Antioxidant vitamins: evidence from biomarkers in humans.
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Antioxidants↗
Biomedical subjects
M R McCall
Publications and source records attributed to M R McCall.
Ldl modified by hypochlorous acid is a potent inhibitor of lecithin-cholesterol acyltransferase activity.
Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily N-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 micromol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B-depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in N-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 micromol/L HOCl, respectively. Addition of 200 micromol/L ascorbate or N-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with </=200 micromol/L HOCl. Protecting the free thiol groups of LCAT with 5,5'-dithio-bis-(2-nitrobenzoic acid) before exposure to HOCl-modified LDL, which inhibits lipid hydroperoxide-mediated inactivation of LCAT, failed to prevent the loss of enzyme activity. Our data indicate that N-chloramines from HOCl-modified LDL mediate the loss of plasma LCAT activity and provide a novel mechanism by which myeloperoxidase-generated HOCl may promote atherogenesis.
Chloramines↗
Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis.
Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low-density lipoprotein (LDL) to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co-precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co-precipitate with apolipoprotein B-100-containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein-deficient plasma, and there was a dose-dependent increase in precipitation following addition of LDL to lipoprotein-deficient plasma. Binding of MPO to LDL could potentially enhance site-directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.
Arteriosclerosis↗
Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection.
Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. These reactive species oxidize the protein, lipid, and antioxidant components of LDL. Modification of apolipoprotein B results in enhanced uptake of LDL by macrophages with subsequent formation of lipid-laden foam cells. Nitric oxide synthases produce nitric oxide and, under certain conditions, superoxide radicals. Numerous other sources of superoxide radicals have been identified in the arterial wall, including NAD(P)H oxidases and xanthine oxidase. Nitric oxide and superoxide readily combine to form peroxynitrite, a reactive nitrogen species capable of modifying LDL. In this review, we examine the reaction pathways involved in LDL oxidation by myeloperoxidase and reactive nitrogen species and the potential protective effects of the antioxidant vitamins C and E.
Animals↗
Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility.
Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (approximately 85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained approximately 18% of the activity) and the d>1.21 g/ml fraction (which contained approximately 32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039-1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Can antioxidant vitamins materially reduce oxidative damage in humans?
Endogenous oxidative damage to proteins, lipids, and DNA is thought to be an important etiologic factor in aging and the development of chronic diseases such as cancer, atherosclerosis, and cataract formation. The pathology associated with these diseases is likely to occur only after the production of reactive oxygen species has exceeded the body's or cell's capacity to protect itself and effectively repair oxidative damage. Vitamin C, vitamin E, and beta-carotene, often referred to as "antioxidant vitamins," have been suggested to limit oxidative damage in humans, thereby lowering the risk of certain chronic diseases. However, epidemiological studies and clinical trials examining the efficacy of antioxidant vitamins, either individually or in combination, to affect disease outcome rarely address possible underlying mechanisms. Thus, in these studies it is often assumed that antioxidant vitamins act by lowering oxidative damage, but evidence in support of this contention is not provided. Therefore, in this review, we examine the scientific evidence that supplementation of humans with vitamin C, vitamin E, or beta-carotene lowers in vivo oxidative damage to lipids, proteins, or DNA based on the measurement of oxidative biomarkers, not disease outcome. With the only exception of supplemental vitamin E, and possibly vitamin C, being able to significantly lower lipid oxidative damage in both smokers and nonsmokers, the current evidence is insufficient to conclude that antioxidant vitamin supplementation materially reduces oxidative damage in humans.
Animals↗
Apolipoprotein A-I promotes cholesterol release and apolipoprotein E recruitment from THP-1 macrophage-like foam cells.
