Acid Low Density Lipoprotein Research Articles

MECHANISMS OF THE INFLUENCE OF SODIUM-GLUCOSE COTRANSPORTER-2 INHIBITORS ON LDL RECEPTOR FUNCTION AND CARDIOVASCULAR RISK IN TYPE 2 DM (literature review)

In the modern world, the prevalence of dysmetabolic conditions, which are accompanied by corruption of lipid metabolism and the distribution of adipose tissue in the body, is increasing, and their consequences include cardiovascular diseases, type 2 diabetes mellitus (T2DM) etc. These pathologies are characterized by dyslipidemia, which reflects an imbalance in the processes of assimilation, transportation, absorption and use by fatty acids’ cells as energy and plastic substrates. A decrease in the relative content of unsaturated fatty acids in low-density lipoproteins (LDL) causes dysfunction of cell membranes, and an increase in serum concentration of LDL means corruption of their absorption by cells, which contributes to the development of atherosclerosis. Absorption of LDL by cells occurs through the interaction of apolipoprotein apoE/B-100 with the membrane receptor of LDL. The cell regulates the supply of lipids and cholesterol by synthesizing these receptors. The expression of LDL receptors is regulated at the level of transcription; particularly, it is stimulated by insulin and suppressed by excess cholesterol, the latter leading to abnormal accumulation of lipids in cells and tissues and the development of pathology in various organs. According to clinical and experimental studies and meta-analyses, drugs from the group of inhibitors of sodium-dependent glucose cotransporter-2 (SGLT2) have a pronounced protective cardiorenal effect in patients with T2DM and in cases of kidney and heart dysfunction. These beneficial effects are associated with improving insulin sensitivity, increasing the level of antiatherogenic HDL cholesterol, reducing the accumulation of lipids in visceral fat, stimulating lipolysis, and switching of oxidation towards the preferential use of lipid substrates. The paradoxical increase in LDL cholesterol is mainly due to less atherogenic large floating particles, and the negative effect is apparently counterweight by the wide range of beneficial pleiotropic effects of gliflozins.

Biological function of endoecology (cleanliness of intercellular environment) realize two biolocical reactions: excretion reaction and inflammation reaction - utilization in vivo, in situ catabolites of the large molecular mass.

For the biological functions of trophology (nutrition) and homeostasis, we believe that the third place is occupied by the function of endoecology: in a single pool of intercellular environment in vivo it must be "pure". The biological function of endoecology, realizes two biological reactions: the reaction of excretion and the reaction of inflammation. The biological reaction of excretion is the filtration of phlogogens of a small mol. mass through the basement membrane of the glomerulus of the nephron and excretion with the final urine. Flogogens are a great mol. mass (more than 70 kDa), catabolites of biological reactions of apoptosis, autophagy must be disposed of in situ, on site. At the stages of phylogenesis, the cells formed structures for the collection and utilization of phlogogens of large mol. mass and exogenous pathogens in tissues. The sequence of biochemical reactions, biological methods of in vivo collection and in situ utilization of phlogogens of large mol. mass and realizes the biological function of endoecology, the biological reaction of inflammation. It functions constantly, every minute, like the biological reaction of excretion. Inflammation is the reaction of maintaining the "cleanliness" of the "piece" of the ocean that every individual has privatized, before beginning to live on land. The inflammation reaction includes congenital, acquired immunity, humoral mediator systems, cytokines and eicosanoids. In the formation of a closed circulatory system, the pool for collecting and utilizing in situ biological catabolites from the bloodstream is located in the late phylogeny of the intima of the arteries of the elastic (muscular-elastic) type. Abuse of herbivorous species Homo sapiens carnivorous (meat) food; blockade of cellular absorption by polyene fatty acids in low density lipoproteins and the absence of acidic hydrolase expression of polyene fatty acids - cholesterol alcohol ethers in monocytes and macrophages - is the cause of the formation of atheroma of intima of the arteries of the elastic type. Atherosclerosis - the pathology of the biological function of trophology, atheromatosis - pathology of the biological function of endoecology. The appointment of patients statins for primary prevention of atherosclerosis, atheromatosis and coronary heart disease, we finally "passed".

