Increased Low-density Lipoprotein Oxidation Research Articles

Relationships of apolipoprotein E genotypes with a cluster of seven in persons with type 2 diabetes.

The objective of the study was to determine if there would be statistically significant differences or trends among apolipoprotein E genotypes (2/2, 2/3, 2/4, 3/3, 3/4, and 4/4) for each member of the cluster of seven associated with type 2 diabetes (T2D). The cluster of seven includes abdominal obesity, hypertension, platelet hyperaggregability, hyperglycemia, dyslipidemia (decreased plasma levels of high-density lipoprotein cholesterol (HDL-C) and increased plasma levels of triglycerides)), increased low-density lipoprotein (LDL) oxidation, and increased inflammation. Forty-six patients with well-controlled T2D participated in the study. Abdominal obesity (assessed by waist circumference), hypertension (measured by manual sphygmomanometry), platelet hyperaggregability (measured by bleeding time), hyperglycemia (by enzymatic kit and spectrophotometry), decreased plasma levels of HDL-C and increased plasma levels of triglycerides (by enzymatic kit and spectrophotometry), increased LDL oxidation (measured by LDL conjugated dienes using spectrophotometry) and increased inflammation measured by C-reactive protein (CRP) (by EIA kit) were determined. All genotypes, except 2/2 were found in the population studied. Abdominal obesity did not vary significantly across the five genotypes. However, glucose levels trended progressively higher going from 2/3 to 2/4 to 3/4 to 4/4. Systolic blood pressure was higher in 3/4 compared to 2/4 and trended higher in 3/4 compared to 3/3. Diastolic blood pressure trended higher in 3/3 vs 2/4 and significantly higher in 3/4 compared to 2/4. Triglycerides trended higher in 3/4 vs 3/3 while HDL-C came close to trending downward in 4/4 compared to 2/4. Bleeding time was unaffected by genotype. Plasma LDL conjugated dienes trended higher in 3/4 vs 2/4 and were significantly higher in 3/4 vs 3/3. CRP trended higher in 4/4 vs 2/3. We can conclude that those with at least one 4 allele in the presence of another allele being 2, 3 or 4 is potentially (in the case of trends) deleterious or is deleterious in terms of hyperglycemia, hypertension (systolic and diastolic blood pressure), dyslipidemia, LDL conjugated dienes and CRP levels.

184. High density lipoproteins lose their antioxidant protective effect during pre-eclampsia

Introduction Pre-eclampsia (PE) is a hypertensive disorder of pregnancy associated with abnormal placentation leading to poor placental perfusion, oxidative stress, and inflammation, which then leads to the dysfunction of the maternal endothelium. High density lipoproteins (HDL) exert protective effects on the vascular endothelium especially an antioxidant effect. However, HDL can become dysfunctional and lose these protective properties. A dyslipidemia characterized by low plasma levels of HDL, elevated triglycerides, and high levels of low density lipoprotein (LDL)-cholesterol associated with an increased LDL oxidation has been described in PE. Objective/hypothesis We hypothesized that HDL become dysfunctional in PE especially with regard to their antioxidant properties and fail to protect the maternal endothelium. Our study is therefore investigating these antioxidant properties. Methods We conducted a case-control study in 10 pregnant women with early severe PE paired for age and gestational age to 10 control healthy pregnant women. We isolated HDL from patients and control women by ultracentrifugation, and then incubated cultured human endothelial cells with the purified HDL, LDL and CuSO4 for 6 h. Then, we measured TBARs (ThioBarbituric Acid Reactive Substances) produced in culture supernatants to evaluate the HDL antioxidant protective effect. Results The TBARs levels in culture supernatants were significantly raised when the endothelial cells were incubated with HDL purified from PE women rather than with HDL purified from control women (respectively 3.24 ± 1.25 μM versus 2.50 ± 0.95 μM, mean ± SD, p = 0.02). These data show that HDL from PE patients have less antioxidant effect than HDL from pregnant controls. Discussion In our study, we found that HDL isolated from PE patients lose their antioxidant properties, which could explain the increase of oxidized LDL previously described in the PE. These dysfunctional HDL could fail to protect the vascular endothelium; such mechanism could be involved in the physiopathology of the PE endothelium dysfunction.