Apolipoprotein E (apoE) is synthesized and secreted by arterial macrophages while apolipoprotein A-I (apoA-I) is present in surrounding interstitial fluids. Both apolipoproteins play important roles in macrophage cholesterol homeostasis by forming lipid complexes (nascent-HDL) with cellular phospholipids (PL) and cholesterol (UC) thereby promoting cholesterol efflux. In this study, we evaluated the relative contributions of apoA-I and endogenously produced apoE in mediating the recruitment of cellular cholesterol. THP-1 human monocytes were differentiated (300 nm phorbol dibutyrate) into macrophages and macrophage-foam cells were generated by cholesterol loading with acetylated LDL (50 microg protein/ml). ApoA-I (10 microg/ml) depleted macrophage-foam cell cholesteryl esters by 50% in 24 h. This reduction was accompanied by a significant increase in the UC/PL mole ratio of nascent HDL (UC/PL = 0.80 +/- 0.15) in the medium compared to complexes isolated from macrophages (UC/PL = 0.59 +/- 0.08). Significantly more (70%) nascent-HDL were formed in incubations of apoA-I with macrophage-foam cells than with macrophages. Medium apoE accumulation paralleled the assembly of apoA-I containing nascent HDL where 2- and 4-fold increases were observed with macrophages and macrophage-foam cells, respectively, compared to incubations in the absence of apoA-I. Despite the increase in medium apoE accumulation, a majority (85%) of particles (11, 9, and 7.4 nm in diameter) from macrophages and macrophage-foam cells possessed apoA-I without apoE. ApoA-I plus apoE particles (13-16 nm) were also formed along with a small quantity of apoE-only particles (19-20 nm). The predominance of apoA-I only particles indicates, however, that the assembly of apoA-I-containing nascent-HDL represents a major metabolic pathway of cellular cholesterol recruitment compared to the endogenous production of apoE.
Apolipoprotein A-I↗
Evidence that apolipoprotein A-IMilano has reduced capacity, compared with wild-type apolipoprotein A-I, to recruit membrane cholesterol.
Human carriers of apolipoprotein (apo) A-IMilano are heterozygous for an Arg173-->Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-IMilano may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-IMilano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-IMilano and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (approximately 2 micrograms/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d = 1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66 +/- 12%) of the lipidated apoA-IMilano was in the homodimer form. Compositional analyses revealed that apoA-IMilano nascent HDL had a significantly lower (P < .001) unesterified cholesterol/phospholipid mole ratio (0.47 +/- 0.10) than wild-type apoA-I complexes (1.29 +/- 0.14), indicating that apoA-IMilano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-IMilano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-IMilano activated LCAT normally. We conclude that, even though apoA-IMilano is a normal activator of LCAT, it is less efficient that wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-IMilano may be unrelated to the initial stages of reverse cholesterol transport.
Animals↗
High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice.
Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.
Animals↗
Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity.
The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.
Apolipoprotein A-I↗
Structural relationships between nascent apoA-I-containing particles that are extracellularly assembled in cell culture.
Apolipoprotein A-I (apoA-I) incubated with CHO cells assembles three major nascent lipid complexes with diameters of 7.3, 9, and 11 nm. Previous studies suggested that the smaller nascent particles were precursors for the larger nascent ones. To test this hypothesis, the 7.3, 9, and 11 nm apoA-I-lipid complexes formed by incubating CHO cells with lipid-free apoA-I were isolated and subsequently each subpopulation was re-incubated with cells in the absence of other subpopulations. The physical-chemical characteristics of each subpopulation were examined before and after re-incubation in an effort to understand relationships. if any, between the different nascent complexes. The 7.3, 9, and 11 nm complexes were unique in that each of the particles had pre-alpha mobility on agarose gels: this rapid migration was not altered by re-incubation with cells. Protein crosslinking studies indicated that the 7.3, 9, and 11 nm complexes possessed 2, 3, and 4 apoA-I molecules per complex, respectively; it is unlikely that the size of the particle and number of apoA-I molecules per particle played a role in the increased negative charge of the particles. The present study shows that smaller particles did not give rise to larger ones upon re-incubation with cells. Rather, the 11 and 9 nm particles both generated smaller discs (the 11 nm giving rise primarily to 9 nm discs and the 9 nm complex giving rise to 7.3 nm discs) suggesting that, during incubation with cells, the complexes are destabilized and remodeled into smaller, not larger, complexes. Surprisingly, the 7.3 nm complexes during re-incubation with cells were extremely stable and did not undergo size alteration. When the 7.3 nm particles were incubated with additional small quantities of lipid-free apoA-I (1-2 microgram/ml), larger discoidal complexes were generated suggesting that the formation of larger particles may be driven by the availability of lipid-free apoA-I.
