)NH<inf>2</inf>. Cyclic dipeptides undergo analogous reactions with cleavage of the ?-carbon to carbonyl-carbon bond and formation of ()C(O)NHR radicals. With substrates with large aliphatic side chains, radicals from side-chain hydroperoxides are also observed. C- terminal decarboxylation and backbone fragmentation are also observed with larger peptides, amino acid homopolymers, and proteins. These observations suggest that ?-carbon alkoxyl radicals may be key intermediates in the fragmentation of proteins in the presence of oxygen. The radicals released in these processes may react further to form O<inf>2</inf>/(-), or redox cycle metal ions. These reactions may be propagating processes during protein chain oxidation.
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Kritharides, Leonard; Kus, Michele; Brown, Andrew J.; Jessup, Wendy K.; Dean, Roger T.Atherosclerosis involves the arterial accumulation of lipid-laden 'foam cells' containing oxidized and unoxidized sterols and their esters (Mattsson- Hulten, L., Lindmark, H., Diczfalusy, U., Bjorkhem, I., Ottosson, M., Liu, Y., Bondjers, G., and Wiklund, O. (1996) J. Clin. Invest. 97, 461-8). Oxidized sterols are probably critical to atherogenesis because they inhibit cholesterol removal from cells and are cytotoxic. We recently reported that there is deficient induction of cellular cholesterol efflux by apolipoprotein A-I, the main initial acceptor of cellular cholesterol from macrophages loaded in vitro with oxidized low density lipoprotein (Kritharides, L., Jessup, W., Mander, E., and Dean, R. T. (1995) Arterioscler. Thromb. 15, 276- 289). There was an even more marked impairment of the release of 7- ketocholesterol which is a major oxysterol in these cells and in human atherosclerotic lesions. Here we show that hydroxypropyl-?-cyclodextrin can induce selective efflux of 7-ketocholesterol. Efflux of 7-ketocholesterol was time and concentration dependent, and the rate of its removal was 50-fold greater for hydroxypropyl-?-cyclodextrin than for apolipoprotein A-I. Over a defined range of concentrations (0-5 mg/ml), efflux of 7-ketocholesterol was preferred over that of cholesterol and occurred without cell toxicity. Efflux of free 7-ketocholesterol was associated with decreased intracellular free and esterified 7-ketocholesterol. Hydroxypropyl-?-cyclodextrin also enhanced efflux of other oxysterols. The physical solubilization of 7-ketocholesterol by the cyclodextrin was much greater than that of cholesterol, in accordance with its differential effects on efflux. These data highlight the importance of extracellular sterol solubilization in the efflux of cellular oxysterols and the mobilization of intracellular free and esterified oxysterol pools in macrophages loaded with oxidized low density lipoprotein. Synthetic sterol- solubilizing agents such as hydroxypropyl-?-cyclodextrin are thus potential prototypes for the further development of oxysterol-removing agents.
