Copper-catalyzed LDL Oxidation Research Articles

Impaired antioxidant action of high density lipoprotein in patients with type 1 diabetes with normoalbuminuria and microalbuminuria

AimsPatients with type 1 diabetes, in the absence of chronic complications, have serum concentrations of high density lipoprotein cholesterol (HDL-C) similar to the general population. However, their HDL particles may be dysfunctional. We aimed to evaluate the antioxidant effect of HDL2 and HDL3 obtained from Caucasian males with type 1 diabetes with normoalbuminuria and microalbuminuria. MethodsTwenty Caucasian men with type 1 diabetes (10 with normoalbuminuria and 10 with microalbuminuria) and 10 healthy Caucasian men participated in the study. Lipoproteins were obtained by density gradient ultracentrifugation. The antioxidant effect of HDL was assessed by measuring lipid hydroperoxide (LOOH) concentration after 3h of pooled LDL oxidation catalyzed by 5μM CuSO4 in the absence or presence of HDL2 or HDL3. ResultsThe control, normoalbuminuria, and microalbuminuria groups had similar HDL-C concentration and estimated glomerular filtration rate. Glycemic control was similar between diabetes groups (HbA1c 8.1±0.9% and 8.3±0.7%, P=0.70), but estimated glucose disposal rate was lower in patients with microalbuminuria (8.0±0.6 and 4.5±1.1mg/kg/min, P<0.01). The relative antioxidant effect of HDL2 from control, normoalbuminuria, and microalbuminuria groups were 92.8±2.4%, 85.4±1.7%, and 74.2±4.6%, respectively (P<0.01), and the HDL3 effect were 95.0±2.2%, 86.4±4.4%, and 75.3±4.2%, respectively (P<0.01). ConclusionBoth HDL2 and HDL3 inhibited LOOH formation in copper-catalyzed oxidation of LDL in vitro. Overall, this antioxidant effect was lower in Caucasian men with type 1 diabetes, and was further compounded in those with microalbuminuria.

The effects of soygerm extracts on blood lipoproteins, antioxidative capacity and urinary estrogen metabolites in postmenopausal women on hormone therapy

Objective To evaluate the effects of soygerm isoflavones extracts on blood lipoproteins, antioxidative capacity and urinary estrogen metabolites in postmenopausal women who receive hormone therapy (HT). Method Thirty-nine volunteers receiving HT were recruited, and 33 completed the study. All subjects received 6 g of soygerm extracts per day for 4 weeks. Blood and urine samples were collected for study at the beginning and at the end of study. Result Plasma HDL-C levels increased markedly with significant decreases of plasma LDL-C/HDL-C ratio and LDL–TG levels. The lag time of conjugated dienes formation prolonged for 9.9% and thiobarbituric acid reactive substances production in copper-catalyzed oxidation of LDL decreased. The differences were statistically significant. Urinary ratio of 2-OHE 1 to 16α-OHE 1 increased without statistical significance. Conclusion Soygerm extracts may improve serum lipid profile in postmenopausal Taiwanese women who receive HT, and probably provide a favorable effect on estrogen metabolism.

Effect of homocysteine, folates, and cobalamin on endothelial cell‐ and copper‐induced LDL oxidation

Oxidation of LDL contributes to endothelial dysfunction and atherosclerosis. This process could be associated with hyperhomocysteinemia, a condition that can be reduced after folic acid treatment. Because a reduction in LDL oxidation may improve endothelial function, we studied the effect of some vitamins (folic acid, 5-methyltetrahydrofolic acid, and vitamin B-12) on LDL oxidation, either in the presence or absence of homocysteine. For this purpose, two in vitro systems were used: an endothelial cell-catalyzed LDL oxidation system and a cell-free copper-initiated LDL oxidation system. The kinetics of copper-catalyzed LDL oxidation was determined by continuous monitoring of the production of conjugated dienes in the reaction medium. TBARS production, a parameter of lipid peroxidation, was also evaluated. In both in vitro systems, only 5-methyltetrahydrofolic acid was able to decrease TBARS production in a concentration-dependent manner, independently of the presence or absence of homocysteine. In the copper-induced LDL oxidation system, vitamin B-12 and 5-methyltetrahydrofolic acid increased the lag time of conjugated diene production by 25 and 47%, respectively, suggesting that both vitamins in this system had antioxidant properties. Folic acid was unable to show antioxidant properties when included in either in vitro system. The results demonstrate that 5-methyltetrahydrofolic acid and vitamin B-12 are important protective agents against LDL oxidative modifications.

