LDL Susceptibility to Oxidative Stress in Smokers.

In 30 cigarette smokers and in 30 sex and age matched nonsmoker controls, susceptibility of LDL to oxidative stress following incubation with copper sulphate (1mM) was measured in terms of thiobarbituric acid reactive substance. Susceptibility of LDL to oxidation in smokers was found to be 0.514131±0.468231picomol MDA/μg protein/hr. LDL susceptibility to oxidation in non-smokers was found to be 0.711726 ± 0.447324picomol MDA/μg protein/hr. These two values were not statistically different (p=0.1) by unpaired T test. This study therefore suggests that cigarette smoke does not increase LDL susceptibility to oxidation.

Effects of olive leaves extract on LDL oxidation induced-CUSO[4] in vitro

Oxidation of low-density lipoprotein [LDL] has been strongly implicated in the pathogenesis of atherosclerosis. The use of some natural antioxidant and herbal medicine may lead to the inhibition of production of oxidized LDL and may decrease both the development and the progression of atherosclerosis. The aim of this study was to investigate the effects of Olive leaves ethanol extract [OLE] on LDL oxidation induced-CuSO[4] quantitatively in vitro. Low-density lipoprotein was incubated with CuSO[4] and the formation of conjugated dienes and thiobarbituric acid reactive substances [TBARS]. Inhibition of this Cu-induced oxidation was studied in the presence of vitamin E and various concentration of OLE. It was demonstrated that OLE reduced the formation of conjugated dienes and TBARS of LDL against oxidation in vitro [P<0.05]. The inhibitory effects of the OLE on LDL oxidation were dose-dependent at concentrations ranging from [2microg/ml] to [200microg/ml]. Moreover, we compared effects of OLE on LDL oxidation with vitamin E as positive control. This study showed that OLE is a source of potent antioxidants and prevented the oxidation of LDL in vitro and it may be suitable for use in food and pharmaceutical applications

Effects of kale (Brassica oleracea L. var. acephala DC) leaves extracts on the susceptibility of very low and low density lipoproteins to oxidation.

Of Brassicaceous plants, kale (Brassica oleraceae L. var. acephala DC) contains polyphenols, flavonoids, isoflavones and glucosinolates and so has antioxidant and anticarcinogenic properties. Antioxidants inhibit negative effects of free radicals and may, therefore, protect tissues against oxidative damage. Oxidation of lipoproteins is a key event in the development of atherosclerosis. In the current study, the levels of total phenolic and flavonoid contents and total antioxidant capacity of methanolic and aqueous extracts of kale leaves were determined. In addition, the susceptibility of isolated lipoproteins — very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to the Cu2+-induced oxidation with various concentrations of metanolic and aqueous extracts was evaluated as t-lag values. Although aqueous extract had higher total antioxidant capacity, methanolic extract had higher total phenolic and flavonoid content (P<0.05). On the other hand, both extracts inhibited lipid peroxidation in both isolated VLDL and LDL. Inhibitory effect of extracts or increasing t-lag values, mainly in methanolic extract was found to be related to increasing the concentration of extracts. It was concluded that because of high antioxidant capacity and phenolic content, kale showed a protective effect on the oxidation of lipoproteins. Therefore, it may be speculated that kale consumption may play an important protective role in the cardiovascular and other related diseases resulting from imbalance of oxidant and antioxidant status.

Efeito de hipolipemiantes sobre a expressão de genes envolvidos no transporte reverso do colesterol / Statin effects on expression of genes involved in reverse cholesterol transport