Hypoxia compounds exercise-induced free radical formation in humans; partitioning contributions from the cerebral and femoral circulation

This study examined to what extent the human cerebral and femoral circulation contribute to free radical formation during basal and exercise-induced responses to hypoxia. Healthy participants (5♂, 5♀) were randomly assigned single-blinded to normoxic (21% O2) and hypoxic (10% O2) trials with measurements taken at rest and 30 min after cycling at 70% of maximal power output in hypoxia and equivalent relative and absolute intensities in normoxia. Blood was sampled from the brachial artery (a), internal jugular and femoral veins (v) for non-enzymatic antioxidants (HPLC), ascorbate radical (A•-, electron paramagnetic resonance spectroscopy), lipid hydroperoxides (LOOH) and low density lipoprotein (LDL) oxidation (spectrophotometry). Cerebral and femoral venous blood flow was evaluated by transcranial Doppler ultrasound (CBF) and constant infusion thermodilution (FBF). With 3 participants lost to follow up (final n = 4♂, 3♀), hypoxia increased CBF and FBF (P = 0.041 vs. normoxia) with further elevations in FBF during exercise (P = 0.002 vs. rest). Cerebral and femoral ascorbate and α-tocopherol consumption (v < a) was accompanied by A•-/LOOH formation (v > a) and increased LDL oxidation during hypoxia (P < 0.043–0.049 vs. normoxia) implying free radical-mediated lipid peroxidation subsequent to inadequate antioxidant defense. This was pronounced during exercise across the femoral circulation in proportion to the increase in local O2 uptake (r = −0.397 to −0.459, P = 0.037–0.045) but unrelated to any reduction in PO2. These findings highlight considerable regional heterogeneity in the oxidative stress response to hypoxia that may be more attributable to local differences in O2 flux than to O2 tension.

LDL fatty acids composition as a risk biomarker of cardiovascular disease

ObjectiveFatty acid composition of Low-density lipoprotein (LDL) particle is an effective factor in LDL oxidation and atherosclerotic plaques formation. This study evaluates the relationship between LDL fatty acid composition and coronary artery disease (CAD). Methods42 men with coronary artery disease (CAD-group) and 40 men without coronary artery disease (non-CAD-group) were selected. LDL fatty acid composition of blood samples was measured by gas chromatography. ResultsOx-LDL was significantly high in CAD-group. Poly unsaturated fatty acids (PUFA) and PUFA/MUFA (Mono unsaturated fatty acids), linoleic acid and arachidonic acid were significantly higher in CAD-group than in non-CAD-group. In CAD-group, a reverse correlation was observed between oleic acid concentrations and ox-LDL levels and a direct correlation was seen between arachidonic acid concentrations and ox-LDL levels. ConclusionComposition of LDL is related to atherosclerosis and CAD. High levels of arachidonic and linoleic acids could increase LDL oxidation and atherosclerotic plaques formation. In addition, LDL arachidonic acid levels could be a better predictor of CAD.

Apolipoprotein B, oxidized low-density lipoprotein, and LDL particle size in predicting the incidence of metabolic syndrome: the Cardiovascular Risk in Young Finns study

