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Showing 1921–1940 of 2058 publications.
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Malle, Ernst; Hazell, Linda J.; Stocker, Roland; Sattler, Wolfgang; Esterbauer, Herrmann; Waeg, GeorgOxidation of LDL is thought to contribute to the early stages of atherogenesis. Because myeloperoxidase is present in atherosclerotic lesions and can produce the strong oxidant hypochlorous acid (HOCl), which converts LDL into its high-uptake atherogenic form in vitro, we raised polyclonal and monoclonal antibodies (MoAbs) against HOCl-modified LDL (HOCl-LDL). Characterization of the polyclonal antihuman HOCl-LDL Abs showed that they cross-reacted strongly with 4-hydroxynomenal-malondialdehyde-, and Cu2+oxidized LDL. Similarly, polyclonal and some monoclonal Abs against aldehyde- anti Cu2+-modified LDL cross-reacted with HOCl-LDL. In contrast to the polyclonal Abs, two selected hybridoma cell line supernatants containing MoAbs raised against HOCl-LDL (MoAb-A and MoAb-B) did not cross- react with either native LDL or aldehyde or Cu2+-modified LDL. MoAb-A (clone IB10A11, subtype IgG1?) recognized an epitope that appeared to be specific for HOCl-LDL and depended on the tertiary structure of the (lipo)protein, as judged by a lack of cross-reactivity with HOCl-modified human and bovine serum albumin and a loss of reactivity associated with lipoprotein denaturation. MoAb-B (clone 2D10G9, subtype lgG2b?), on the other hand, gave identical titration curves with HOCl-LDL and HOCl-modified albumins, suggesting that this antibody recognized epitopes that are commonly generated on proteins that have been oxidized with HOCl. Thus, MoAb-A and MoAb-B may be useful tools for the investigation of a possible role for HOCl- mediated damage to (lipo)proteins in atherosclerosis and other inflammatory diseases.
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Adams, Mark R.; Forsyth, Cecily J.; Jessup, Wendy K.; Robinson, Jacqui T.C.; Celermajer, David S.Objectives.: Our aim was to assess the effect of oral l-arginine on endothelial or platelet physiology in humans. Background.: l-Arginine is the substrate for nitric oxide synthesis, and in cholesterol-fed rabbits, oral l-arginine improves endothelium-dependent dilation, inhibits platelet aggregation and reduces atheroma. In hypercholesterolemic humans, intravenous l-arginine immediately improves endothelium-dependent dilation; however, the vascular effects of oral l-arginine in healthy humans have not previously been investigated. Methods.: In a prospective, double-blind, randomized crossover trial, 12 healthy young men 27 to 37 years old took l-arginine (7 g three times daily) or placebo for 3 days each, separated by a washout period of 7 to 14 days. Results.: After l-arginine, plasma levels of arginine (mean SEM 303 36 vs. 128 12?mol/liter, p = 0.01) and urea (6.7 0.5 vs. 5.2 0.2 mmol/liter, p < 0.01) were higher than levels measured after placebo, and platelet aggregation in response to adenosine diphosphate was markedly impaired (37 12% vs. 81 3%, p = 0.02). The inhibition of platelet aggregation correlated with the plasma level of l-arginine (r = 0.74, p = 0.01), and it could be completely or partially reversed by ex vivo incubation with N-monomethyl-l-arginine, a specific nitric oxide synthase inhibitor. Platelet cyclic guanosine monophosphate levels were higher after oral l-arginine than at baseline (1.91 0.46 vs. 1.38 0.40 pmol/109 platelets, p = 0.04). No changes were seen in fasting lipid levels, heart rate, blood pressure, endotheliumdependent dilation of the brachial artery (measured in response to reactive hyperemia, using external vascular ultrasound) (6.1 0.7% vs. 6.5 0.7%, p = NS) or in plasma levels of nitrosylated proteins (a marker of in vivo nitric oxide production) (3.5 0.5 vs. 3.3 0.4 ?mol/liter, p = NS) 1 to 1.5 h after the last dose of l-arginine. Conclusions.: In these healthy young adult men, oral l-arginine inhibited platelet aggregation by way of the nitric oxide pathway. However, it had no effect on systemic hemodynamic variables, plasma nitrosylated protein levels or endothelium-dependent dilation. Therefore, at certain doses, oral l-arginine may result in a relatively platelet-specific increase in nitric oxide production. 1995 American College of Cardiology.