Cocoa Products Decrease Low Density Lipoprotein Oxidative Susceptibility but Do Not Affect Biomarkers of Inflammation in Humans

Flavonoids and related polyphenolics with antioxidant and anti-inflammatory activities may play a role in the prevention of cardiovascular disease by decreasing oxidative stress and inflammation. We wished to determine the effects of cocoa extract supplementation on markers of oxidative stress and inflammation. Healthy subjects (n = 25) were studied at baseline, after cocoa supplementation (36.9 g of dark chocolate bar and 30.95 g of cocoa powder drink) for 6 wk and after a 6-wk washout period. Fasting blood and early morning urine were collected at the three time points. Two indices of flavonoid intake, total phenols and oxygen radical absorbance capacity of plasma, were measured after an overnight fast. Neither was affected by supplementation. Measures of oxidative stress included copper-catalyzed LDL oxidation kinetics and urinary F(2) isoprostanes. LDL oxidizability was lower after chocolate supplementation as evidenced by a longer lag time (P < 0.05) of conjugated diene formation (101.0 +/- 20.7 min) compared with baseline (91.3 +/- 18.0 min) and washout (96.4 +/- 7.5 min) phases. There was no effect of chocolate on urinary F(2) isoprostane levels or on markers of inflammation including the whole-blood cytokines, interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha, high sensitivity C-reactive protein and P-selectin. In conclusion, cocoa products supplementation in humans affects LDL oxidizability, but not urinary F(2) isoprostanes or markers of inflammation.

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (−)-epigalocatechin gallate (EGCG)=(−)-epicatechin gallate (ECG)>(−)-epicatechin (EC)=(+)-catechin (C)>(−)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.

Apple juice inhibits human low density lipoprotein oxidation

Dietary phenolic compounds, ubiquitous in vegetables and fruits and their juices possess antioxidant activity that may have beneficial effects on human health. The phenolic composition of six commercial apple juices, and of the peel (RP), flesh (RF) and whole fresh Red Delicious apples (RW), was determined by high performance liquid chromatography (HPLC), and total phenols were determined by the Folin-Ciocalteau method. HPLC analysis identified and quantified several classes of phenolic compounds: cinnamates, anthocyanins, flavan-3-ols and flavonols. Phloridzin and hydroxy methyl furfural were also identified. The profile of phenolic compounds varied among the juices. The range of concentrations as a percentage of total phenolic concentration was: hydroxy methyl furfural, 4–30%; phloridzin, 22–36%; cinnamates, 25–36%; anthocyanins, n.d.; flavan-3-ols, 8–27%; flavonols, 2–10%. The phenolic profile of the Red Delicious apple extracts differed from those of the juices. The range of concentrations of phenolic classes in fresh apple extracts was: hydroxy methyl Furfural, n.d.; phloridzin, 11–17%; cinnamates, 3–27%; anthocyanins, n.d.-42%; flavan-3-ols, 31–54%; flavonols, 1–10%. The ability of compounds in apple juices and extracts from fresh apple to protect LDL was assessed using an in vitro copper catalyzed human LDL oxidation system. The extent of LDL oxidation was determined as hexanal production using static headspace gas chromatography. The apple juices and extracts, tested at 5 μM gallic acid equivalents (GAE), all inhibited LDL oxidation. The inhibition by the juices ranged from 9 to 34%, and inhibition by RF, RW and RP was 21, 34 and 38%, respectively. Regression analyses revealed no significant correlation between antioxidant activity and either total phenolic concentration or any specific class of phenolics. Although the specific components in the apple juices and extracts that contributed to antioxidant activity have yet to be identified, this study found that both fresh apple and commercial apple juices inhibited copper-catalyzed LDL oxidation. The in vitro antioxidant activity of apples support the inclusion of this fruit and its juice in a healthy human diet.