To test whether serum apolipoprotein B (apoB) and low-density lipoprotein (LDL) particle characteristics (oxidation and mean particle size) predict the incidence of metabolic syndrome (MetS). The 6-year follow-up study included 1429 adults (baseline mean age 31.5). Lipids, apoB, and apoA1 were measured at baseline in 2001. LDL oxidation was measured with monoclonal antibody-based enzyme-linked immunosorbent assay (oxLDL-prot) and with a method measuring oxidized lipids in LDL (oxLDL-lipids). Mean LDL particle size was calculated from proton nuclear magnetic resonance spectroscopy data. Increased concentrations of both oxLDL-measures were associated with increased apoB levels but not with LDL particle size. The odds ratios (95% confidence intervals) for MetS incidence during a 6-year follow up by quartiles of apoB were 2.0 (1.0-3.8) for the second quartile, 3.1 (1.7-5.7) for the third quartile, and 4.2 (2.3-7.6) for the fourth quartile. This association remained after adjusting for age, sex, body mass index, homeostasis model assessment for insulin resistance, C-reactive protein, smoking, LDL cholesterol, oxidized LDL measures (p ≤ 0.01) in addition to risk factors comprising the MetS (p = 0.03). OxLDL-prot and oxLDL-lipids levels were not independently associated with incident MetS after adjusting for apoB. Mean LDL particle size was not associated with the incidence of MetS. ApoB is associated with increased risk of MetS incidence. We found no clear evidence to suggest that increased LDL oxidation or small mean LDL particle size would facilitate the development of MetS.

비정제유 첨가가 참치유 보강 에멀젼의 산화방지활성에 미치는 영향

This study was conducted to evaluate the effects of adding unrefined oil on the antioxidant activity of a tuna oil-enriched emulsion by determining DPPH radical scavenging activity, reducing power, and inhibition of low-density lipoprotein (LDL) oxidation in vitro. The emulsion consisted of tocopherol-stripped canola (18.3 g) and tuna (9.1 g) oil, one of the unrefined oils (4.6 g), such as extra virgin olive, mustard, perilla, or sesame oil, 0.5% acetic acid (64 g), and egg yolk powder (4 g). The control emulsion contained only canola (21.4 g) and tuna oil (10.6 g), as oil sources,with the same composition of the remaining ingredients. The emulsion with added unrefined oil, particularly mustard oil, showed higher radical scavenging activity and reducing power than those of the control emulsion. The radical scavenging activity and reducing power of the emulsion with added unrefined oil were higher at 1,000 ppm than at 500 ppm thus, the effect was concentration-dependent. Adding sesame or perilla oil to the tuna oil-enriched emulsion resulted in higher inhibition of LDL oxidationwhereas adding olive oil increased LDL oxidation. The results clearly showed that adding roasted mustard, sesame, or perilla oil improved the antioxidant activity of a tuna oil-enriched emulsion by increasing free radical scavenging activity, reducing power, and inhibiting LDL oxidation. The results also suggest that adding unrefined oils produces a healthier fish oil-enriched salad dressing recipe.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: Implication in hemodialysis mediated atherogenic LDL modification

Objective: Al3+ stimulates Fe2+ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al3+ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al3+ on LDL oxidation.Materials and methods: Using different LDL modifying systems (Fe2+, Cu2+, free radical generating compounds, human endothelial cells, hemin/H2O2 and HOCl), the influence of Al3+ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.Results: Al3+ could stimulate the oxidizability of LDL by Fe2+, but not in the other systems tested. Al3+ and Fe2+ were found to bind to LDL and Al3+could compete with Fe2+ binding to the lipoprotein. Fluorescence polarization data indicated that Al3+ does not affect the phospholipid compartment of LDL.Conclusions:The results indicate that increased LDL oxidation by Fe2+ in presence of Al3+ might be due to blockage of Fe2+ binding sites on LDL making more free Fe2+ available for lipid oxidation.