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Kritharides, Leonard; Jessup, Wendy K.; Dean, Roger T.The oxidation of low-density lipoprotein (LDL) may be important in the pathogenesis of atherosclerosis. However, the interactions between cells and metals in promoting LDL oxidation are inadequately understood. A sensitive high-performance liquid chromatography analysis of cholesterol, cholesteryl esters, and their oxidation products was used to identify and accurately measure LDL oxidation achieved in thiol-free Hanks' balanced salt solution (HBSS) at pH 7.4. Mouse peritoneal macrophages inhibited LDL oxidation when incubated in HBSS containing either 10 ?Miron or 1 ?Mcopper, but were markedly prooxidant in the presence of both metals. The prooxidant effect of macrophages in the presence of both iron and copper did not require the provision of added disulfides or thiols. Both Fe2+and macrophages were demonstrated to independently reduce Cu2+to Cu1+in HBSS, indicating that the direct reduction of copper by cells or iron may underlie the observed promotion of LDL oxidation by macrophages in this system. We conclude that macrophages can either promote or inhibit metal-mediated LDL oxidation and that externally supplied thiols are not essential to the promotion of LDL oxidation by cells. The presence of both iron and copper may be particularly important for macrophages to promote LDL oxidationin vivo. 1995 Academic Press, Inc.
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Thomas, Shane R.; Neuil, Ji? Mohr, Detlef; Stocker, RolandThe oxidation of low-density lipoproteins (LDLs) is now commonly implicated as an important early event in atherogenesis. The resulting interest in LDL antioxidation has focused on ?-tocopherol, the biologically and chemically most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. We review advances made in our understanding of the molecular action of a-tocopherol in radical-mediated oxidation of isolated human LDL and how the vitamin's antioxidant activity is enhanced or even dependent on the presence of suitable reducing species, which are referred to as coantioxidants.
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Fu, Shanlin; Gebicki, Silvia; Jessup, Wendy K.; Gebicki, Janusz M.; Dean, Roger T.In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. ?-hydroperoxyvaline, (2S,3S)-?-hydroperoxyvaline and (2S,3R)-?-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on ?-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.
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Iheanacho, Eugene N.; Hunt, Nicholas H.; Stocker, RolandIt has been suggested that the host antimalarial response depends in part on phagocyte-derived oxidants and that the parasite itself exerts an oxidative stress on its erythrocytic environment. Intraerythrocytic malaria parasites are particularly susceptible to being damaged by oxidative drugs, several of which are under development as chemotherapeutic agents. Thus the antioxidant status and associated regulatory mechanisms of the blood during malaria infection are of great interest. The important antioxidant ascorbate (AH-) and isoascorbate (IAH-), an isomer that does not occur naturally in animals, were found to have similar redox properties. We therefore assessed the usefulness of IAH- as a marker for studies of AH- handling in vivo and in vitro under normal conditions and in murine malaria infection. DHIA added to whole blood from normal or Plasmodium vinckei- infected mice in vitro was rapidly taken up into blood cells and reduced to IAH-. Intracellular IAH- derived from the exogenous DHIA was released into the plasma by blood cells from malaria-infected mice but not those from normal mice. Uptake and reduction of DHIA had no effect on plasma or cellular levels of AH- under these conditions. IAH- injected IV- into either normal or P. vinckei-infected mice was rapidly cleared in both cases and led to an increase in plasma levels of AH-; this suggested displacement of the latter from some intracellular site, presumably not associated with blood cells. DHIA administered as an intravascular bolus into either normal or malaria-infected mice was rapidly reduced. However, in contrast to the in vitro situation, the concentration of plasma IAH- derived from the injected DHIA was approximately the same in both the infected and control animals. The IAH- so formed disappeared quickly from the plasma. Intravenous injection of DHIA into malaria-infected mice caused a rapid, prolonged increase in the proportion of plasma vitamin C in the form of DHA, whereas in uninfected mice there was a transient decrease in plasma DHA followed by normalisation. The changes in plasma AH- and DHA following IV injection of a single dose of DHA closely paralleled those seen after DHIA administration. These observations indicate that: (i) blood cells from normal and malaria-infected mice take up and reduce DHIA in a similar fashion, but they have different ways of handling the resulting IAH-; (ii) cells other than blood cells are important in the reduction of plasma DHIA and DHA in vivo; (iii) malaria-infected mice are less capable of handling oxidative challenge than normal ones; (iv) in some circumstances IAH- and DHIA may be useful nonisotopic markers for studies of vitamin C handling in vitro and in vivo. 1995.