Animals↗
Gas-phase cigarette smoke inhibits plasma lecithin-cholesterol acyltransferase activity by modification of the enzyme's free thiols.
Cigarette smoking is associated with an increased risk of premature atherosclerosis. The underlying mechanisms responsible for this association are unknown. Recent work from this laboratory has shown that ex vivo exposure to plasma to gas-phase cigarette smoke (CS) produces a rapid inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and modified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-phase of an equivalent of 1/8 of a cigarette (one 'puff') at 15 min intervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to controls; supplementation of plasma with glutathione produced a dose-dependent protection of LCAT activity where at the highest concentration (1 mM) 78% protection was observed. A similar protection was obtained with N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apolipoproteins were crosslinked after 3 h exposure of plasma to CS; crosslinking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl arginine, and aminoguanidine failed to protect LCAT and HDL indicating a specificity with regard to the ability of free thiols to buffer the deleterious components of CS which inhibited LCAT and crosslinked HDL-apolipoproteins. Since LCAT contains two free cysteine residues (Cys-31 and -184) near the active site of the enzyme, we tested whether pretreatment of plasma with the reversible sulfhydryl modifying compound, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-induced inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcysteine↗
Inhibition of lecithin-cholesterol acyltransferase and modification of HDL apolipoproteins by aldehydes.
Experimental evidence suggests that aldehydes generated as a consequence of lipid peroxidation may be involved in the pathogenesis of atherosclerosis. It is well documented that aldehydes modify LDL: however, less is known concerning the effects of aldehydes on other plasma and interstitial fluid components. In the present study, we investigated the effects of five physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal [HNE], and malondialdehyde [MDA]) on two key constituents of the antiatherogenic reverse cholesterol transport pathway, lecithin-cholesterol acyltransferase (LCAT) and HDL. Human plasma was incubated for 3 hours at 37 degrees C with each one of the five aldehydes at concentrations ranging from 0.16 to 84 mmol/L. Dose-dependent decreases in LCAT activity were observed. The short-chain (acrolein) and long-chain (HNE) alpha,beta-unsaturated aldehydes were the most effective LCAT inhibitors. Micromolar concentrations of these unsaturated aldehydes resulted in significant reductions in plasma LCAT activity. The short- and longer-chain saturated aldehydes acetaldehyde and hexanal and the dialdehyde MDA were considerably less effective at inhibiting LCAT than were acrolein and HNE. In addition to inhibiting LCAT, aldehydes increased HDL electrophoretic mobility and cross-linked HDL apolipoproteins. Cross-linking of apolipoproteins A-I and A-II required higher aldehyde concentrations than inhibition of LCAT. The alpha,beta-unsaturated aldehydes acrolein and HNE were fourfold to eightfold more effective cross-linkers of apolipoproteins A-I and A-II than the other aldehydes studied. These data suggest that products of lipid peroxidation, especially unsaturated aldehydes, may interfere with normal HDL cholesterol transport by inhibiting LCAT and modifying HDL apolipoproteins.
Aldehydes↗
Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT.