A eficácia das estatinas em reduzir o risco de eventos coronarianos não é completamente explicada por seus efeitos em diminuir colesterol de lipoproteína de baixa densidade (LDL-C). Um dos seus efeitos adicionais pode ser decorrente da modificação na concentração de lipoproteína de alta densidade (HDL), reconhecida como ateroprotetora, principalmente por seu papel no transporte reverso do colesterol (TRC). Os transportadores de membrana do tipo ATP-binding cassette, ABCA1 e ABCG1, e o scavenger receptor BI (SRBI) são proteínas importantes envolvidas no TRC e seus genes são regulados por vários fatores de transcrição, entre eles os liver-x-receptors (LXRs). Com a finalidade de avaliarmos os efeitos dos hipolipemiantes sobre expressão dos transportadores ABC e do receptor SRBI, a expressão de RNAm do ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) e NR1H2 (LRXβ) foi avaliada por PCR em tempo real em células das linhagens HepG2 (origem hepática) e Caco-2 (origem intestinal) tratadas com atorvastatina ou sinvastatina (10 µM) e/ou ezetimiba (até 5 µM) por até 24 horas. Além disso, a expressão desses genes também foi avaliada em células mononucleares do sangue periférico (CMSP) de 50 pacientes normolipidêmicos (NL) e 71 hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4semanas, n=48) ou sinvastatina e/ou ezetimiba (10mg/dia/4 ou 8 semanas, n=23). A possível associação entre os polimorfismos ABCA1 C-14T e R219K e a expressão de RNAm em CMSP também foi avaliada por PCR-RFLP. O SCARB1 foi o gene mais expresso nas células HepG2 e Caco-2, seguido por NR1H2, NR1H3, ABCG1 e ABCA1 em HepG2 ou por ABCA1 e ABCG1 em Caco-2. O tratamento com estatinas (1 ou 10 µM) ou ezetimiba (5 µM), por 12 ou 24 horas, aumentou a expressão de RNAm do ABCG1, mas não de ABCA1 e SCARB1, em células HepG2. Ainda nesta linhagem, o aumento na transcrição dos genes NR1H2 e NR1H3 foi observado somente com a maior concentração de atorvastatina (10 µM) e, ao contrário, o tratamento com ezetimiba...

The efficacy of statins in reducing the risk of coronary events is not completely explained by their effects in decreasing cholesterol low-density lipoprotein (LDL-C). One of their additional effects may result from the change in concentration of high-density lipoprotein (HDL), recognized as atheroprotective, mainly for the role in reverse cholesterol transport (RCT). The membrane transporters, as ATP-binding cassette, ABCA1 and ABCG1, and scavenger receptor BI (SRBI) are important proteins involved in the RCT and their genes are regulated by various transcription factors, including the liver-X-receptors (LXRs) . In order to evaluate the effects of lipid lowering on expression of ABC transporters and SRBI receptor, the mRNA expression of ABCA1, ABCG1, SCARB1, NR1H3 (LXRα) and NR1H2 (LRXβ) was assessed by real time PCR in HepG2 (hepatic origin) and Caco-2 (intestinal origin) cells treated with atorvastatin or simvastatin (10 µM) and/or ezetimibe (up to 5 µM) for 24 hours. Furthermore, the expression of these genes was evaluated in peripheral blood mononuclear cells (PBMC) of 50 normolipidemic (NL) and 71 hypercholesterolemic (HC) patients treated with atorvastatin (10mg/d/4 weeks, n = 48) or simvastatin and/or ezetimibe (10mg/d/4 or 8 weeks, n = 23). The possible association between ABCA1 C-14T and R219K polymorphisms and mRNA expression in PBMC was also evaluated by PCR-RFLP. SCARB1 was the most expressed in HepG2 and Caco-2 cells, followed by NR1H2, NR1H3, ABCG1 and ABCA1 in HepG2 or by ABCG1 and ABCA1 in Caco-2. The treatment with statins (1 or 10 µM) or ezetimibe (5 µM) for 12 or 24 hours, increased mRNA expression of ABCG1 but not ABCA1 and SCARB1 in HepG2 cells. Moreover, in HepG2 cells, atorvastatin also upregulated NR1H2 and NR1H3 only at 10.0 µM, meanwhile ezetimibe downregulated NR1H2 but did not change NR1H3 expression. In Caco-2 cells, atorvastatin or simvastatin treatment for 12 or 24 hours reduced the amount of ABCA1 transcript and did not ...

Synthetic nanoemulsion resembling a protein-free model of 7-ketocholesterol containing low density lipoprotein: In vitro and in vivo studies

7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorpórate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.

Modification of platelet shape change parameter by modified phospholipids in oxidized LDL.

BACKGROUND: It is believed that the oxidatively modified lipoproteins play a critical role in activating platelets and is a contributing factor in the etiology of a number of cardiovascular-related diseases. OBJECTIVE: Identify the active component(s) of oxidized LDL that initiated shape change in plasma-free human platelets prepared by a gel filtration method Material and Method: Shape change parameter of platelets was monitored following exposure platelets to LDL, copper sulfate-oxidized LDL, and different types of lipids extracted of the corresponding LDL. RESULTS: Oxidized LDL, but not native LDL, increased the shape-change parameter in a concentration-dependent manner Specifically, phosphatidyl serine from oxidized LDL was responsible for this effect. CONCLUSION: Oxidized phospholipids generated during the oxidative modification of LDL are likely to be the active components responsible for changes in platelet function.

