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PubMed1992

Metabolism in vivo of [14C]oleic acid and [3H]retinol of lipid-poor and lipid-rich chyle.

[3H]Retinol and [14C]oleic acid labelled fresh chyle was obtained from thoracic duct cannulated rats. The labelled compounds were fed dispersed in either a small amount of egg phosphatidylcholine to produce a lipid-poor chyle, or in a soy bean lipid emulsion to produce a lipid-rich chyle. Small amounts (100 microliters, 24 and 172 micrograms triacylglycerol, respectively) of fresh labelled chyle preparations were injected i.v. into fed recipient animals, which were killed after 10, 20 or 30 min. At 20 min, more [3H]retinyl ester remained in plasma in the rats injected with lipid-rich than in those injected with lipid-poor chyle. The difference was, however, smaller than the difference in the hepatic uptake of 3H. Both the uptake of 3H by the liver and the hydrolysis of [3H]retinyl ester after the uptake, was faster in the group that had been injected with the lipid-poor chyle. The 3H/14C ratios of the serum and liver lipids in relation to that of the injected material did not differ between the two groups, indicating that the proportion of the [14C]triacylglycerol that underwent hydrolysis before clearance of remnants by the liver did not differ. Particularly in the heart, but also in adipose tissue, lungs and kidneys the 3H radioactivity after injecting lipid-rich chyle was highest at 10 min and then decreased with time, being similar in the two groups at 30 min. The results suggest that the formation of remnants from lipoproteins formed after a fat meal requires a longer time for the interaction with endothelial-bound lipoprotein lipase. The uptake by the spleen was also 6-9-fold higher than in the group receiving lipid-poor chyle, indicating that the reticuloendothelial system participates in the metabolism of chyle lipoproteins after a fat meal.

Adipose Tissue↗
PubMed1991

Lipids in cells of atherosclerotic and uninvolved human aorta. III. Lipid distribution in intimal sublayers.

The distribution, content, and composition of tissue and cellular lipids in intimal layers of unaffected and atherosclerotic human aorta were studied. Aortic tissue was divided into medial and intimal layers; the intimal layer was further separated into elastic-hyperplastic and musculo-elastic sublayers. Cells were isolated from both intimal layers by enzyme digestion. The lipids extracted from whole tissue and cells were separated by TLC and analyzed by scanning densitometry. The highest content of phospholipids (PhL), triglycerides (TG), cholesterol (C), and cholesteryl esters (CE) was detected in the elastic-hyperplastic layer of atherosclerotic plaque. However, taking into account that the elastic-hyperplastic layer of intima in lesioned areas was thickened, the lipid content per volume unit of both sublayers in fatty streaks and in plaques was equal. In the media underlying an atherosclerotic plaque, an increase in CE rather than in other lipid classes occurred. In the intima, an overall increase in PhL, TG, C, and CE content was found to display a constant ratio between these lipid classes, similar to that of low density lipoproteins (LDL). Cells isolated from atherosclerotic lesions had a higher lipid content than cells from areas of unaffected intima. However, the increase in the content of different lipid classes was not proportional, compared with tissue lipids. The content of PhL was the same, while an increase in TG, C, and CE was observed. The major contribution to excess cellular lipid accumulation in cells from atherosclerotic lesions was made by CE.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2004

Comparison between the effects of soy milk and non-fat cow milk on lipid profile and lipid peroxidation in patients with primary hypercholesterolemia.