Mechanism of high-density lipoprotein subfractions inhibiting copper-catalyzed oxidation of low-density lipoprotein

Objective: To investigate the role of HDL subfractions, HDL 2 and HDL 3, on the oxidation of LDL catalyzed by 5 μM Cu 2+ ion, and to illustrate the mechanism of the generation of conjugated diene and thiobarbituric acid reactive substances (TBARS) during LDL oxidation. Methods: LDL was incubated for 8 h with 5 μM Cu 2+ ion in phosphate-buffered saline (PBS) alone, or in the presence of HDL 2, HDL 3, HSA, BSA, or transferrin. Meantime, LDL was incubated for 24 h with 10 μM Ni 2+ ions in PBS. The amount of conjugated diene and TBARS in each sample of LDL were measured. Results: (a) HDL 2 and HDL 3 could inhibit the generation of conjugated diene, but could not inhibit the generation of TBARS; (b) the transferrin containing HDL 3 shows the ability of inhibiting the generation of both conjugated diene and TBARS; (c) the transferrin presented in blood exhibits the inhibitory effect on the generation of conjugated diene and TBARS, however, when the transferrin is saturated with Fe 3+ ion, it could not inhibit the generation of TBARS; (d) HSA and BSA could prevent the generation of conjugated diene and TBARS; (e) Ni 2+ ion could induce the generation of conjugated diene, but the amount of TBARS was much smaller than that induced by Cu 2+ ion. Conclusion: HDL 2 and HDL 3 play important role in the copper-catalyzed oxidation of LDL; it is absolutely necessary to require chelation of Cu 2+ ion for inhibiting generation of TBARS; whereas, inhibition of conjugated diene can be fulfilled either by chelating Cu 2+ ion, or the free radicals scavenger.

Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein

Cardiovascular disease is the leading cause of morbidity and mortality in Westernized populations. Evolving lipoprotein risk factors include LDL oxidation and lipoprotein(a) [lp(a)]. Several lines of evidence support a role for oxidatively modified LDL in atherogenesis and its in vivo existence. There are both direct and indirect measures of oxidative stress. The most relevant direct measure of lipid peroxidation is urinary F2 isoprostanes. The most common indirect measure of LDL oxidation is quantifying the lag phase of copper-catalyzed LDL oxidation by assaying conjugated diene formation. Lp(a) is increased in patients with cardiovascular and cerebrovascular disease. However, not all prospective studies have confirmed a positive relationship between Lp(a) and cardiovascular events. Lp(a) appears to present three major problems: standardization of the assay, establishing its role in atherogenesis, and the lack of an effective therapy that can substantially lower Lp(a) concentrations. Thus, at the present time, Lp(a) concentrations should not be recommended for the general population but be reserved for patients with coronary artery disease without established risk factors, young patients with coronary artery disease or cerebrovascular disease, or a family history of premature atherosclerosis and family members of an index patient with increased concentrations of Lp(a). Although both LDL oxidation and Lp(a) are evolving risk factors for cardiovascular disease, more data are needed before they become part of the established lipoprotein repertoire.

HDL 3 exerts more powerful anti-oxidative, protective effects against copper-catalyzed LDL oxidation than HDL 2

Objective: To evaluate which HDL subfraction, HDL 2 or HDL 3, exerts the greater preventive effect on the Cu 2+-induced LDL oxidation. Methods: LDL was incubated for 6 h with 2.5 μM Cu 2+ in phosphate-buffered saline alone, or in the presence of HDL 2 or HDL 3 at various protein concentrations. Each sample was subjected to agarose gel electrophoresis, and the amount of lipid hydroperoxide in each sample of LDL was measured. Results: There was no significant difference in the levels of LPO between the LDL and LDL + HDL 2 cases, whereas a significant reduction was apparent with LDL + HDL 3. Both HDL 2 and HDL 3 significantly inhibited oxidative modification of LDL, as assessed by electrophoretic mobility, in a concentration dependent manner, but this effect was much more pronounced with HDL 3. Conclusion: HDL 3 may play an important role in the prevention of atherosclerosis in vivo, more effectively inhibiting oxidation of LDL than HDL 2.

In vivo action of 15-lipoxygenase in early stages of human atherogenesis.