Lp(a) and LDL2: differences in fatty acid composition and collagen lattice contraction activity

Lp (a) and LDL2 were used for detailed fatty acid analyses and tested in an in vitro model promotion of fibroblast-mediated collagen lattice contraction to determine possible compositional and functional differences between these two apoB-containing lipoprotein species. Autologous Lp (a) was more saturated with respect to fatty acid composition than LDL2 in triglyceride and cholesterol ester lipid classes and had differences in the fatty acid content of phospholipids. Functionally, LDL2 promoted rapid fibroblast-mediated contraction while Lp (a) was significantly less active in promoting rapid contraction on a protein per weight basis. These studies suggest a synthetic route for Lp(a) diverging from the majority of other apoB-containing lipoproteins and significant activity of LDL2 in a collagen lattice contraction system.

Oxidative stress and metabolic control in non-insulin dependent diabetes mellitus.

The aim of this study was to evaluate conjugated dienes in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and its metabolic control. To achieve good metabolic control in addition to dietary management oral hypoglycemic agents such as glibenclamide, gliclazide and metformin were given to patients. Human plasma low-density lipoproteins (LDL) were delipidised and triglycerides (LDL-TG) and cholesterol esters (LDL-CE) were separated. Conjugated dienes in LDL-TG and LDL-CE of subjects with NIDDM (n = 90) and normal glucose tolerance (NGT) (n = 30) were measured using second derivative of uv absorption spectrum. Hypoglycemic agents lowered substantially concentration of cis, trans (c, t) and trans, trans (t, t) conjugated dienes in LDL-CE and LDL-TG. The duration of NIDDM has shown significant correlation (p < 0.001) with conjugated dienes in LDL-TG. Concentration of c, t and t, t-conjugated dienes in LDL-CE and LDL-TG were found significantly higher in subjects with NIDDM than NGT (p < 0.001). In conclusion, NIDDM, status of metabolic control and duration of diabetes have strong positive relation with oxidative stress.

Alterations in dense LDL subfractions in normolipidemic IDDM patients

Low density lipoproteins (LDL) of human plasma, consist of a continuum of particles subclasses with distinct physicochemical, immunological and hydrodynamic characteristics. Such structural differences are intimately linked to atherogenesis. The current sludy was designated to investigate the LDL subclasses profile in 12 normolipidemic IDDM patients, and compare it with 11 healthy controls. Four plasma LDL subfractions were isolated by sequential ultracentrifugation in the density range (1.025- 1.063 g/ml) and were characterized by their content of free and esterified cholesterol, triglycerides, phospholipids, and proteins. The net electrical charge was evaluated. Plasma concentration of the two denser LDL subfractions were higher in IDDM patients vs control subjects, due to an increase in cholesterol (free and esterified and phospholipids, while more buoyant subfractions in the two groups were not diferent. In IDDM patients the LDL profile was skewed towards the dense subclasses LDL-III and LDL-IV, being significant this increase for LDL-IV: 22.3 ñ 5.2 vs 18.3 ñ 4.0 per cent, p<0.05, XñDS. In the healthy controls the LDL profile was skewed toward the lighter subclasses (LDL-I and LDL-II), being significant for LDL-II: 30.0 ñ 4.3 vs 23.3 ñ 4.2 per cent, p<0.005. Diabetic patients, even those who are normolipidemic, present increased risk of premature atherosclerosis. This suggest that normal values in lipid and lipoprotein profile can mask deeper alterations, such as changes in the composition and distribution of the denser subclasses, whose characteristics make them potentially more atherogenic. Despite the apparently normolipidemic status, dense LDL particles considered to be atherogenic, are increased in IDDM patients.