OBJECTIVE: This study assessed whether the consumption of soy milk could add significantly to the lipid profile and lipid peroxidation in comparison with non-fat milk. METHODS: A double-blind, randomized, crossover study was conducted on 60 outpatients with primary hypercholesterolemia following a lipid-lowering diet for at least 6 wk. Lipid profile was obtained at baseline and at 6 and 12 wk, with the patients randomly assigned to receive initially 1 L/d of soy milk or non-fat cow milk for 6 wk. Lipid peroxidation was estimated by plasma thiobarbituric reactive substances. Apolipoprotein E genotypes were examined by polymerase chain reaction restriction fragment length polymorphism. RESULTS: The soy milk diet was associated with low-density lipoprotein cholesterol reduction (baseline = 157 +/- 5 mg/dL; soy milk = 148 +/- 4 mg/dL; non-fat cow milk = 158 +/- 4 mg/dL; P < 0.05, soy milk versus other treatments) and with high-density lipoprotein cholesterol increase (baseline = 58 +/- 2 mg/dL; soy milk = 62 +/- 2 mg/dL; non-fat cow milk = 57 +/- 2 mg/dL; P < 0.05, soy milk versus other treatments). In addition, plasma thiobarbituric reactive substances were reduced by the soy milk diet (baseline = 1.82 +/- 0.12 nM/L; soy milk = 1.49 +/- 0.09 nM/L; non-fat cow milk = 1.91 +/- 0.11 nM/mL; P < 0.05, soy milk versus non-fat cow milk). Changes in lipid profile were not influenced by APOE genotypes. CONCLUSIONS: These results indicate that soy milk as part of a lipid-lowering diet has beneficial effects in improving lipid profile and reducing lipid peroxidation.

Adult↗
PubMed2000

Effects of normal alcohols and isoflurane on lipid headgroup dynamics in nicotinic acetylcholine receptor-rich lipid vesicles.

The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.

Alcohols↗
PubMed2006

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

Amino Acid Substitution↗
PubMed2006

Dynamics imaging of lipid phases and lipid-marker interactions in model biomembranes.

Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions.

Anisotropy↗
PubMed1998

Rapid automated determination of lipid hydroperoxide concentrations and total antioxidant status of serum samples from patients infected with HIV: elevated lipid hydroperoxide concentrations and depleted total antioxidant capacity of serum samples.

Excessive production of oxygen free radicals causes the oxidation of circulating or membrane lipids, proteins, and DNA. Patients infected with HIV usually have severe malnutrition in the AIDS stage of disease. Therefore, they may be at higher risk of oxidative stress. We measured lipid hydroperoxide concentration, antioxidant status, cholesterol, triglyceride, iron, ceruloplasmin, and transferrin concentrations in the serum samples of 14 patients infected with HIV and compared our results with the results from 14 volunteers who served as controls. Lipid hydroperoxide concentrations in serum samples were measured by a colorimetric assay in which hemoglobin catalyzes the reaction of lipid hydroperoxide with a methylene blue derivative, yielding methylene blue. The total antioxidant capacity of serum was measured by the ability of serum to inhibit the formation of ferrylmyoglobin by metmyoglobin and hydrogen peroxide. Both assays were automated on the Syva-30R analyzer (Behring, San Francisco, Calif). We measured serum cholesterol and triglyceride concentrations by using the Vitro 950 analyzer (Johnson & Johnson, Rochester, NY). The lipid hydroperoxide concentrations were significantly elevated (mean, 1.44; SD, 0.95 micromol/L) in patients with HIV compared with control subjects (mean, 0.25; SD, 0.24 micromol/L). In contrast, the total antioxidant capacity was significantly lower in patients with HIV (mean, 1.04; SD, 0.13 mmol/L of trolox equivalent) compared with control subjects (mean, 1.66; SD, 0.09 mmol/L). We observed a fair correlation between serum lipid hydroperoxide concentrations and serum triglyceride concentrations in patients with AIDS. The correlation between serum hydroperoxide concentration and antioxidant status of serum was relatively poor. The lipid hydroperoxide assay was linear, from 0.1 micromol/L to 50 micromol/L. The within-run and between-run coefficients of variation were 3.5% and 4.5%, respectively, at a lipid hydroperoxide concentration of 2.5 micromol/L. The total antioxidant capacity assay was linear, from 0.1 to 2.5 mmol/L of trolox equivalent. The within-run and between-run coefficients of variation were 1.4% and 4.2% for the standard, with a target total antioxidant capacity of 1.5 mmol/L of trolox equivalent. We conclude that our automated assays for determination of total antioxidant status of serum and lipid hydroperoxide products may be helpful screening tests followed by measuring individual antioxidants, such as tocopherol, ascorbic acid, and other antioxidants for patients with severe deficiency of antioxidant status.