Oxidative stress in atherosclerosis‐prone mouse is due to low antioxidant capacity of mitochondria

Atherosclerotic disease remains a leading cause of death in westernized societies, and reactive oxygen species (ROS) play a pivotal role in atherogenesis. Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Here, we show that mitochondria from atherosclerosis-prone, hypercholesterolemic low-density lipoprotein (LDL) receptor knockout mice have oxidative phosphorylation efficiency similar to that from control mice but have a higher net production of ROS and susceptibility to develop membrane permeability transition. Increased ROS production was observed in mitochondria isolated from several tissues, including liver, heart, and brain, and in intact mononuclear cells from spleen. In contrast to control mitochondria, knockout mouse mitochondria did not sustain a reduced state of matrix NADPH, the main source of antioxidant defense against ROS. Experiments in vivo showed faster liver secretion rates and de novo synthesis of triglycerides and cholesterol in knockout than in control mice, suggesting that increased lipogenesis depleted the reducing equivalents from NADPH and generated a state of oxidative stress in hypercholesterolemic knockout mice. These data provide the first evidence of how oxidative stress is generated in LDL receptor defective cells and could explain the increased LDL oxidation, cell death, and atherogenesis seen in familiar hypercholesterolemia.

Contribution of superoxide to reduced antioxidant activity of glycoxidative serum albumin

Hyperglycemia increases oxidative stress in various tissues and leads to diabetic cardiovascular complication. Dyslipidemia, such as an increase in oxidized low-density lipoprotein (LDL), is well recognized in diabetic patients with hyperglycemia. However, the mechanism by which hyperglycemia causes the increased LDL oxidation remains unclear. Albumin is the most abundant protein in the circulation, and can function as an antioxidant. Therefore, we examined whether glycoxidative modification inhibits the antioxidant activity of albumin to LDL oxidation and clarified the mechanism by which this modification may suppress its antioxidant activity. Human serum albumin (HSA) was incubated in phosphate-buffered saline with and without glucose at 37 degrees C for up to 8 weeks under aerobic conditions (referred to as glycoxidation (goHSA) and oxidation (oHSA), respectively). Metal chelator-treated, nonoxidative HSA (chHSA) and freshly prepared HSA (fHSA) were used as controls. N(epsilon)-(carboxymethyl)lysine (CML), a glycoxidative product, was determined by enzyme-linked immunosorbent assay. Oxidation was estimated by measuring the thiols of the HSA molecule. Copper-mediated oxidation of LDL was conducted in the presence or absence of modified HSAs at 37 degrees C for 6 days. Malondialdehyde and negative charge of LDL were measured. To clarify the mechanism of reduced antioxidant activity of HSA, we examined firstly the binding activity of modified HSAs to copper, and secondly the effects of free radical scavengers on the formation of malondialdehyde. CML was formed in goHSA in a time- and concentration-dependent manner. Both goHSA and oHSA significantly decreased the contents of free thiol groups compared to ch- and fHSAs. The antioxidant activity of goHSA to LDL oxidation was the lowest among various modified HSAs. The oHSA showed a moderate decrease in antioxidant activity. The binding activity of go- and oHSAs to copper was lower than that of ch- and fHSAs. The formation of MDA from LDL oxidation in the presence of goHSA was completely inhibited by Tiron (1,2-dihydroxy-3,5-benzenedisulfonic acid) and superoxide dismutase. In contrast, catalase and mannitol had no effect. Our results indicate that in vitro glycoxidation of HSA induced a marked loss of antioxidant activity of this molecule to copper-mediated oxidation of LDL, which may be caused by the generation of superoxide.

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.

Red wine protects diabetic patients from meal-induced oxidative stress and thrombosis activation: a pleasant approach to the prevention of cardiovascular disease in diabetes.