The "Unexpected Role" of Vitamin E in Free Radical-Induced Hemolysis of Human Erythrocytes: α-Tocopherol-Mediated Peroxidation

Vitamin E involves a group of tocopherols and tocotrienols, in which alpha-tocopherol with the highest biological activity plays a more efficient role in advanced lesions with aged oxidized tissues. However, the results of the present study reveal that a large amount of endogenous alpha-tocopherol in human low-density lipoprotein (LDL) in the absence of any other antioxidants may initiate additional free radical propagation under low concentration of free radical initiator (i.e., 2,2'-azobis(2-amidinopropane hydrochloride) [AAPH], a water-soluble free radical source) to peroxide polyunsaturated fatty acids in LDL in the manner of alpha-tocopherol-mediated peroxidation (TMP). Whether the addition of high concentration of exogenous alpha-tocopherol to human erythrocytes under low concentration of AAPH can also drive TMP is the concern in this research work. Moreover, the hemolysis extent of human erythrocytes peroxidized by AAPH is followed easily by the determination of the hemoglobin outside the erythrocytes. A series of observations on various concentrations of AAPH-induced hemolysis in the presence of various concentrations of exogenous alpha-tocopherol demonstrates that the high concentration of exogenous alpha-tocopherol, coupled with low concentration of AAPH, can initiate TMP in the free-radical-induced peroxidation of human erythrocytes system as well. This result provides direct evidence to support TMP theory and expands its application into in vitro erythrocyte system.

Effect of genistein supplementation on tissue genistein and lipid peroxidation of serum, liver and low-density lipoprotein in hamsters

The aim of this study was to investigate the effects of genistein supplementation in a vitamin E-deficient diet on the genistein concentrations and the lipid oxidation of serum, liver and low-density lipoprotein (LDL) of hamsters. Thirty-six male hamsters were randomly divided into three groups and fed a vitamin E-deficient semisynthetic diet (AIN-76) containing different levels of genistein, i.e., G0 (control group, genistein-free diet), G50 (50 mg genistein/kg diet) and G200 (200 mg genistein/kg diet) for 5 weeks. The concentrations of genistein in serum and liver significantly increased with the increase of genistein supplementation. The vitamin E contents in LDL were higher in hamsters fed G50 or G200 diets than in hamsters fed genistein-free diet. Genistein supplementation to hamsters significantly reduced the propagation rate during conjugated diene formation of LDL oxidation, and the lag time of LDL oxidation in hamsters fed G200 diets was significantly lower than that of G0 diets. In addition, genistein supplementation significantly raised serum total antioxidant capacity and decreased the thiobarbituric acid-reactive substances (TBARS) of LDL and liver in hamsters. However, no significant differences in TBARS were found in serum, irrespective of genistein addition. On the other hand, the relative contents of polyunsaturated fatty acids in LDL were decreased after genistein supplementation. There was a negative correlation between lag time and P/S ratio, and a positive correlation between lag time and vitamin E contents. These data demonstrate that genistein supplementation markedly increased its concentrations in body tissues and reduced oxidative stress of lipid oxidation of serum, liver and LDL.

Differential recruitment of the coactivator proteins CREB-binding protein and steroid receptor coactivator-1 to peroxisome proliferator-activated receptor gamma/9-cis-retinoic acid receptor heterodimers by ligands present in oxidized low-density lipoprotein.

Peroxisome proliferator-activated receptor gamma (PPARgamma) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPARgamma activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxy and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), as well as 5-hydroxy-, 12-hydroxy- and 15-hydroxyeicosatetraenoic acid (5-, 12- and 15-HETE respectively). PPARgamma was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)- and 12(S)-HETE as well as 15-deoxy-Delta(12,14)-prostaglandin J(2) also activated PPARgamma but were less potent. Interestingly, the effect of the lipoxygenase products 13(S)-HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-Delta(12,14)-prostaglandin J(2) was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPARgamma/9-cis retinoic acid receptor heterodimer complexes.