Antioxidants↗
PubMed2004

High-polarity Mycobacterium avium-derived lipids interact with murine macrophage lipid rafts.

Cholesterol- and sphingolipid-rich membrane microdomains (lipid rafts) are widely recognized as portals for pathogenic micro-organisms. A growing body of evidence demonstrates mobilization of host plasma cell membrane lipid rafts towards the site of contact with several pathogens as well as a strict dependence on cholesterol for appropriate internalization. The fate of lipid rafts once the pathogen has been internalized and the nature of the pathogen components that interact with them is however less understood. To address both these issues, infection of the J774 murine cell line with Mycobacterium avium was used as a model. After demonstrating that M. avium induces lipid raft mobilization and that M. avium infects J774 by a cholesterol-dependent mechanism, it is shown here that mycobacterial phagosomes harbour lipid rafts, which are, at least in part, of plasma cell membrane origin. On the other hand, by using latex microbeads coated with any of the three fractions of M. avium-derived lipids of different polarity, we provide evidence that high-polarity, in contrast to low-polarity and intermediate-polarity, mycobacterial lipids or uncoated latex beads have a strong capacity to induce lipid raft mobilization. These results suggest that high-polarity mycobacterial lipid(s) interact with host cell cholesterol-enriched microdomains which may in turn influence the course of infection.

Animals↗
PubMed1998

Autoxidation of rat brain homogenate: evidence for spontaneous lipid peroxidation. Comparison with the characteristics of Fe2+- and ascorbic acid-stimulated lipid peroxidation.

Aerobically-incubated brain homogenates are known to undergo autoxidation characterized by spontaneous TBARS production, presumably as a result of lipid peroxidation. However, TBARS measurement alone, because of its lack of specificity, is not sufficient to demonstrate the occurrence of lipid peroxidation in complex biological systems. This study, undertaken to determine whether or not spontaneous oxidation of rat brain homogenate is due to lipid peroxidation, measured different specific markers of this process (fatty acids, lipid aldehydes and the formation of fluorescence products) and studied changes in alpha-tocopherol. Incubation of rat brain homogenates at 37 degrees C under air led to spontaneous TBARS formation, which was accompanied by lipid aldehydes and lipid fluorescence products as well as polyunsaturated fatty acid (PUFA) degradation. Alpha-tocopherol was also consumed. On the whole, these results demonstrate that autoxidation of brain homogenate is a spontaneous lipid peroxidation process. When homogenates were exposed to Fe2+ and ascorbic acid-induced oxidative stress, lipid peroxidation was enhanced. However, spontaneous and stimulated peroxidation showed similar patterns not characteristic of classical lipid peroxidation, i.e. without the lag and accelerating phases typical of a propagating chain reaction. PUFA degradation was limited despite stimulation of peroxidation.

Animals↗
PubMed2001

Relationships among total lipid, lipid classes, and polychlorinated biphenyl concentrations in two indigenous populations of ribbed mussels (Geukensia demissa) over an annual cycle.

Two indigenous ribbed mussel (Geukensia demissa) populations were sampled approximately every four weeks during 1997 to investigate the relationships among concentrations of total lipid, lipid classes, and polychlorinated biphenyls (PCBs). One population was located in a highly contaminated area near a Superfund site (New Bedford Harbor, MA, USA), while the other population was located at a relatively clean site (West Island, Fairhaven, MA, USA). Mussel tissue total PCB concentrations (quantified as the sum of 18 congeners) from the contaminated site were two orders of magnitude greater than those at the clean site. Total lipid and triacylglycerol (TG) also were higher at the contaminated site. No significant relationship (p > 0.05) was observed between total PCBs and total lipid at either location; however, the correlations at both sites increased when total PCBs were compared with total TG or, to a lesser extent, total nonpolar lipid. Principal component analysis and linear discriminatory analysis indicated that the two mussel populations could be distinguished by the proportions of their lipid classes, particularly the concentration of nonpolar lipids, which consisted mainly of TG. These results suggest that the standard method of normalizing organism PCB concentrations to total lipid may not be appropriate as a routine practice, especially when the organism has a relatively low total lipid content (<6% dry wt in this study).