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Kritharides, Leonard; Jessup, Wendy K.; Mander, Erin L.; Dean, Roger T.Although oxidized low-density lipoprotein (Ox-LDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I-containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells. Furthermore, the subcellular distributions of both cholesterol and 7-ketocholesterol in individual subcellular organelle fractions were identical to that of free cholesterol in nonloaded cells, indicating that these sterols in OxLDL-loaded cells are not selectively sequestered in lysosomes. 7-Ketocholesterol is released much less efficiently than cholesterol from OxLDL-loaded cells. In addition, OxLDL-loaded cells release cholesterol less efficiently than do cells derived from AcLDL. It is possible that this impairment of efflux from OxLDL-loaded cells influences the generation and persistence of the foam cell phenotype in vivo and may therefore contribute to the atherogenicity of OxLDL.
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Sattler, Wolfgang; Christison, Julie K.; Stocker, RolandExposure of isolated high-(HDL) and low-density lipoproteins (LDL) to aqueous peroxyl radicals generated from a thermo-labile azo-compound resulted in immediate formation of cholesteryllinoleate hydroxide (Ch18:2-OH) in addition to hydroperoxides of cholesteryllinoleate (Ch 18: 2- OOH) and phospholipids. Ch 18: 2- OH was also formed in peroxyl radical-oxidizing human plasma devoid of ascorbate or low molecular weight compounds or isolated lipoproteins in the presence of desferrioxamine. In contrast, peroxyl radical-mediated oxidation of HDL or LDL lipid extracts or detergent-solubilized lipoproteins resulted in the formation of Ch 18:2-OOH without concomitant formation of Ch 18:2-OH. Heat treatment of the isolated lipoproteins prior to oxidation greatly reduced Ch18:2-OH formation. Compared to the concentrations of Ch18:2-OOH accumulating, formation of Ch18:2-OH was more pronounced in oxidizing HDL than LDL isolated from the same blood donor. The levels of Ch18: 2-OH detected after prolonged oxidation periods were independent of the radical flux to which the lipoproteins were exposed. In the absence of peroxyl radical generator, [3H]Chl8: 2-OOH associated with HDL was converted readily and in a biphasic manner into [3H]Chl8: 2-OH upon incubation at 37 but not 4C. LDL-associated [3H]Ch18: 2-OOH were also reduced, albeit with an initial reaction rate -10 times slower than that observed with labelled HDL. Together, the results show that cholesterylester hydroxides are formed during (peroxyl) radical-mediated oxidation of isolated intact HDL and LDL under transition metal-free conditions. The findings suggest the presence of a hydroperoxide reducing activity in isolated human lipoproteins, particularly HDL. 1995.