Molecular mechanism of low density lipoprotein oxidation by 12-lipoxygenase-overexpressing macrophages

Oxidative modification of low-density lipoprotein (LDL) leads to foam cell formation in atherosclerosis. The involvement of 12- and 15-lipoxygenases in LDL oxidation has been implicated by enzymological and knockout mouse studies. However, the molecular basis of oxidized LDL formation in vivo has not yet been elucidated. We constructed expression vectors pEF-BOS for porcine leukocyte and human platelet 12-lipoxygenases exhibiting distinct activity toward esterified fatty acids in LDL. Macrophage-like J774A.1 cells were transfected with these expression vectors, transcription of which was driven by elongation factor-1α promoter. The established clones showed 12-lipoxygenase activity, while the original J774A.1 cells did not show any detectable enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with LDL, their oxidation was markedly increased over mock-transfected cells as determined by the thiobarbituric acid reactive substance (TBARS). Furthermore, the cells expressing porcine 12-lipoxygenase generated more oxygenated products than the platelet enzyme. Most of the oxygenated derivatives were of an esterified form, and the major product was identified to be 13 S-hydroxy-octadeca-9 Z,11 E-dienoic acid. In addition, the oxidized LDL generated by the 12-lipoxygenase-expressing cells was recognized by a scavenger receptor as assessed by macrophage degradation assay. These results suggest that the intracellular 12-lipoxygenase of macrophages play an essential role in the oxygenation of esterified fatty acids in LDL.

News on the cradle of atherosclerosis

The processes initiating atherosclerosis that eventually lead to all the well-known complications of the disease have long been debated. Now, Borén and coworkers provide convincing data on the first steps of atherogenesis [ 1. Skålén K. et al. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis. Nature. 2002; 417: 750-754 Crossref PubMed Scopus (714) Google Scholar ]. Their findings indicate that the central event in the development of atherosclerosis is the retention of low-density lipoprotein (LDL) by proteoglycans present in the subendothelium. In particular, stretches of basic amino acids in LDL bind to the negatively charged sulfate groups of the proteoglycans.

Effect of ingestion of virgin olive oil on human low-density lipoprotein composition.

To measure the incorporation of oleic acid and antioxidants (phenols and vitamin E) to low density lipoprotein (LDL) after acute and short-term ingestion of virgin olive oil. To study whether this incorporation contributes to an increase in LDL resistance to oxidation. Department of Food and Nutrition, University of Barcelona, Spain and Department of Lipids and Cardiovascular Epidemiology, IMIM, Barcelona, Spain. Sixteen healthy volunteers aged 25-65 y. To observe the change in the fatty acid profile, vitamin E, phenolic compounds and LDL oxidation-related variables after the postprandial phase and after daily ingestion of olive oil for one week. Few changes were observed in the postprandial phase. However, after a week of olive oil consumption there was an increase in oleic acid (P=0.015), vitamin E (P=0.047), phenolics (P=0.021) and lag time (P=0.000), and a decrease in the maximum amount of dienes (P=0.045) and oxidation rate (P=0.05). After ingestion of virgin olive oil, an increase in antioxidants and oleic acid in LDL was observed as well as an improvement of LDL resistance to oxidation. Our results support the idea that daily ingestion of virgin olive oil could protect LDL from oxidation. This study was supported by a research grant from Spain (ALI 97-1607-C02-02).

Fatty acid composition of low-density lipoprotein in renal transplant recipients treated with cyclosporine.

THERE IS A HIGH prevalence of cardiovascular disease among long-term renal transplant recipients. Its etiology is multifactorial, though hyperlipidemia has been identified as one of the most important risk factors. The atherogenic capacity of the lipoproteins is conditioned by the fatty acid composition of their lipid component. The balance in the concentration of polyunsaturated and monounsaturated fatty acids in low-density lipoproteins (LDL) influences the susceptibility of the LDL particles to oxidation. The fatty acid content of the various lipid fractions has not been extensively studied in renal transplant recipients. The aim of the study was to analyze the fatty acid distribution in the LDL fraction of renal transplantation patients treated with cyclosporine-neoral.

Long chain polyunsaturated fatty acids may account for higher low-density lipoprotein oxidation susceptibility in Lithuanian compared to Swedish men