Animals↗
PubMed2003

The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules.

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.

Fluorescent Dyes↗
PubMed1990

Lipid peroxidation-induced changes in physical properties of annular lipids in rat brain synaptosomal membranes.

The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.

Animals↗
PubMed1995

Effect of tamoxifen on erythrocyte membrane lipids, lipid peroxides, and antioxidative enzymes in breast cancer women.

Fasting blood samples were taken from 64 tamoxifen-treated postmenopausal women with early stage breast cancer. The levels of erythrocyte lipid peroxidation and the status of erythrocyte detoxifying enzymes were analyzed in untreated and treated patients for 3 months and 6 months with tamoxifen. Erythrocyte membrane lipid peroxidation and membrane cholesterol, phospholipid were also determined in all the patients. The 3 months and 6 months tamoxifen-treated patients showed significantly decreased levels of erythrocyte, erythrocyte membrane lipid peroxide with concomitantly increased levels of detoxifying enzymes when compared with baseline values of untreated women. Erythrocyte membrane cholesterol and phospholipid levels were markedly decreased in tamoxifen-treated patients than in untreated women. An interesting finding of this study indicates that the lipid peroxide, as well as, the lipid lowering efficacy of tamoxifen, was increased in patients with greater levels of baseline lipid and lipid peroxides in their erythrocyte membrane. These results indicate that tamoxifen is a potent suppressor of lipid peroxide formation through the favorable effects on membrane lipids and protective enzyme system.

Aged↗
PubMed1993

Pyruvate is a lipid precursor for rat lymphocytes in culture: evidence for a lipid exporting capacity.

Since acetyl-CoA produced through pyruvate dehydrogenase reaction is poorly oxidized by the Krebs cycle in rat lymphocytes, the fate of acetyl units was investigated in these cells. The results presented here show that 24-h cultured lymphocytes actively synthesize lipids from [3-14C]pyruvate. Furthermore, a considerable amount of these lipids have shown to be exported into the culture medium. Experiments with [1-14C] acetate as a lipid precursor showed a close similarity with the rates of incorporation of [3-14C] pyruvate into the same lipid fractions. Treatment of lymphocytes with the mitogen concanavalin A (Con A) markedly enhanced [1-14C] acetate incorporation into a variety of lipids, but the lectin did not affect [3-14C] pyruvate incorporation. The results suggest that lymphocytes convert pyruvate into lipids via the acetyl-CoA pathway and that Con A interferes in lymphocyte lipogenesis but does not seem to affect the pyruvate dehydrogenase reaction. The ability to incorporate pyruvate into certain lipids may have an important role for the rapidly dividing capacity of lymphocytes since the human cancer strain HeLa 155 (a quickly proliferating cell line) also exhibits this feature by converting much more [3-14C] pyruvate into lipids than do lymphocytes. In addition, comparative experiments with lymphocytes, peritoneal macrophages and HeLa cells indicate that pyruvate may provide precursors for cells with active lipid producing and exporting capacities.

Acetates↗
PubMed1980

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B.

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.

Acholeplasma laidlawii↗
PubMed1982

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes.

Molecules analogous to biological and synthetic lipids have been prepared conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10(3)-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.

Alkynes↗
PubMed1995

Lipids and oxidised lipids in human atherosclerotic lesions at different stages of development.