Lipoproteína de baja densidad rica en triglicéridos y actividad de lipasa hepática en pacientes diabéticos insulino-dependientes / Low density lipoprotein rich in triglycerides and hepatic lipose activity in insulin-dependent diabetic patients

Genetic hepatic lipase (HL) deficiency is associated with low density lipoprotein (LDL) rich in triglycerides (TG), whose affinity for B:E receptors is decreased. In rats, experimental hypoinsulinemia produces HL deficiency. However, the relation between human insulin-dependent Diabetes Mellitus (IDDM), HL activity and the characteristics of LDL have not been studied. The objective of our study is to evaluate the relation between HL activity and the chemical composition of LDL in treated IDDM patients. Subjects were 15 IDDM patients and 15 controls (C), matched for sex and body mass index (BMI). The IDDM patients were classified by the WHO criteria, were free of nephropathy and hypothyroidism, and received no medication except insulin. Controls were clinically healthy and normolipidemic with no family history of diabetes. The IDDM group was divided into two subgroups: subgroup IDDM-A (n = 9) with HL values > 4.3 and IDDM-B (n = 6) with HL < than 4.2 mu-moles glycerol/ml h. The HL in IDDM was lower than in C (p < 0.001). Table 1 shows clinical data. Blood samples were drawn after 12 h fasting. Percentage of HbAlc and plasma concentrations of glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol and TG were assayed. LDL was separated by sequential ultracentrifugation at densities of 1.019-1.063 g/ml and its chemical composition was analyzed. The most relevant results were: plasma TG concentration was higher in IDDM than in C (p < 0.05) (Table 2), although average values DMID not exceed the reference values of 200 mg/dl. The TG-LDL were higher in IDDM than in C: 24.8 +/- 2.7 vs 17.5 +/- 1.1 mg/dl plasma, media +/- SE, (p < 0.02). This difference reflected the values of IDDM-B, whose plasma concentrations of TG-LDL were higher than in C: 32.3 +/- 3.6 vs 17.5 +/- 1.1 mg/dl (p < 0.001), and also higher than in IDDMA (p < 0.02). (Table 3). The chemical composition of LDL in IDDM-B contained a higher percentage of TG than C: 8.5 +/- 0.7 vs 6.8 +/- 0.3 percent (p < 0.05), a lower percentage of cholesterol than IDDM-A: 39.0 +/- 1.7 vs 45.2 +/- 2.2 percent (p < 0.05) and also a larger percentage of proteins than IDDM-A: 28.9 +/- 1.9 vs 20.8 +/- 1.0 percent (p < 0.01). The correlations between TG/cholesterol and HL activity in IDDM were r = -0.53 (p < 0.05) and in IDDM-B, r = -0.81 (p = 0.05). The noteworthy result of this study is the modification of the LDL particle in IDDM, rich in TG in patients with low HL activity. Anomalies in the chemical composition of LDL like those described decrease the uptake of this particle by its physiological B:E receptors. It has recently been demonstrated that LDL is an indissoluble association of lipids and apoproteins, and that both act simultaneously to hold the apoB in a spatial position that expresses normal epitopes. It has been described that particles of LDL rich in TG and poor in cholesterol, shows low affinity for LDL receptors in human fibroblasts. Also in IDDM the interaction of LDL rich in TG with B:E receptors is decreased. This might be one more mechanism contributing to the accelerated atherosclerosis of these patients. Our results suggest that there may be a threshold of HL activity for the complete hydrolysis of the TG of LDL, for the normalization of the TG/cholesterol relation and for the conformation of typical LDL particles.

Biochemical modifications of low density lipoprotein particles in diabetic patients

Modified forms of low density lipoprotein [LDL] are associated with increased atherogenicity in diabetic patients. Therefore, biochemical characteristics of LDL particles from 17 type I and 26 type II diabetic patients in the form of glycosylation, sialic acid content, thiobarbituric acid reactive substances [TBARS] as well as lipid composition were studied. The study also included 10 healthy control subjects. The study revealed that LDL from diabetic patients contained decreased amount of sialic acid, increased amounts of fructosyl lysine and TBARS compared with controls. The degree of glycosylation and sialic acid content were correlated with status of diabetic control as determined by blood glucose level and total fructosamine. Furthermore, the lipid composition of LDL from diabetic patients was changed, where triglycerides were increased and cholesterol was decreased compared with healthy subjects. Altogether, diabetic patients' LDL were characterized by a lowered lipid/protein ratio. In conclusion, the cholesterol accumulating effect of diabetic patient's blood sera is mainly related to atherogenic low density lipoprotein fraction, which is modified in various ways, by increased non-enzymatic glycosylation, desialylation and alterations in lipid composition. This multiple-modified LDL may contribute to the premature atherosclerosis development in diabetes mellitus