Improved Measurement of Low-Density-Lipoprotein Susceptibility to Copper-Induced Oxidation: Application of a Short Procedure for Isolating Low-Density Lipoprotein

1992 ◽  
Vol 38 (10) ◽  
pp. 2066-2072 ◽  
Author(s):  
H A Kleinveld ◽  
H L Hak-Lemmers ◽  
A F Stalenhoef ◽  
P N Demacker
Keyword(s):  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Thiobarbituric Acid ◽  
Lag Time ◽  
Butylated Hydroxytoluene ◽  
Ldl Oxidation ◽  
Low Density ◽  
Short Run

Abstract Low-density-lipoprotein (LDL) oxidation may provide the crucial link between plasma LDL and atherosclerotic-lesion formation. Oxidation can be induced in vitro by incubating LDL with cells or metal ions and can be measured by continuously monitoring conjugated-diene absorbance at 234 nm. Measurement of LDL oxidizability was improved by performing the assay with 0.05 g of LDL-protein per liter of phosphate buffer containing 1 mumol of EDTA, by initiating oxidation by adding CuCl2 (5 mumol/L) at 30 degrees C, and by using a short-run ultracentrifugation method for isolating LDL, which reduced the time needed for obtaining purified LDL and thus reduced in vitro oxidation. LDL apolipoprotein analysis and oxidizability determination showed that this method is better than the longer sequential-isolation procedure. Adding butylated hydroxytoluene (BHT) to plasma as an antioxidant unpredictably increased the LDL oxidation lag time, making BHT unsuitable as an antioxidant. Adding EDTA appeared to be sufficient to prevent in vitro oxidation. Additionally, the diene production correlated highly with the concentration of thiobarbituric acid-reactive substances (r = 0.97). No relation between the vitamin E content of LDL and the oxidation lag time was found.

scholarly journals Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production

1994 ◽  
Vol 301 (2) ◽  
pp. 421-428 ◽  
Author(s):  
B Garner ◽  
R T Dean ◽  
W Jessup
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Direct Methods ◽  
Thiobarbituric Acid ◽  
Human Monocyte ◽  
Ldl Oxidation ◽  
Human Macrophage ◽  
Low Density ◽  
Oxidatively Modified

There is growing evidence that oxidatively modified low-density lipoprotein (LDL) accumulates in the atherosclerotic intima of arteries. Cells present in the intima (including the monocyte/macrophage) are capable of oxidizing LDL in vitro, but the mechanisms by which this occurs are unknown. Several reports have claimed a crucial role for superoxide as a cell-derived radical species capable of enhancing the rate of LDL oxidation. We have used a sensitive h.p.l.c. system with chemiluminescence detection to measure LDL cholesteryl ester hydroperoxides at early stages of LDL oxidation. During the initial stages of LDL oxidation, there is at least a 2 h delay before human monocyte-derived macrophages enhance this process. Stimulation of these cells to produce large fluxes of superoxide does not increase the rate of LDL oxidation or decrease the delay of its onset. Prior exposure of LDL to a high flux of superoxide does not increase its susceptibility to oxidation by human monocyte-derived macrophages. We also show that the thiobarbituric acid-reactive substances (TBARS) assay does not always correlate with more direct methods of assessing LDL oxidation and confirm recent reports that superoxide dismutase only partially inhibits cell-mediated LDL oxidation. We conclude that superoxide does not play a major role in human monocyte-derived macrophage-mediated LDL oxidation under the conditions that we describe.

scholarly journals Inhibition of in vitro macrophage-induced low density lipoprotein oxidation by thyroid compounds

2003 ◽  
Vol 177 (1) ◽  
pp. 137-146 ◽  
Author(s):  
L Oziol ◽  
P Faure ◽  
N Bertrand ◽  
P Chomard
Keyword(s):  
Cell Viability ◽  
Antioxidant Capacity ◽  
Density Lipoprotein ◽  
Thiobarbituric Acid ◽  
Human Monocyte ◽  
Ldl Oxidation ◽  
Low Density ◽  
Redox Systems