Objectives: Mortality in coronary heart disease among middle-aged men is four times higher in Lithuania than in Sweden. Traditional risk factors cannot account for this difference. We earlier reported that low-density lipoprotein (LDL) in Lithuanian men showed a lower resistance to oxidation, measured as LDL lag time during copper oxidation, than that in Swedish men. Serum concentrations of several fat-soluble antioxidant vitamins were lower among Lithuanian men. The aim of this study was to investigate whether differences in LDL fatty acid composition could account for the difference in LDL oxidation susceptibility between men in the two countries. Methods: This cross-sectional study included randomly selected healthy 50-year-old men from Vilnius, Lithuania (n = 50) and Linköping, Sweden (n = 50). Main outcome measures were fatty acids in LDL, phospholipid (PL) and cholesterol ester (CE) fractions of LDL and LDL oxidation susceptibility. Results: The mean proportions of PL 20:5n3 (eicosapentaenoic acid, EPA) were higher in Vilnius men (2.09 - 1.05 vs. 1.53 - 0.58%, p = 0.004). LDL lag time was shorter in Vilnius men, mean - SD (75.4 - 13.6 vs. 89.5 - 13.1 mins, p < 0.0001) than in Linköping men. Mean serum n -tocopherol was lower in Vilnius men (0.07 - 0.05 vs. 0.12 - 0.04 w g/mmol, p < 0.0001) but f -tocopherol did not differ. In a multiple regression analysis controlled for city, high PL-EPA, low alpha-tocopherol, and high plasma triglycerides significantly contributed to a short LDL lag time, r 2 = 0.53. Conclusions: Fat quality, i.e. poly unsaturated fatty acids, especially LDL-EPA, plasma triglycerides and antioxidative vitamins may partly account for the increased LDL oxidation susceptibility found in Vilnius men compared with Linköping men.

Oxidation of Linoleic Acid in Low-Density Lipoprotein: An Important Event in Atherogenesis.

Contrary to earlier views the main oxidation products of low-density lipoprotein (LDL) are derived from linoleic acid and not arachidonic acid, as determined by GC/MS investigations of the in vitro oxidation of LDL samples. A similar product spectrum, in which epoxyhydroxyoctadecenoic acids such as 1 and 2 have been identified for the first time, is obtained from minimally oxidized (that is, aged) LDL. Since this is still recognized by the LDL receptor, it is concluded that toxic oxidation products are introduced in endothelial cells in vivo and cause damage there.

Dietary habits affect the susceptibility of low-density lipoprotein to oxidation.

To study, if there are differences in the fatty acid composition of low-density lipoprotein (LDL) in people eating three different long-standing habitual diets: vegetarian, high fish intake, or high saturated fat (milk fat) diet as a control group, and to study if these differences influence the oxidation susceptibility of LDL. Cross-sectional study using blood samples and a validated dietary frequency questionnaire with illustrations. Helsinki University Central Hospital, Finland. The effect of three different types of long-standing diets of different fatty acid content (a strict vegetarian diet, n=11; a high fish intake diet, n=9; and a high saturated fat (milk fat) diet, controls, n=7) on the serum and LDL fatty acid content, and on the susceptibility of LDL to oxidation in vitro, was studied in healthy normocholesterolemic volunteers who had been on these diets for years. Oxidation of LDL was carried out by using CuSO4 as a pro-oxidant. There were no statistically significant differences in the serum lipids or lipoproteins, though the vegetarian group exhibited lowest mean values of total, high-density lipoprotein (HDL) and LDL cholesterol levels. Both the serum and LDL eicosapentaenoic, docosapentaenoic and docosahexaenoic acid proportions were highest in the fish and lowest in the vegetarian groups. Linoleic acid was highest among the vegetarians. In the fish group, the vitamin A concentration in serum was higher than in vegetarians and controls and beta-carotene lower than in controls, but in alpha-tocopherol, or lycopene concentrations there were no statistically significant differences. The lag phase of LDL oxidation was shortest (116 min) in the fish group and longest (165 min) in the vegetarian group, and the control group was between them (129 min). The mean oxidation percentage after 2.5 h of copper-induced oxidation was highest (44%) in the fish group and lowest (22%) in the vegetarian group and intermediate (31%) in the control group. Long-term dietary habits predict the fatty acid composition of serum and LDL, and influence the susceptibility of LDL to oxidation. In the fish group with the highest content of omega-3 fatty acids in LDL, the oxidation susceptibility of LDL was highest. In the vegetarian group with less omega-3 fatty acids in LDL, the LDL was more resistant to oxidation. Helsinki University Central Hospital.

Essential involvement of 12-lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages.

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with 400 microg.mL-1 LDL in Dulbecco's modified Eagle's medium at 37 degrees C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.

Strong increase of 9-hydroxy-10,12-octadecadienoic acid in low density lipoprotein after a hemorrhagic shock.