Oxidative modification of low density lipoprotein has been suggested as patho-physiologically relevant process in atherogenesis and the lipid peroxidizing enzyme 15-lipoxygenase may be involved. For experimental evidence on the in vivo action of this enzyme in the time course of plaque formation we analyzed the lipid extracts of lesional areas representing various stages of human atherogenesis for the occurrence of specific 15-lipoxygenase products. In advanced human lesions the degree of oxygenation of the lesion lipids measured as hydroxy linoleic acid/linoleic acid ratio varied between 0.2 and 3.2%. Here an unspecific pattern of oxygenated lipids that did not differ from the pattern formed during copper-catalyzed LDL oxidation was detected. In both cases an enantiomer ratio (S/R-ratio) of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) of approximately 1:1 was found. In young human lesions which were obtained from the collection of the pathological determinants of atherosclerosis in youth (PDAY) program the hydroxy linoleic acid/linoleic acid ratio was much smaller (variation between 0.05 and 0.6%), and a significant share of specific 15-lipoxygenase products was detected (S/R-ratio of 13-hydroxy linoleic acid of 54 +/- 3.1/46 +/- 3.1 [mean +/- SD]). These data suggest that the 15-lipoxygenase is enzymatically active on endogenous substrates in young human lesions and thus, may be of patho-physiological importance for early atherogenesis. In advanced human plaques the 15-lipoxygenase may be functionally silent and specific lipoxygenase products formed in earlier stages may be decomposed or superimposed by large amounts of nonenzymatic lipid peroxidation products.

The role of oxidative stress in the long-term glycation of LDL.

Advanced glycation is a major pathway for the posttranslational modification of plasma and tissue proteins. The initiating reaction is the nonenzymatic addition of sugars such as glucose to the primary amino groups of proteins, i.e., mainly to lysine residues. These "early" Schiff base and Amadori products then undergo a series of inter- and intramolecular rearrangements to produce the "late" products termed advanced glycation end products (AGEs). Incubation of LDL with glucose or glucose-6-phosphate produces AGE moieties on both the lipid and apolipoprotein B components. In addition, we tried to generate AGE-LDL by reaction with AGE-peptides (< 10 kD) obtained by enzymatic digestion of long-term glycated fibronectin as a model for connective tissue AGE-peptides. AGE-formation can be assessed by monitoring of fluorescence (370/440 nm) which is easily differentiated from the much lower autofluorescence of oxidized low density lipoproteins (oxLDL). Alternatively, AGE formation was detected by an AGE-specific ELISA using antibodies elicited in rabbits against bovine AGE-RNAse. In the present study we investigated the influence of oxidative stress on the long-term glycation of LDL and the modulation of LDL-oxidation by AGE-modification. We observed (a) that the rate of AGE formation is reduced by BHT/EDTA both on LDL and serum albumin (glycation vs. glycoxidation), (b) long-term glycated LDL is more readily oxidized than unglycated LDL, (c) oxLDL is more prone to AGE-modification, (d) AGE-modification of LDL strongly alters its epitope spectrum and (e) that aminoguanidine at higher concentrations (1-10 mM) inhibits copper-catalyzed LDL oxidation in the way of a classical antioxidant.

Principal phenolic phytochemicals in French Syrah and Grenache Rhone wines and their antioxidant activity in inhibiting oxidation of human low density lipoproteins

&lt;p style="text-align: justify;"&gt;Wine contains natural plant phenolic antioxidants that may protect circulating lipoproteins from oxidative damage. By inhibition of the copper-catalyzed oxidation of LDL we determined the activity of thirteen Rhône coast wines exclusively from Syrah and Grenache varieties. About 50 p. cent of the wines were made with a long maceration process. Major monomeric phenolic compounds and procyanidin dimers were analyzed in each sample by HPLC and correlated with relative LDL antioxidant activity. Correlations obtained can be grouped in 3 classes: catechin (r = 0.75), procyanidins, BI, B2, B3 (r = 0.43-0.55), malvidin-3-glucoside and cyanidin (r = 0.43), gallic acid, myreeitin, rutin (r = 0.2-0.4). On the same basis total phenol contents of wines gave a correlation with LDL antioxidant activity of r = 0.72. Comparison, at the same total phenol concentration, with different red California wines shows that antioxidant activity of French Syrah and Grenache range between that of Merlot (56-65 p. cent) and Cabernet Sauvignon (37-45 p.cent). In contrast Syrah wines made with a short extraction process gave lower inhibition of LDL oxidation of 16 p. cent which is less than white Califomia wines averaging 36 p. cent. Activity of each wine phenolic compound can play a role in protecting LDL against oxidation.&lt;/p&gt;

Effect of combined supplementation with alpha-tocopherol, ascorbate, and beta carotene on low-density lipoprotein oxidation.