Oxidized low density lipoproteins (LDL) are highly suspected of initiating the atherosclerosis process. Thyroid hormones and structural analogues have been reported to protect LDL from lipid peroxidation induced by Cu2+ or the free radical generator 2,2'-azobis-'2-amidinopropane' dihydrochloride in vitro. We have examined the effects of thyroid compounds on macrophage-induced LDL oxidation. Human monocyte-derived macrophages (differentiated U937 cells) were incubated for 24 h with LDL and different concentrations (0-20 microM) of 3,5,3'-triiodo-l -thyronine (T3), 3,5,3',5'-tetraiodo-L-thyronine (T4), 3,3',5'-tri-iodo-l -thyronine (rT3), the T3 acetic derivative (3,5,3'-tri-iodothyroacetic acid; TA3) or L-thyronine (T0) (experiment 1). Cells were also preincubated for 24 h with 1 or 10 microM of the compounds, washed twice, then incubated again for 24 h with LDL (experiment 2). Oxidation was evaluated by measurement of thiobarbituric acid-reactive substances (TBARS) and cell viability by lactate deshydrogenase release. In experiment 1, T0 had no effect, whereas the other compounds decreased LDL TBARS production, but T3 and TA3 were less active than T4 and rT3 (IC50: 11.0 +/- 2.6 and 8.1 +/- 0.8 vs 1.4 +/- 0.5 and 0.9 +/- 0.3 microM respectively). In experiment 2, the compounds at 1 microM had no effect; at 10 microM, T3 and rT3 slightly reduced LDL TBARS production, whereas TA3 and T4 inhibited it by about 50% and 70% respectively. TBARS released by the cells were also highly decreased by T3, T4, rT3 and TA3 in experiment 1, but only by T3 (30%) and T4 (70%) in experiment 2. Cell viability was not affected by the compounds except slightly by TA3 at 10 microM. The data suggested that the physico-chemical antioxidant capacity of thyroid compounds was modulated by their action on the intracellular redox systems of macrophage. Overall cellular effects of T3 led to a reduction of its antioxidant capacity whereas those of T4 increased it. Thus T4 might protect LDL against cellular oxidation in vivo more than T3.

Folate: In Vitro and in Vivo Effects on VLDL and LDL Oxidation

2007 ◽  
Vol 77 (1) ◽  
pp. 66-72 ◽  
Author(s):  
McEneny ◽  
Couston ◽  
McKibben ◽  
Young ◽  
Woodside
Keyword(s):  
Folic Acid ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Folic Acid Supplementation ◽  
Serum Folate ◽  
Ldl Oxidation ◽  
Low Density ◽  
Acid Supplementation

Raised total homocysteine (tHcy) levels may be involved in the etiology of cardiovascular disease and can lead to damage of vascular endothelial cells and arterial wall matrix. Folic acid supplementation can help negate these detrimental effects by reducing tHcy. Recent evidence has suggested an additional anti-atherogenic property of folate in protecting lipoproteins against oxidation. This study utilized both an in vitro and in vivo approach. In vitro: Very-low-density lipoprotein (VLDL) and low density lipoprotein (LDL) were isolated by rapid ultracentrifugation and then oxidized in the presence of increasing concentrations (0→ μmol/L) of either folic acid or 5-methyltetrahydrofolate (5-MTHF). In vivo: Twelve female subjects were supplemented with folic acid (1 mg/day), and the pre- and post-VLDL and LDL isolates subjected to oxidation. In vitro: 5-MTHF, but not folic acid, significantly increased the resistance of VLDL and LDL to oxidation. In vivo: Following folic acid supplementation, tHcy decreased, serum folate increased, and both VLDL and LDL displayed a significant increase in their resistance to oxidation. These results indicated that in vitro, only the active form of folate, 5-MTHF, had antioxidant properties. In vivo results demonstrated that folic acid supplementation reduced tHcy and protected both VLDL and LDL against oxidation. These findings provide further support for the use of folic acid supplements to aid in the prevention of atherosclerosis.

Paraoxonase-1 (PON-1) genotype and activity and in vivo oxidized plasma low-density lipoprotein in Type II diabetes

2005 ◽  
Vol 109 (2) ◽  
pp. 189-197 ◽  
Author(s):  
Mike J. Sampson ◽  
Simon Braschi ◽  
Gavin Willis ◽  
Sian B. Astley
Keyword(s):  
Type Ii Diabetes ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Paraoxonase 1 ◽  
Ldl Oxidation ◽  
Phenyl Acetate ◽  
Low Density ◽  
Type Ii

The HDL (high-density lipoprotein)-associated enzyme PON (paraoxonase)-1 protects LDL (low-density lipoprotein) from oxidative modification in vitro, although it is unknown if this anti-atherogenic action occurs in vivo. In a cross-sectional study of 58 Type II diabetic subjects and 50 controls, we examined the fasting plasma LDL basal conjugated diene concentration [a direct measurement of circulating oxLDL (oxidatively modified LDL)], lipoprotein particle size by NMR spectroscopy, PON-1 polymorphisms (coding region polymorphisms Q192R and L55M, and gene promoter polymorphisms −108C/T and −162G/A), PON activity (with paraoxon or phenyl acetate as the substrates) and dietary antioxidant intake. Plasma oxLDL concentrations were higher in Type II diabetic patients (males, P=0.048; females, P=0.009) and unrelated to NMR lipoprotein size, PON-1 polymorphisms or PON activity (with paraoxon as the substrate) in any group. In men with Type II diabetes, however, there was a direct relationship between oxLDL concentrations and PON activity (with phenyl acetate as the substrate; r=0.611, P=0.0001) and an atherogenic NMR lipid profile in those who were PON-1 55LL homozygotes. Circulating oxLDL concentrations in vivo were unrelated to PON-1 genotypes or activity, except in male Type II diabetics where there was a direct association between PON activity (with phenyl acetate as the substrate) and oxLDL levels. These in vivo data contrast with in vitro data, and may be due to confounding by dietary fat intake. Male Type II diabetic subjects with PON-1 55LL homozygosity have an atherogenic NMR lipid profile independent of LDL oxidation. These data do not support an in vivo action of PON on LDL oxidation.