Oxidative stress and thrombosis have been reported to be increased in diabetic patients and involved in the pathogenesis of cardiovascular complications. It has been demonstrated in diabetic patients that consumption of a meal is accompanied by the generation of an oxidative stress and of a hypercoagulable state. It is well recognized that red wine shows antithrombotic activity and that its ingestion increases plasma antioxidant capacity in man. In this study the possibility that red wine consumption may reduce the oxidative stress and thrombosis produced postprandially in diabetic patients has been evaluated. Twenty type 2 diabetic patients were studied during fasting consumption of 300 mL of red wine, or during a meal accompanied, or not, by red wine ingestion. Plasma glucose, insulin, triglycerides, total plasma radical-trapping capacity, activated factor VII and prothrombin fragments 1 + 2 were measured in basal state and at 60, 120 and 180 min after the start of each experiment. Low-density lipoprotein (LDL) oxidation was also evaluated at baseline and after 120 min Plasma glucose, insulin, triglycerides and LDL oxidation significantly increased, while the total plasma radical-trapping parameter significantly decreased during the meal test. Consumption of red wine in the fasting state significantly increased total plasma radical-trapping parameter activity, while wine ingestion with a meal counterbalanced the decrease of total plasma radical-trapping parameter and the increase of LDL oxidation. Meal consumption induced an increase in plasma prothrombin fragments 1 + 2 and activated factor VII in diabetic patients. Wine ingestion with the meal significantly reduced the production of both prothrombin fragments 1 + 2 and activated factor VII. Fasting consumption of red wine alone did not show effects on coagulation or LDL oxidation. This finding confirms that in the absorptive phase free radicals are produced in diabetic patients, which reduce serum antioxidant defences, increase LDL oxidation and activate the coagulation system. Red wine consumption during a meal significantly preserves plasma antioxidant defences and reduces both LDL oxidation and thrombotic activation. The consumption of a moderate amount of red wine during meals may have a beneficial effect in the prevention of cardiovascular disease in diabetic patients.

Antibodies against oxidised low-density lipoprotein in hypopituitary patients with growth hormone deficiency.

Growth hormone-deficient hypopituitary patients on conventional replacement therapy have increased mortality and morbidity from atherosclerotic cardiovascular disease. Oxidation of low-density lipoprotein (LDL) is a key event in the development and progression of atherosclerosis. Antibodies against oxidatively modified LDL may reflect in vivo oxidation processes. The aim of this study is to determine the effect of growth hormone deficiency on oxidised-LDL antibody titres in panhypopituitary patients taking conventional replacement therapy. Twenty-one GH deficient, adult panhypopituitary patients and 17 age, sex and body mass index-matched healthy controls were studied. After an overnight fast, anthropometric parameters were measured and body composition was determined by a bioelectrical impedance analyser. Venous blood samples were obtained for the measurements of biochemical parameters. Antibodies to oxidised-LDL were analysed by an ELISA system in the patients' and controls' serum. No significant difference was observed between the oxidised-LDL antibody titres in hypopituitary patients and controls (395.4+/- 183.2 mU/ml and 393.2 +/- 186.2 mU/ml respectively, p = NS). A significant positive correlation was observed between oxidised-LDL antibody titres and total cholesterol concentrations in the patients (r = 0.449, p < 0.05). No significant correlation was observed between oxidised-LDL antibody titres and anthropometric/biochemical variables in the controls. It is concluded that relatively increased LDL oxidation may not contribute to the progression of atherosclerosis in hypopituitary patients.

Increased fruit and vegetable consumption within the EU: potential health benefits

In this EU (DG XII) funded project, a Core human study was conducted in Ireland, Northern Ireland, Spain, France and The Netherlands. Oxidative and antioxidant status, vegetable and fruit consumption, and carotenoid intake of volunteers from different countries was compared. Response to increased carotenoid intake was determined using a range of measurements. Attention was paid to whether the antioxidant capability of β-carotene, lutein and lycopene demonstrated in vitro, was apparent in relation to increased oxidation resistance of low-density-lipoprotein (LDL). Other (Complementary) studies were undertaken and included determination of: protective effects of carotenoid-rich foods against LDL and DNA oxidative damage; carotenoid absorbability; barriers to increased vegetable consumption; and carotenoid content of fruits and vegetables frequently consumed in Europe. Our results demonstrated that carotenoid supplementation did not increase LDL oxidation resistance. However, increased consumption of carotenoid-rich fruits and vegetables did increase LDL oxidation resistance, and higher plasma concentration of total and specific carotenoids (pre-supplementation) was associated with lower DNA damage and higher repair activity.