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Lau, W.; Devery, Jannine M.; Geczy, Carolyn L.In the early development of atherosclerotic plaque, monocytes are recruited to the arterial intima where they accumulate lipid and become foam cells. The recently described murine chemotactic S100 protein, CP-10, may have an important role in this process. Intraperitoneal injection of CP- 10<inf>42-55</inf> (chemotactic hinge region peptide) into mice caused a sustained leukocyte recruitment with a sixfold increase in monocyte numbers over 24 h. CP-10<inf>42-55</inf>-elicited monocyte/macrophages accumulated significantly increased cholesteryl esters in response to acetylated LDL, both in vivo and in vitro and this was associated with a twofold increase in scavenger receptor expression. By contrast, thioglycollate-and macrophage colony- stimulating factor-elicited macrophages expressed levels of scavenger receptor similar to those on resident macrophages and did not exhibit enhanced acetylated LDL loading in vitro. The leukocyte integrin Mac-1 (CD11b/CD18) and its ? subunit (CD18), but neither lymphocyte function- associated antigen-1 nor very late activation antigen-4, were upregulated on monocyte/macrophages elicited by CP-10<inf>42-55</inf>, thioglycollate, and macrophage colony-stimulating factor. Cholesteryl ester accumulation in vitro was significantly enhanced by adhesion, which appeared to involve macrophage activation via ligation of Mac-1. The initial events of monocyte recruitment and adhesion to the vessel wall may be important in macrophage foam cell development, and CP-10 or related S100 proteins may contribute to the early inflammatory events of atherogenesis by stimulating these events.
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Kohar, Indrajati; Baca, Manuel; Suarna, Cacang; Stocker, Roland; Southwell-Keely, Peter T.The products of oxidation of the a-tocopherol model compound, 2,2,5,7,8-pentamethyl-6-chromanol (PH) by t-butyl hydroperoxide in chloroform varied with the amount of water present. In the presence of a trace of water, the main products were the spirodimer (PSD) and spirotrimer (PST). As the content of water increased, the main product became 2-(3-hydroxy-3-methylbutyl)-3,5,6-trimethyl-1,4-benzoquinone (PQ). Oxidation ofPH in aqueous liposome suspension also producedPQ as the major product. These results suggested that, in aqueous solutions, the major oxidation product ofPH would bePQ and of ?-tocopherol (TH) would be ?-tocopheryl quinone (TQ). The ease of reduction ofPQ andTQ was studied in chemical and biological systems.PQ, TQ, and ubiquinone-10 (UQ) were rapidly reduced to their respective hydroquinones (PQH<inf>2</inf>,TQH<inf>2</inf>, andUQH<inf>2</inf>) atpH 7.3 by NADH plus FAD. Whole blood reducedPQ rapidly at 37C toPQH<inf>2</inf> but did not reduceTQ toTQH<inf>2</inf>. Human peripheral blood mononuclear cells took upTQ from a bovine serum albumin complex and reduced it to TQH<inf>2</inf>. Ingestion ofTQ (350 mg) by one of us (PSK) resulted in the formation ofTQH<inf>2</inf> during a 5 h period. These results demonstrate that several biological systems are able to reduceTQ toTQH<inf>2</inf> and that it is a reaction that may occur normally in vivo. 1995 Elsevier Science Ltd.
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Shen, Jie; Devery, Jannine M.; King, Nicholas J.C.West Nile Virus (WNV) infection of human embryonic fibroblasts can induce expression of ICAM-1 by two distinct mechanisms. An early and direct mechanism occurs within 2 hr of virus infection which is cytokine independent, and an indirect mechanism occurs within 24 hr of virus infection and is regulated by the release of IFN-type 1. Virus-inactivated, conditioned supernatants removed from WNV-infected fibroblast cultures at 4 hr did not alter ICAM-1 expression on fresh, untreated fibroblasts, whereas conditioned supernatants from 24-hr-infected cultures induced small increases in ICAM-1 expression after incubation for 24 hr but not after 4 hr. These studies also demonstrate that the expression of ICAM-1 on fibroblasts in response to flavivirus is cell-cycle dependent. WNV can only induce increased ICAM-1 expression in quiescent fibroblasts in G<inf>o</inf> phase. In contrast, induction of ICAM-1 after exposure to types 1 and 2 IFN is not cell-cycle dependent. Other viruses, including double-stranded DNA viruses, vaccinia, and adenovirus 2 and 5 and the single, positive-stranded RNA alphavirus, Semiliki Forest virus, did not induce ICAM-1 expression on fibroblasts after 24 hr. Another alphavirus, Ross river, was able to induce ICAM-1 but only by the indirect mechanism of type 1 IFN-dependent release. The closely related flavivirus, Kunjin, induced increased ICAM-1 expression in a manner similar to WNV. The ability of flaviviruses to induce increased ICAM-1 expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infection/replication. 1995 Academic Press. All rights reserved.