scholarly journals The Effect of Quail Egg and Hen Egg Consumption on Low-Density Lipoprotein Oxidation and Small Dense Low-Density Lipoprotein

2020 ◽  
Author(s):  
Raveenan Mingpakanee ◽  
Chatchanok Chaisitthichai ◽  
Nattaporn Wichitamporn ◽  
Paradee Sappittayakorn ◽  
Suparnnikar Phongphanwatana
Keyword(s):  
Plasma Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lag Time ◽  
Plasma Triglyceride ◽  
Ldl Oxidation ◽  
Low Density ◽  
Egg Consumption ◽  
Hen Egg ◽  
Lipoprotein Profiles

Objective: The aim of the study was to investigate the effect of quail egg and hen egg supplements on lipoprotein profiles, low-density lipoprotein (LDL) oxidation and small dense LDL cholesterol (sd-LDL-C) in young healthy people, compared with hen eggs. Material and Methods: Twenty-three healthy volunteers (11 men and 12 women) were randomly assigned to consume 3 whole hen eggs per day (hen group, n=11) (total cholesterol 633 mg) or 9 quail eggs per day (quail group, n=12) (total cholesterol 459 mg) for 30 days. The plasma cholesterol and plasma triglyceride concentrations and lipoprotein fractions (Triglyceride-rich lipoprotein; TRL, LDL and high-density lipoprotein; HDL) were determined at baseline and after the 30-day period of egg consumption. The LDL oxidation (lag time) was measured by the increase of conjugated diene production. Sd-LDL-C was calculated from the major lipid and lipoprotein parameters. Results: In the quail group, plasma triglyceride (TG) and LDL-TG were significantly decreased, whereas the plasma cholesterol and HDL-C were unchanged. There was no alteration in lipoprotein profiles in the hen group. The LDL lag time of the quail group was longer than at baseline. There were no significant changes in sd-LDL-C levels in both groups during the study.Conclusion: Quail egg and hen egg consumptions for 30 days did not change the lipoprotein profiles, sd-LDL as well as the LDL-oxidation, which not modified the cardiovascular disease risk factor.

scholarly journals Low-density lipoprotein oxidation, antioxidants, and atherosclerosis: a clinical biochemistry perspective

1996 ◽  
Vol 42 (4) ◽  
pp. 498-506 ◽  
Author(s):  
I Jialal ◽  
S Devaraj
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Direct Methods ◽  
Ldl Oxidation ◽  
Low Density ◽  
Indirect Measures ◽  
Oxidatively Modified

Abstract Cardiovascular disease is the leading cause of mortality in westernized populations. An increased concentration of plasma low-density lipoprotein (LDL) cholesterol constitutes a major risk factor for atherosclerosis. Several lines of evidence support a role for oxidatively modified LDL in atherosclerosis and for its in vivo existence. Antioxidants have been shown to decrease atherosclerotic lesion formation in animal models and decrease LDL oxidation; the evaluation of LDL oxidation in vivo is therefore very important. However, there is a paucity of methods for direct measurement of LDL oxidation. Of the direct methods currently available, the preferred ones seem to be the measurement of F2-isoprostanes, autoantibodies to epitopes on oxidized LDL, and the assessment of antioxidant status. Of the indirect measures, the most uniformly accepted procedure is examining the oxidative susceptibility of isolated LDL by monitoring conjugated diene formation.

scholarly journals Low-density lipoprotein susceptibility to in vitro oxidation in healthy smokers and nonsmokers

1996 ◽  
Vol 42 (4) ◽  
pp. 524-530 ◽  
Author(s):  
R Siekmeier ◽  
P Wülfroth ◽  
H Wieland ◽  
W Gross ◽  
W März
Keyword(s):  
Body Mass ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
Thiobarbituric Acid ◽  
Low Density ◽  
Apo B ◽  
Lag Times