Data continue to accumulate supporting a proatherogenic role for oxidized low-density lipoprotein (Ox-LDL). Antioxidant micronutrients such as ascorbate, alpha-tocopherol, and beta carotene, levels of which can be favorably manipulated by dietary measures without side effects, could be a safe approach in inhibiting LDL oxidation. In fact, in vitro studies have shown that all three antioxidants can inhibit LDL oxidation. The present study was undertaken to ascertain both the safety and antioxidant effect of combined supplementation with alpha-tocopherol, ascorbate, and beta carotene on LDL oxidation. The effect of combined supplementation with alpha-tocopherol (800 IU/d) plus ascorbate (1.0 g/d) and beta carotene (30 mg/d) on copper-catalyzed LDL oxidation was tested in a randomized, placebo-controlled study in two groups of 12 male subjects over a 3-month period. Blood samples for the lipoprotein profile, antioxidant levels, and LDL isolation were obtained at baseline and at 3 months. Neither placebo nor combined antioxidant therapy resulted in any side effects or exerted an adverse effect on the plasma lipoprotein profile. Compared with placebo, combined antioxidant therapy resulted in a significant increase in plasma ascorbate and lipid standardized alpha-tocopherol and beta carotene levels (2.6-, 4.1-, and 16.3-fold, respectively). At baseline, there were no significant differences in the time course curves and kinetics of LDL oxidation as evidenced by the thiobarbituric acid reacting substances (TBARS) assay and the formation of conjugated dienes. However, at 3 months, combined supplementation resulted in a twofold prolongation of the lag phase and a 40% decrease in the oxidation rate. The combined antioxidant group was also compared with a group that received 800 IU of alpha-tocopherol only. Although the combined antioxidant group had significantly higher ascorbate and beta carotene levels than the group supplemented with alpha-tocopherol alone, there were no significant differences between the two groups with respect to LDL oxidation kinetics. Combined supplementation with ascorbate, beta carotene, and alpha-tocopherol is not superior to high-dose alpha-tocopherol alone in inhibiting LDL oxidation. Hence, alpha-tocopherol therapy should be favored in future coronary prevention trials involving antioxidants.

Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein.

Much data has accumulated supporting a proatherogenic role for oxidized low density lipoprotein (Ox-LDL). Micronutrient antioxidants such as alpha-tocopherol, the principal lipid-soluble antioxidant, assume potential significance because levels can be manipulated by dietary measures without resulting in side effects. Co-incubation of LDL in vitro with alpha-tocopherol inhibits its oxidative modification. Hence the effect of dietary supplementation with alpha-tocopherol on the time course of copper-catalyzed oxidation of LDL was tested in a randomized placebo-controlled single-blind study. Two groups of 12 male subjects were given either placebo or alpha-tocopherol (800 IU/day) for a period of 12 weeks. Alpha-tocopherol therapy did not result in any side effects or exert an adverse effect on the plasma lipid and lipoprotein profile. While the lipid standardized alpha-tocopherol levels were similar at baseline, the supplemented group had 3.3-fold and 4.4-fold higher levels compared to placebo at 6 and 12 weeks, respectively. In the 15 subjects in whom both plasma and LDL alpha-tocopherol levels were quantitated, there was a significant correlation (r = 0.79, P less than 0.0001). At baseline there were no significant differences in the time course curves of thiobarbituric acid-reacting substances (TBARS) activity or conjugated diene formation between the alpha-tocopherol and placebo groups. However, at both 6 and 12 weeks the mean levels of TBARS activity and conjugated diene formation were lower in the alpha-tocopherol group; the most significant differences were manifest at the 3-h time point. Also at both 6 and 12 weeks the mean rate of oxidation was lower in the alpha-tocopherol group.2+_