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Kritharides, Leonard; Jessup, Wendy K.; Dean, Roger T.The extent to which cells can oxidize LDL may be underestimated because of the use of standard and arbitrary 24 hour in vitro incubations of cells with LDL. Such incubations have resulted in inconsistent results regarding the ability of cell-mediated LDL oxidation to generate relatively advanced oxidation products such as 7-ketocholesterol (7-KC). We studied prolonged oxidation of low density lipoprotein (LDL) by mouse peritoneal macrophages using HPLC measurement of cholesterol, cholesteryl esters and their oxidation products 7-KC and cholesteryl linoleate hydroperoxide (CL-OOH). Cell-mediated oxidation in Ham's F10 consistently followed the successive stages previously described during 24 hour-10 ?M copper-mediated LDL oxidation, always generating 7-KC if allowed to proceed for sufficient time. The degree of inhibition of LDL oxidation achieved by metal chelators EDTA and DTPA at more advanced stages of cell-mediated LDL oxidation was not predictable from the published effects of such chelators upon early stages of metal-mediated and cell-mediated LDL oxidation. EDTA and DTPA only incompletely prevented the consumption of cholesteryl esters and the loss of preformed CL-OOH when added after cell-mediated LDL oxidation was established, while effectively concurrently inhibiting the generation of 7-KC. These data indicate that progressive cell-mediated peroxidation of LDL cholesteryl esters and decomposition of CL-OOH may be less dependent upon a continuing supply of redox active metals than is the generation of 7-KC. In addition, they confirm the plausibility of prolonged cell-mediated oxidation of LDL as a source of oxysterols found in human atherosclerotic plaque, and imply that active redox cycling of metals is particularly important for their generation in vivo. 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Ashton, Anthony Wayne; Dawes, Joan; Chesterman., Colin N.Acidic and basic fibroblast growth factor (aFGF and bFGF respectively) are closely related mitogens (55% homology) of the heparin binding growth factor family. Reports of the relative potency of these growth factors and the ability of heparin to potentiate the activity of bFGF are conflicting. We have examined the effect of heparin and human recombinant aFGF and bFGF on basal and thrombin challenged release of metabolites from cultured human umbilical vein endothelial cells (HUVEC). Culture supernatant was assayed for thrombospondin, prostacyclin and PAI-1 and cell lysates were analysed for t-PA. aFGF and bFGF were equipotent in regulating the release of all metabolites studied, except thrombin stimulated release of PGI<inf>2</inf> where bFGF was more potent than aFGF in the absence of heparin. Heparin potentiated the mitogenic and metabolic effects of both bFGF and aFGF. However, heparin was not essential for the expression of the biological activity of FGF. 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Christison, Julie K.; Rye, Kerry Anne; Stocker, RolandThis study examines the cholesteryl ester transfer protein (CETP)- mediated exchange of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) and cholesteryl linoleate hydroxide (Ch18:2-OH) between low density lipoprotein (LDL) and high density lipoprotein (HDL). When [3H]Ch18:2-OOH-and [3H]18:2- OH-labeled LDL were incubated at 37C for 0-24 h with unoxidized HDL and purified CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the HDL. Similarly, when incubations were carried out with [3H]Ch18:2-OOH-and [3H]Ch18:2-OH- labeled HDL unoxidized LDL, and CETP, Ch18:2-OOH and Ch18:2-OH accumulated in the LDL. Comparable results were obtained for the CETP-mediated transfer of [3H]Ch18:2-OH alone from LDL to HDL. Transfer to HDL of oxidized cholesteryl linoleate from [3H]Ch18:2-OOH- and [3H]Ch18:2-OH-labeled LDL was comparable to that of unoxidized cholesteryl linoleate (Cb18:2). However, the rate of transfer of [3H]Ch18:2-OOH and [3H]Ch18:2-OH from LDL to HDL increased linearly as the molar ratio of acceptor (HDL) to donor (oxidized LDL) particles in the incubation increased from 0.5:1 to 10:1. This increased rate of exchange was accompanied by an increased proportion of the oxidized Ch18:2 being present as the hydroxide rather than hydroperoxide. Further increases in the molar ratio of HDL to oxidized LDL particles neither affected the transfer rate nor the extent of reduction of Ch18:2-OOH to Ch18:2-OH. We therefore conclude that i) CETP mediates bidirectional transfers of Ch18:2- OOH and Ch18:2-OH between HDL and LDL; ii) CETP does not distinguish between Ch18:2-OOH, Ch18:2-OH, and Ch18:2 as it mediates their exchange between HDL and LDL; and iii) association with HDL hastens the reduction of Ch18:2-OOH to Ch18:2-OH.