9-hydroxy-10,12-octadecadienoic acid (9-HODE) is generated by lipid peroxidation (LPO) processes in comparison to other marker compounds in at least 10 fold amount. A 10-25 fold increase of this new marker compound in relation to age matched healthy individuals was observed in the low density lipoprotein (LDL) fraction of ten patients suffering from a hemorrhagic shock. The 9-HODE values dropped to normal levels after recovery. Similarly the 9-HODE content in LDL of patients which had to undergo orthopedic surgery--replacement of their arthritic hip joints by endoprosthesis--were investigated. The rather high HODE values dropped also after recovery reflecting obviously the disappearance of inflammatory processes associated with arthritis.

Human Low Density Lipoprotein as a Target of Hypochlorite Generated by Myeloperoxidase

The aim of this study was to further clarify which part of human low density lipoprotein (LDL) is attacked by the MPO/H 2O 2/Cl −-system and which reactive oxygen species is responsible for the attack. Therefore the influence of this system on the modification of the lipid and protein moiety of LDL was studied in vitro. Using the monochlorodimedone assay it was found that HOCl is produced in micromolar quantities in the absence of LDL and is rapidly consumed by LDL in a concentration dependent manner. The consumption of HOCl was reflected in the formation of HOCl-specific epitopes on apo B-100 as determined by an antibody raised against HOCl-modified LDL. The absorbency at 234 nm was applied to measure continuously the extent of modification of LDL. The general kinetic pattern of the absorbency measurement consisted of a lag phase where no LDL modification was observed, followed by a rapid increase of absorbency and a plateau phase. Finally the absorbency decreased due to LDL precipitation. Time dependent absorption spectra indicated that this kinetic pattern is mainly caused by light scattering due to particle aggregation rather than by a specific absorption at 234 nm due to conjugated diene formation. In agreement with this finding a low rate of thiobarbituric acid reactive substances (TBArS) formation was observed after a lag phase. The aggregation of LDL occurs most likely by modification of apo B-100, which was determined fluorimetrically in terms of LDL-tryptophan destruction in presence of the MPO/H 2O 2/Cl −-system. The kinetic course of tryptophan fluorescence generally consisted of a rapid decrease leveling off into a low plateau phase. Gas chromatographic determinations of linoleic acid in LDL in presence of the MPO system showed that this polyunsaturated fatty acid (PUFA) is easily attacked by HOCl. Consistent with this finding NMR spectra of HOCl modified LDL indicated a complete disappearance of bis-allylic methylene groups. Since lipid peroxidation products only partially account for this loss of PUFAs, other reactions of HOCl with unsaturated lipids—probably chlorohydrin formation—must be involved. Summarizing, although the rate of lipid peroxidation is low, both the lipid and the protein moiety of LDL are readily modified by the MPO system. It appears that the immediate consequence of apo B-100 modification is its aggregation. It is concluded that MPO, which has been detected in atherosclerotic lesions, is able to contribute to the modification of LDL into a form recognizable for uncontrolled uptake by macrophages.

Oxidation of Free Fatty Acids in Low Density Lipoprotein by 15-Lipoxygenase Stimulates Nonenzymic, α-Tocopherol-mediated Peroxidation of Cholesteryl Esters

15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15-lipoxygenase (rhLO) can be ascribed largely to alpha-tocopherol (alpha-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A2 (PLA2) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE-O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu2+ ions. Analyses of the regio- and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. alpha-Tocopheroxyl radical (alpha-TO.) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA2. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing alpha-TO. and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.

The dynamic reduction of Cu(II) to Cu(I) and not Cu(I) availability is a sufficient trigger for low density lipoprotein oxidation

Copper (II) is one of the most widely employed experimental models to induce in vitro low density lipoprotein (LDL) oxidation. It is generally assumed that Cu(I), generated from active reduction of Cu(II), is the real trigger for the peroxidation of polyunsaturated fatty acids in LDL. We have employed isolated human LDL challenged with Cu(II) and the Cu(I) chelator bathocuproine disulfonic acid (BC) to test the validity of this hypothesis. At lower Cu(II)/LDL molar ratios, BC completely inhibited copper-mediated LDL oxidation evaluated either as thiobarbituric acid reactive substances (TBARS) production or changes in apo B100 electrophoretic mobility. On the contrary, at higher Cu(II)/LDL molar ratios, BC did not prevent LDL oxidation but rather markedly stimulated both the generation of TBARS and the increase of apo B100 electronegativity. These oxidative modifications were completely prevented by the Cu(II) chelator EDTA. Furthermore, the BC-Cu(I) complex alone was neither redox active nor active inducer of TBARS generation. These findings indicate that, under these experimental conditions, [1]Cu(I) is not necessary to promote LDL oxidation, [2]the availability of Cu(II) is a prerequisite and [3]some of the reaction(s) involved in Cu(II) reduction may play an essential role in initiating LDL oxidation.