Lipids and oxidised lipids were analysed by GC and GC-MS in human necropsy samples of normal artery and individual atherosclerotic lesions, from aorta and common carotid artery, including fatty streaks, intermediate lesions and advanced lesions. Age-related increases were seen for linoleate, oleate and cholesterol in normal artery, but not in lesions. Each category of lesion was much richer than normal artery in all the lipids measured and in oxidised lipids (oxysterols and hydroxyoctadecadienoic acids), although a degree of overlap existed between the compositions of the various categories of lesion. 26-Hydroxycholesterol and 7 beta-hydroxycholesterol levels were extremely low or undetectable in normal artery, but significantly higher in each of the categories of lesions. The generally wide variation in lipid composition of individual lesions within each category, and the fact that a few individual lesions showed no detectable 26-hydroxycholesterol or 7 beta-hydroxycholesterol, suggested that the lipid oxidation in lesions and therefore perhaps the progression of lesions may be intermittent. Fatty streaks showed the highest concentration of 7 beta-hydroxycholesterol relative to cholesterol, and the lowest ratio of linoleate to oleate, suggesting that this type of lesion experiences the greatest concentration of free radical activity. Levels of the enzymatic product 26-hydroxycholesterol were approximately proportional to cholesterol in all the categories of lesions. 26-Hydroxycholesterol was significantly more abundant in advanced lesions than in intermediate lesions or fatty streaks. 26-Hydroxycholesterol levels were higher in macrophage-rich intermediate and advanced lesions than in their fibrous counterparts. This distinction between macrophage-rich and fibrous lesions was also true for most of the other lipid components, consistent with the involvement of macrophages in lipid accumulation, lipid oxidation and lesion development.

Aging↗
PubMed1995

Diquat-dependent protein carbonyl formation. Identification of lipid-dependent and lipid-independent pathways.

In a previous report on diquat-dependent oxidative damage in rat hepatic microsomes, protein oxidation, as measured by protein carbonyl (PC) formation, was observed in addition to lipid peroxidation (LP). Both phenomena were antioxidant sensitive. Inhibition of PC formation was somewhat surprising given the proposed mechanism of metal-catalyzed protein oxidation. Studies reported here examined diquat-dependent PC formation in greater detail. In rat hepatic microsomes, diquat-dependent thiobarbituric acid-reactive substances (TBARS) and PC formation were time and concentration dependent. In this system, LP was inhibited completely by U-74006F or U-78517G, whereas PC formation was inhibited only partially by these antioxidants. In an essentially lipid-free system consisting of purified rat hepatic cytochrome P450 reductase, BSA and an NADPH-generating system, PC formation was also observed, but was not antioxidant-sensitive. Under these conditions, minimal diquat-dependent TBARS formation was observed. The observation of relative antioxidant insensitivity is consistent with H2O2 (generated during the diquat redox cycle) catalyzing protein oxidation via a site-specific, metal-catalyzed mechanism. Thus, different pathways would appear to be involved in diquat-dependent PC formation in lipid-containing and lipid-free systems. Carbon tetrachloride induces LP following reductive activation to the trichloromethyl free radical, a pathway not directly involving H2O2 generation. In the microsomal system, CCl4 induced TBARS and PC formation, both of which were completely inhibitable by antioxidants. Taken together, these data suggest that diquat induces PC formation by lipid-dependent (antioxidant-sensitive) and lipid-independent (antioxidant-insensitive) pathways. In microsomes, both pathways contribute to diquat-dependent PC formation. Data for the lipid-independent pathway are consistent with the mechanism of metal-catalyzed protein oxidation proposed by Stadtman and colleagues (reviewed in Free Radic Biol Med 9: 315-325, 1990), while the lipid-dependent pathway is likely secondary to LP itself--via a Michael-type addition reaction between hydroxyalkenals and protein sulfhydryl groups, amino groups or other protein nucleophiles. The latter pathway is also responsible for carbon tetrachloride-dependent PC formation. Additional studies are in progress to further characterize the lipid-independent mechanism.

Animals↗