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Fu, Shanlin; Hick, Larry A.; Sheil, Margaret M.; Dean, Roger T.We have previously demonstrated the formation of two reactive moieties on proteins during free radical attack: hydroperoxides, and 3,4-dihydroxyphenylalanine (DOPA). Here we have undertaken the structural elucidation of the hydroperoxides of valine, the amino acid which is most susceptible to peroxidation. Exposure of L-valine to free radicals generated by radiolysis in an oxygen-saturated system produced three valine hydroperoxides. Upon treatment with sodium borohydride these were reduced to their corresponding hydroxides, which can be separated and purified by high performance liquid chromatography (HPLC). Based on spectroscopic data from high resolution chemical ionization (CI) mass spectrometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton (1H) nuclear magnetic resonance (NMR) and carbon-13 (13C) NMR studies, the three valine hydroxides have been identified as ?-hydroxyvaline [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-?-hydroxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R)-?-hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyvalines provides a sensitive and accurate method for quantitative measurement. This method enabled hydroxyvalines to be detected in the hydrolysates of a tripeptide (glutamylvalinyl-phenylalanine) and a protein (bovine serum albumin) that had been gamma-radiolysed and treated with sodium borohydride. Hydroxyvaline may be useful as a marker in studying protein oxidation in some biological systems under oxidative stress. 1995.
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Suarna, Cacang; Dean, Roger T.; May, James; Stocker, RolandWe assessed the antioxidant status and contents of unoxidized and oxidized lipids in freshly obtained, homogenized samples of both normal human iliac arteries and carotid and femoral atherosclerotic plaque. Optimal sample preparation involved homogenization of human atherosclerotic plaque for 5 minutes, which resulted in recovery of most of the unoxidized and oxidized lipids without substantial destruction of endogenous vitamins C and 87% and 43% recoveries of added standards of ?-tocotrienol and isoascorbate, respectively. The total protein, lipid, and antioxidant levels obtained from human plaque varied among donors, although the reproducibility of replicates from a single sample was within 3%, except for ubiquinone-10 and ascorbate, which varied by 20% and 25%, respectively. Plaque samples contained significantly more ascorbate and urate than control arteries, with no discernible difference in the vitamin C redox status between plaque and control materials. The concentrations of ?-tocopherol and ubiquinone-10 were comparable in plaque samples and control arteries. However, approximately 9 mol percent of plaque ?-tocopherol was present as ?-tocopherylquinone, whereas this oxidation product of vitamin E was not detectable in control arteries. Coenzyme Q<inf>10</inf> in plaque and control arteries was only detected in the oxidized form ubiquinone-10, although coenzyme Q<inf>10</inf> oxidation may have occurred during processing. The most abundant of all studied lipids in plaque samples was free cholesterol, followed by cholesteryl oleate and cholesteryl linoleate (Ch18:2). Approximately 30% of plaque Ch18:2 was oxidized, with 17%, 12%, and 1% present as fatty acyl hydroxides, ketones, and hydroperoxides, respectively. In comparison, 7-ketocholesterol was detected at an ?75-fold lower concentration. Normal arteries contained similar levels of protein as atherosclerotic arteries, much less free cholesterol, and no detectable amounts of unoxidized or oxidized cholesteryl esters. Together, these results demonstrate the coexistence in human plaque of large amounts of oxidized cholesteryl esters with significant concentrations of ascorbate and vitamin E in their reduced, antioxidant-active form. We conclude that compared with healthy human arteries, advanced atherosclerotic plaques are not deficient in the antioxidant vitamins C and E, despite the occurrence of massive lipid oxidation.