Effect of low density lipoprotein fatty acid composition on copper-induced peroxidation: 1H-nuclear magnetic resonance analysis

Oxidation of low density lipoprotein (LDL) has been implicated in atherogenesis. An increased content of oleic acid in LDL and the substitution of monounsaturated for polyunsaturated fatty acids in the diet reduce LDL oxidation. With 1H-NMR analysis, all LDL modifications, including the production of copper-induced aldehyde products, can be evaluated simultaneously. The aim of the present study was to investigate whether the fatty acid composition of LDL affected the NMR evaluation of aldehyde compounds. The LDL of the samples utilized were rich in oleic fatty acid (26.9%). After 48 h of exposure to copper sulfate, the mean production of malonyldialdehyde (MDA) by LDL was 31.2 nmol/mg of protein. Moreover, in the present study NMR did not reveal large amounts of peroxidative compounds since the nanomolar amounts of MDA produced after exposure to copper sulfate could not be detected. This study also demonstrated that the fatty acid composition (i.e. the oleic:linoleic acid ratio) must be taken into account in the evaluation of LDL peroxidation by NMR. In particular, a high concentration of oleic acid may limit the formation, of large amounts of peroxidative compounds generated after exposure to the oxidant copper sulfate.

Increase in hydroxy fatty acids in human low density lipoproteins with age

The content of hydroxy fatty acids in low density lipoprotein (LDL) of healthy volunteers aged between 22 and 87 years without any signs of atherosclerosis or other age-dependent diseases was investigated. The level of hydroxy fatty acids obtained from LDL increases during life time: clinically healthy persons between 56 to 66 years showed a 3- to 4-fold increase compared to young volunteers. This level increased in samples of probands aged 68 to 74 years compared to samples of young people for a factor of 10–20 and in samples of probands aged 78 to 87 years for a factor of 30–40. These hydroxy acids — generated mainly from hydroperoxy acids of linoleic acid and only partly from arachidonic acid — are obviously parameters of the LDL oxidation stage. About 90%, of the total amount of hydroxy fatty acids were free fatty acids. The distribution patterns of the monohydroxy derivatives of linoleic and arachidonic acid indicate that they originate mainly from autocatalytic processes. The individual level of hydroxy acids is probably an indicator of the biological age.

Predominance of esterified hydroperoxy-linoleic acid in human monocyte-oxidized LDL.

Low density lipoprotein that was oxidized by activated human monocytes was analyzed to determine the identity of oxidized fatty acids present and the conditions required for their formation. The oxidized lipids were also analyzed under conditions allowing preservation of their oxidation state. Using reversed-phase high performance liquid chromatography (HPLC) analysis of native and saponified lipid extracts of oxidized low density lipoprotein (LDL), we found that the major fatty acid oxidation product was esterified hydroperoxyoctadecadienoic acid (HPODE), the oxidized product of the most abundant polyunsaturated fatty acid in human LDL, linoleic acid. Although some esterified hydroxyoctadecadienoic acid (HODE) was also detected, the reduction of HPODE to HODE did not appear to be monocyte-dependent. Essentially all of the HPODE was found to be esterified with the majority being esterified to cholesterol followed by phospholipids and generally following the abundance of esterified linoleic acid within the lipid classes. The percent of cholesteryl linoleate converted to cholesteryl HPODE and cholesteryl HODE at the end of the 24-h incubation was determined to be approximately 13.5%. The formation of oxidized esterified linoleic acid in the LDL was shown to require immunological activation of the human monocytes, a previously observed requirement for general LDL oxidation in this culture system. The oxidized esterified linoleic acid was present in the supernatant with the LDL and was not cell-associated. HPODE formation on LDL was prevented by including superoxide dismutase (SOD) or eicosatetraynoic acid (ETYA) during the 24-h coincubation of activated monocytes with LDL whereas indomethacin was without effect. The analysis of the lipid oxidation products in oxidized LDL can provide insight into the mechanisms involved in oxidation of LDL by activated human monocytes.