Mechanism of Dacron-activated monocytic cell oxidation of low density lipoprotein

Purpose: Oxidized lipids are believed to contribute to atherogenesis and may play a role in the development of anastomotic intimal hyperplasia in prosthetic vascular grafts. This study examines the hypothesis that clinically relevant graft material activates monocytes to oxidize low density lipoprotein (LDL). Methods: LDL and Dacron or expanded polytetrafluoroethylene (ePTFE) graft material were incubated in the presence of U937 cells, a monocytic cell line. LDL oxidation was measured by conjugated dienes, lipid peroxides, thiobarbituric acid–reacting substances, and electrophoretic mobility. Cell production of superoxide was measured by ferricytochrome c reduction. Metal ion requirement was assessed with the metal chelators, ethylenediaminetetra-acidic acid, deferoxamine, and bathocuproinedisulfonic acid. To determine whether human monocytes were capable of being activated by Dacron graft material to oxidize LDL, freshly isolated peripheral blood monocytes were also studied. Results: Incubation of LDL with U937 cells and Dacron increased LDL oxidation by 5- to 20-fold. LDL incubated with ePTFE or U937 cells alone resulted in minimal oxidation. Dacron graft increased U937 cell production of superoxide by 4-fold, whereas ePTFE had no effect. Superoxide dismutase inhibited Dacron-activated U937 cell oxidation of LDL by greater than 50%, which indicates a role for superoxide. Ethylenediaminetetra-acidic acid, deferoxamine, and bathocuproinedisulfonic acid each inhibited Dacron-activated U937 cell oxidation of LDL. Human peripheral blood monocytes were activated by Dacron graft material to oxidize LDL; superoxide dismutase inhibited Dacron-activated human monocytic oxidation of LDL, which suggests a role for superoxide. Conclusion: These results suggest that Dacron graft material activates monocytes to oxidize LDL by a mechanism that involves superoxide and requires iron and copper ions. Our work suggests a mechanism by which lipids that have been deposited within implanted vascular grafts may become oxidized. Oxidized lipids may contribute to the cellular dysfunction that results in anastomotic intimal hyperplasia and graft failure. (J Vasc Surg 2000;31:171-80.)

Increased oxidizability of low-density lipoproteins in hypothyroidism.

Hypothyroidism leads to an increase of plasma low-density lipoprotein (LDL) cholesterol levels. Oxidation of LDL particles changes their intrinsic properties, thereby enhancing the development of atherosclerosis. T4 has three specific binding sites on apolipoprotein B; furthermore it inhibits LDL oxidation in vitro. We therefore hypothesized that T4 deficiency not only results in elevated LDL-cholesterol levels but also in increased LDL oxidation. Ten patients with overt hypothyroidism were studied when untreated (TSH 76 +/- 13 mU/L, T4 40 +/- 6 nmol/L) and again when they were euthyroid for at least 3 months during T4 treatment (TSH 2.7 +/- 0.5 mU/L, T4 115 +/- 11 nmol/L). Plasma lipids and lipoproteins and the oxidizability and chemical composition of LDL were determined. The transition from the hypothyroid to the euthyroid state was associated with a decrease (mean +/- SE) of plasma total cholesterol (5.8 +/- 0.3 vs. 4.8 +/- 0.2 mmol/L, P < 0.005), LDL cholesterol (3.8 +/- 0.3 vs. 2.9 +/- 0.2 nmol/L, P < 0.005) and apolipoprotein B (1.2 +/- 0.1 vs. 0.9 +/- 0.1 g/L, P < 0.005); plasma high-density lipoprotein cholesterol, apolipoprotein A-1, and triglycerides did not change. The actual content of dienes in LDL particles was increased in hypothyroidism, with a decrease after T4 suppletion [median (range) = 257 (165-346) vs. 188 (138-254) nmol/mg LDL protein, P < 0.005; reference range 140-180]. The lag time, an estimate of the resistance of LDL against oxidation in vitro, was shortened when hypothyroid but normalized after T4 treatment [29 (19-90) vs. 77 (42-96) min, P < 0.005; reference range 67-87]. The density, the relative fatty acid content, and the vitamin E content of LDL particles did not change. In conclusion, the hypothyroid state is not only associated with a quantitative increase of LDL particles, but it also changes their quality by increasing LDL oxidizability.