Abstract We analyzed the susceptibility of low-density lipoproteins (LDL) to oxidation in 17 healthy smokers (43.3 +/- 16.8 pack-years) and 19 healthy nonsmokers, matched for age (smokers: 52 +/- 7 years; nonsmokers: 53 +/- 7 years), gender, and relative body mass. Cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, and apolipoprotein (apo) B were not different between smokers and nonsmokers; apo A-I was slightly lower in smokers (one-tailed P = 0.066). To study whether LDL from smokers were prone to in vitro oxidation than LDL from nonsmokers, we measured the time kinetics of diene formation and the production of malondialdehyde during oxidation of LDL in vitro. In smokers and nonsmokers, respectively, the mean (+/-SD) lag times (tinh) of diene formation were 111 +/- 26 and 100 +/- 27 min, the peak rates of diene formation (Vmax) were 5.99 +/- 2.34 and 6.34 +/- 2.30 mmol x min-1 x g-1, and the amounts of dienes produced during the propagation phase (dmax) were 250 +/- 264 and 248 +/- 56 mmol x g-1. Neither the malondialdehyde content of LDL (measured as thiobarbituric acid-reactive substances) before oxidation nor the amount of malondialdehyde generated during oxidation (smokers: 57.0 +/- 14.2 micromol x g-1; nonsmokers: 63.2 +/- 15.2 micromol x g-1 indicated any statistically significant effect of smoking. When nonsmokers and smokers were considered together, the amount of malondialdehyde generated during oxidation correlated with age (nonparametric rs = 0.405), body mass index (r2 = 0.573), and concentrations of apo B (rs = 0.480), cholesterol (rs = 0.448), triglycerides (rs = 0.436), and LDL cholesterol (rs = 0.398). Our data show that smoking is not associated with increased oxidizability of LDL in healthy men and women at ages 42-63 years.

Gemfibrozil metabolite inhibits in vitro low-density lipoprotein (LDL) oxidation and diminishes cytotoxicity induced by oxidized LDL

Metabolism ◽  
2000 ◽  
Vol 49 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Mitsunobu Kawamura ◽  
Shigeru Miyazaki ◽  
Tamio Teramoto ◽  
Keiko Ashidate ◽  
Hisako Thoda ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Ldl Oxidation ◽  
Low Density

scholarly journals Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis

1994 ◽  
Vol 300 (1) ◽  
pp. 243-249 ◽  
Author(s):  
J V Hunt ◽  
M A Bottoms ◽  
K Clare ◽  
J T Skamarauskas ◽  
M J Mitchinson
Keyword(s):  
Glucose Concentration ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Protein Glycation ◽  
Ldl Oxidation ◽  
Low Density ◽  
Catalysed Oxidation ◽  
Induce Lipid Peroxidation ◽  
High Concentrations

The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.

scholarly journals Antioxidative Effects and Inhibition of Human Low Density Lipoprotein OxidationIn Vitroof Polyphenolic Compounds inFlammulina velutipes(Golden Needle Mushroom)

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Mohammad Azizur Rahman ◽  
Noorlidah Abdullah ◽  
Norhaniza Aminudin
Keyword(s):  
Low Density Lipoprotein ◽  
Radical Scavenging ◽  
Density Lipoprotein ◽  
Thiobarbituric Acid ◽  
Polyphenolic Compounds ◽  
Oxidative Modification ◽  
Ldl Oxidation ◽  
Total Phenolic ◽  
Low Density ◽  
Antioxidative Effects

Dietary polyphenolic compounds mediate polynomial actions in guarding against multiple diseases. Atherosclerosis is an oxidative stress driven pathophysiological complication where free radical induced oxidative modification of low density lipoprotein (LDL) plays the ground breaking role. Mushrooms have been highly regarded for possessing an antioxidant arsenal. Polyphenolic compounds present in dietary mushrooms seem pertinent in withstanding LDL oxidation en route to controlling atherosclerosis. In this study, the antioxidative effect of five solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ) ofFlammulina velutipeswas evaluated. M : DCM fraction showed the most potent 2,2-diphenyl-1-picrylhydrazyl radical scavenging effect with IC50of 0.86 mg/mL and total phenolic content of 56.36 gallic acid equivalent/g fraction. In LDL oxidation inhibitory tests, M : DCM fraction at 1 µg/mL concentration mostly lengthened the lag time (125 mins) of conjugated diene formation and inhibited the formation of thiobarbituric acid reactive substances (48.71%, at 1 mg/mL concentration). LC-MS/MS analyses of M : DCM fraction identified the presence of polyphenolic substances protocatechuic acid,p-coumaric, and ellagic acid. These chain-breaking polyphenolics might impart the antioxidative effects ofF. velutipes. Thus, mushroom-based dietary polyphenolic compounds might be implicated in slowing down the progression of atherosclerosis.

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