Studies were carried out to determine whether apolipoprotein (apo) A-II, like apoA-I, can recruit phospholipid and cholesterol from cell membranes, thereby forming nascent apoA-II-specific HDL. ApoA-II and apoA-I were purified from plasma and each was incubated with CHO cells at a concentration of 10 micrograms/ml. Lipid-containing complexes were isolated from the medium in both cases; the composition of the apoA-II- and apoA-I-specific complexes were similar where percent protein, phospholipid, and cholesterol were 35 +/- 3, 38 +/- 2, and 25 +/- 1 for apoA-II, respectively, and 40 +/- 2, 35 +/- 1, and 24 +/- 2 for apoA-I, respectively. On a per mole of apolipoprotein basis, apoA-I recruited significantly more phospholipid and cholesterol than dimeric apoA-II suggesting that apoA-I with its greater number of alpha helices binds more lipid. By electron microscopy, nascent apoA-II- and apoA-I-specific particles were predominantly discoidal in morphology. ApoA-II complexes were unique in their nondenaturing polyacrylamide gradient gel size distribution as six distinct populations of particles with diameters of 8.1, 9.3, 10.4, 11.8, 13.1, and 14.6 nm were routinely noted, compared with apoA-I which formed only three major populations with diameters of 7.3, 9.2, and 11.0 nm. Nascent apoA-I complexes incubated with purified lecithin:cholesterol acyltransferase (LCAT) were transformed into predominantly 8.4 nm particles. The latter is similar in size to plasma HDL3a, LpA-I particles, suggesting that extracellularly assembled apoA-I-lipid complexes can directly give rise to a major plasma LpA-I subpopulation upon interaction with LCAT. Unlike apoA-I, apoA-II-lipid complexes could not serve as substrates for LCAT and did not undergo transformation. This study also demonstrates, for the first time, that apoA-II and apoA-I show a preference in phospholipid recruitment from membranes. Although phosphatidylcholine is the major phospholipid removed by both apolipoproteins, apoA-II preferentially recruits phosphatidylethanolamine (PE) as its second most abundant phospholipid while apoA-I recruits sphingomyelin. As PE is usually associated with the inner leaflet of the membrane, it is likely that dimeric apoA-II, compared with apoA-I, can penetrate farther into the membrane and extract PE. This ability of apoA-II to insert more deeply into the lipid milieu may explain the known ability of apoA-II to resist dissociation from the mature HDL particle.
Animals↗
Copper and gas-phase cigarette smoke inhibit plasma lecithin:cholesterol acyltransferase activity by different mechanisms.
Cigarette smokers have reduced levels of plasma high density lipoprotein (HDL) compared to nonsmokers and are at risk of premature cardiovascular disease. Previous work from this laboratory has shown that exposure of human plasma to gas-phase cigarette smoke (CS) inhibited the activity of lecithin:cholesterol acyltransferase (LCAT), the enzyme that catalyzes the formation of cholesteryl ester in HDL and thereby promotes HDL maturation. As CS contains free radicals that could potentially oxidize plasma lipoproteins, we examined the involvement of lipid peroxidation in LCAT inhibition. Results obtained with CS were compared with those obtained by initiating lipid peroxidation with copper ions. Exposure of dialyzed human plasma to an equivalent of one-eighth of a cigarette at 15-min intervals resulted in a progressive loss of LCAT activity (50 and 90% reductions by 1 and 6 h, respectively). A similar pattern of LCAT inhibition was produced with copper (0.5 mM) where 50 and 97% reductions were observed at 1 and 6 h, respectively. To determine whether LCAT inhibition was related to lipid peroxidation, lipoprotein fractions corresponding to VLDL-IDL, LDL, and HDL were isolated from plasma exposed to CS or copper and analyzed for changes in TBARS, the polyunsaturated fatty acid arachidonate relative to palmitate (20:4/16:0 ratio), and vitamin E concentrations. Exposure of plasma for 6 h to CS had no effect on the levels of TBARS and 20:4/16:0 ratio; however, 6 h copper treatment (0.5 mM) caused a 3.0-, 4.0-, and 1.4-fold increase in TBARS and a 17, 25, and 13% reduction in the 20:4/16:0 ratio in VLDL-IDL, LDL, and HDL fractions, respectively. In addition, a complete depletion of lipoprotein vitamin E was observed with CS, whereas copper decreased vitamin E levels by approximately 50% in each fraction. Supplementation of plasma with either vitamin C (85 microM) or butylated hydroxytoluene (BHT, 0.45 mM) was unable to protect LCAT from CS. In contrast, BHT completely protected LCAT activity from inhibition by copper. We conclude that unlike copper, CS-induced inhibition of plasma LCAT activity was unrelated to free radical-induced lipid peroxidation. The inhibition of LCAT activity by cigarette smoke may contribute to the development of atherosclerosis by impairing HDL metabolism and the reverse cholesterol transport process.