The effects of ascorbate and dehydroascorbate on the oxidation of low-density lipoprotein.

Ascorbate at concentrations of 60-100 microM inhibits the modification of freshly prepared low-density lipoprotein (LDL) by macrophages. With 'moderately oxidized' LDL (produced by prolonged storage in a refrigerator), however, ascorbate does not inhibit LDL modification by macrophages and actually modifies the LDL itself in the absence of macrophages [Stait and Leake (1994) FEBS Lett. 341, 263-267]. We have now shown that dehydroascorbate can modify both 'fresh' LDL and moderately oxidized LDL in a dose-dependent manner to increase its uptake by macrophages. The modification of moderately oxidized LDL by ascorbate and dehydroascorbate or of 'fresh' LDL by dehydroascorbate is dependent on the presence of iron or copper. In 'fresh' LDL, ascorbate inhibited conjugated-diene formation by copper. In moderately oxidized LDL, the number of conjugated dienes present was decreased rapidly in the presence of copper and ascorbate. Dehydroascorbate decreased the lag phase and increased the rate of copper-induced conjugated-diene formation in 'fresh' LDL (although in some experiments it inhibited the formation of conjugated dienes). The ascorbate-modified moderately oxidized LDL was taken up by macrophages by their scavenger receptors, as the uptake was inhibited by polyinosinic acid or fucoidan. Ascorbate and dehydroascorbate therefore have the potential to increase LDL oxidation under certain conditions, but whether or not they do so in vivo is unknown.

Ceruloplasmin Enhances Smooth Muscle Cell- and Endothelial Cell-mediated Low Density Lipoprotein Oxidation by a Superoxide-dependent Mechanism

Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

Macrophage-mediated oxidation of extracellular low density lipoprotein requires an initial binding of the lipoprotein to its receptor.

Cells of the arterial wall including macrophages were shown to oxidize low density lipoprotein (LDL) in vitro. Upon incubation of LDL with J-774.A1 macrophage-like cell line for 18 h in the presence of 1 microM CuSO4, extensive macrophage-mediated oxidation of the LDL fatty acids and cholesterol moieties was demonstrated. Similar results were found with mouse peritoneal macrophages or human monocyte-derived macrophages. Several lines of evidence suggest that LDL binding to the LDL receptor on macrophages is required for the cell-mediated oxidation of LDL. 1) Incubation of the cells in the presence of monoclonal antibody to the LDL receptor (IgG-C7), substantially inhibited lipoprotein oxidation. 2) Pretreatment of LDL with monoclonal antibodies to the LDL receptor binding domains on the LDL apoB-100 (mAbs B1B6 and B1B3) inhibited cell-mediated oxidation of LDL by 52-95%. 3) Down-regulation of the macrophage LDL receptors (by preloading the cells with cholesterol) reduced LDL oxidation by 42%. 4) Up-regulation of the LDL receptor (by macrophage incubation in serum-free medium) was associated with 80% elevation in LDL oxidation. 5) Macrophage activation with lipopolysaccharide up-regulated the LDL receptors and was associated with up twofold increase LDL oxidation. 6) Human monocyte-derived macrophages from a patient with homozygous familial hypercholesterolemia, which lack the LDL receptor, failed to oxidize the LDL. 7) On using acetylated LDL or methylated LDL, which do not bind to the LDL receptor, macrophage-mediated oxidation of the lipoprotein did not occur. The binding of LDL to the macrophage LDL receptor under oxidative stress induced the oxidation of extracellular unbound LDL as demonstrated by cell-mediated lipid peroxidation of mAb B1B6-treated LDL by cells that were preincubated with native LDL. Furthermore, macrophage conditioned medium (MCM) that was obtained after 5 h of cells preincubation with native LDL under oxidative stress (1 microM CuSO4), followed by lipoprotein removal and a further 18 h of cell incubation (but not MCM that was similarly obtained without cell preincubation with LDL), was found to contain oxidized linoleic and arachidonic acids and was able to induce LDL lipids peroxidation. In conclusion, macrophage-mediated oxidation of LDL requires an initial binding of the lipoprotein to the LDL receptor on the cell surface under oxidative stress. This interaction leads to the formation and release of cellular oxidized polyunsaturated fatty acids that can oxidize the LDL molecule extracellularly.