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Van Reyk, David M.; Brown, Andrew J.; Jessup, Wendy K.; Dean, Roger T.Removal of adventitious redox-active metals from buffers by treatment with Chelex resin is a widely used procedure in free radical research. Use of a new batch of Chelex-100 resin in our laboratory coincided with a sudden inability to oxidise low-density lipoprotein with copper. We found that copper-mediated oxidation of ascorbate in water treated with the same batch of Chelex was inhibited when compared with untreated water and water treated with a different batch of the resin. Washing the Chelex removed the inhibitory effect suggesting that material was leaching from the resin. The washing procedure for Chelex-100 described is simple and can be scaled up. Oxidation of ascorbate with low concentrations of copper can be used to test the quality of batches of the resin. 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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Zammit, Adrian; Dawes, JoanDermatan sulfate is a naturally occurring antithrombotic glycosaminoglycan. The antithrombin activity of several dermatan sulfate preparations has been measured in whole human plasma and found to be ~55% of that in purified systems. Kinetic studies under pseudo-first-order conditions indicated that the reduction in antithrombin activity of dermatan sulfate in plasma compared with that in buffer was due to noncompetitive inhibition with respect to dermatan sulfate. Analysis of the protein profile bound to immobilized dermatan sulphate showed that on a molar basis, histidine-rich glycoprotein and apolipoprotein E were the most abundant proteins specifically bound, together with significant amounts of fibrinogen and vitronectin. Addition of these proteins to the purified system showed that only fibrinogen inhibited the antithrombin activity of dermatan sulfate and that it did so in a concentration-dependent manner over the physiologic range of plasma fibrinogen levels. These results indicate that the anticoagulant activity of dermatan sulfate may be modulated in human plasma by fibrinogen.
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Bannon, Paul Gerard; Dean, Roger T.; Dawes, JoanA method is described for the isolation and culture of large numbers of nonadherent quiescent human peripheral blood monocytes for adhesion/migration studies. Approximately 1.008 cells/white cell concentrate with > 95% viability are isolated by countercurrent centrifugal elutriation. These cells are initially activated by the isolation procedure, but after a minimum culture time post isolation of 48 hours and a rest period after radiola-belling of at least 3 hours quiescent cells are routinely obtained, with low basal levels of adhesion and a rapid, reproducible superoxide response to activation with phorbol myristate acetate. 1995 Kluwer Academic Publishers.
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Bowry, Vincent W.; Mohr, Detlef; Cleary, Janelle A.; Stocker, RolandOxidation of low density lipoprotein (LDL) may be involved in the development of atherosclerosis. It has recently been shown that ?- tocopherol (?-TOH) can act either as an antioxidant or prooxidant for isolated low density lipoprotein (LDL). In the absence of an effective co- antioxidant, ?-TOH is a prooxidant and this activity is evidently due to reaction of the ?-tocopheroxyl radical (?-TO) with the LDL's polyunsaturated lipids (Bowry, V. B., and Stocker, R. (1993) J. Am. Chem. Soc. 115, 6029-6045). Herein we examined the effectiveness of selected natural and synthetic radical scavengers as co-antioxidants for inhibiting peroxyl radical-induced peroxidation in LDL that is devoid of ubiquinol-10 (an effective endogenous co-antioxidant) but still contains most of its natural complement of ?-TOH. Various quinols, catechols, and aminophenols, as well as ascorbate, 6-palmityl ascorbate, and bilirubin, were very effective co-antioxidants under our test conditions, whereas ordinary phenolic antioxidants, including short-tailed ?-TOH homologues, were less effective. Reduced glutathione, urate, and Probucol were ineffective. These findings confirm that the prooxidant activity of ?-TOH in LDL relies heavily on the segregation of water-insoluble radicals (particularly ?-TO') into individual LDL particles, since it was those compounds that are expected to either irreversibly reduce ?-TO' or accelerate the diffusion of radicals between particles which most effectively inhibited the tocopherol-mediated phase of peroxidation. Theoretical and practical implications of these findings are discussed, as is their relevance to the 'LDL oxidation' hypothesis of atherogenesis.