Ascorbic Acid↗
The role of apolipoprotein A-I-containing lipoproteins in atherosclerosis.
The inverse relationship between HDL and apolipoprotein A-I concentrations and the risk for premature atherosclerosis is well established, but the mechanism whereby apolipoprotein A-I offers protection is still somewhat elusive. Recent studies suggest that a specific subpopulation within the lipoprotein (AI) subclass may be more effective than others in promoting cholesterol efflux from cells. In addition, it appears that the lipid-free form of apolipoprotein A-I may have an important role in the antiatherosclerotic process. Unique new functions of apolipoprotein A-I-containing particles in modulating cytokines and lipid hydroperoxide transport, together with their role in antiatherogenesis, are also discussed. Current research with transgenic mice, however, indicates that apolipoprotein A-II must be taken into consideration in understanding the development of atherosclerosis, because it appears to be a potent antagonist for the protective properties of apolipoprotein A-I.
Animals↗
Modification of LCAT activity and HDL structure. New links between cigarette smoke and coronary heart disease risk.
The mechanism(s) through which smoking influences the progression of atherosclerosis is poorly understood. Recent evidence suggests that oxidants present in the gas phase of cigarette smoke are involved. We exposed human plasma to the filtered gas phase of cigarette smoke to assess its effects on plasma components involved in the antiatherogenic reverse cholesterol transport pathway. In our model, freshly isolated plasma (24 mL) was exposed to filtered air or gas-phase cigarette smoke for up to 6 hours at 37 degrees C. Lecithin-cholesterol acyltransferase (LCAT) activity was dramatically inhibited by cigarette smoke. A single 15-minute exposure to the smoke from an eighth of a cigarette was sufficient to reduce LCAT activity by 7%; additional exposures resulted in further decreases in activity. At 6 hours, only 22% of control LCAT activity remained in plasma exposed to smoke. Compared with control, gas-phase cigarette smoke-exposed plasma possessed high-density lipoprotein (HDL) with increased (16%) negative charge and with cross-linked apolipoproteins AI and AII. These data demonstrate that gas-phase cigarette smoke can inhibit a key enzyme (LCAT) and modify an integral lipid transport particle (HDL) that are essential components for the normal function of the reverse cholesterol transport pathway. Gas-phase cigarette smoke-induced modification of the reverse cholesterol transport pathway may provide a new mechanistic link between cigarette smoke and coronary heart disease risk.
Adult↗
Re-uptake of nascent low-density lipoproteins by HepG2 cells.
The kinetics and specificity of the interaction of nascent HepG2 LDL with the HepG2 LDL receptor were examined. 125I-Labeled HepG2 LDL and plasma LDL were bound by HepG2 cells in a specific and saturable manner at 4 degrees C. Competition studies with HepG2 LDL and plasma LDL indicated that both ligands bound to the same receptor. Scatchard analyses of the specific 4 degrees C-binding data revealed a Kd of 75 nM for HepG2 LDL and a Kd of 30 nM for plasma LDL suggesting that HepG2 LDL bind less efficiently to the HepG2 LDL receptor than plasma LDL. Binding, internalization and degradation studies carried out at 37 degrees C indicated that HepG2 cells are capable of catabolizing their own nascent LDL; however, under normal experimental conditions re-uptake of nascent LDL is quantitatively insignificant.
Binding, Competitive↗