Dietary Olive Oil Reduces Low-Density Lipoprotein Uptake by Macrophages and Decreases the Susceptibility of the Lipoprotein to Undergo Lipid Peroxidation

Low-density lipoprotein (LDL) oxidation and macrophage cholesterol accumulation are both involved in atherogenesis. Recently it was shown that feeding rabbits or humans with an oleic-acid-rich diet reduced the susceptibility of their LDL to in vitro oxidation. Since olive oil is highly enriched with oleic acid, we tested the effect of olive oil supplementation (50 g/day) to the diet of 10 healthy male subjects, during a 2-week period, on macrophage uptake of their LDL and on the propensity of their LDL to oxidation (with copper ions). Olive oil supplementation to the diet resulted in LDL enrichment with oleic acid (C18:1) and sitosterol. No effect on plasma cholesterol levels was found, but the LDL cholesteryl ester content was reduced (16%) whereas its unesterified cholesterol was increased (41%). Even after 1 week of the olive oil diet, the LDL susceptibility to in vitro oxidation was significantly reduced (p < 0.01). Macrophage uptake of LDL was studied by analysis of cellular cholesterol content and by analysis of the macrophage cholesterol esterification rates. LDL obtained after 1 and 2 weeks of the olive oil diet demonstrated reduced cellular uptake in comparison with LDL obtained before the supplementation of olive oil, by 50 and 61%, respectively. The LDL resistance to oxidation was shown by a reduction in its peroxide, malondialdehyde and conjugated diene content by 73, 28 and 32%, respectively. LDL incubation with oleic acid for the period of its oxidation with copper ions demonstrated a dose-dependent inhibition of lipoprotein oxidation by up to 72% as opposed to linoleic and arachidonic acids (50 microM) which increased LDL oxidation by 22 and 72%, respectively. Sitosterol, in a similar incubation system, inhibited LDL oxidation by up to 26%. We conclude that olive oil supplementation to the diet modifies LDL lipid composition and enriches the lipoprotein with oleic acid and sitosterol. The antiatherogenic properties of this modified lipoprotein may be related to its resistance to in vitro peroxidation and its reduced uptake by macrophages.

Low density lipoprotein oxidation by stimulated neutrophils and ferritin.

Low density lipoprotein (LDL) oxidation mediated by phorbol myristate acetate (PMA)- and formylmethionylleucylphenylalanine (FMLP) -stimulated human neutrophils was enhanced by 70% in the presence of ferritin. Iron released from ferritin by the superoxide anion generated in the respiratory burst of stimulated neutrophils is shown to be involved in lipoprotein oxidation. Ascorbate (100 microM), superoxide dismutase (10 micrograms/ml) and uric acid (430 microM) showed inhibitory effects of 30% [corrected], 70% and 50% on LDL oxidation, respectively. Ceruloplasmin (2.7 microM) potentiated LDL oxidation by stimulated neutrophils and ferritin, both alone and in the presence of methionine. Methionine (1 mM) and catalase (30 micrograms/ml) increased LDL oxidation by stimulated neutrophils and ferritin. These data suggest that LDL oxidation by stimulated neutrophils and ferritin may be relevant in inflammation when both neutrophils and ferritin are increased.