Lipid Hydroperoxides In Low Density Lipoprotein Research Articles

Paraoxonase and Coronary Heart Disease Risk

Organophosphates were first synthesized in the 1930s as insecticides1 and were subsequently shown to have direct neurotoxic effects in mammals, as well. The neurotoxicity is derived from their ability to inhibit acetylcholinesterase by covalently modifying the active-site serine group in the enzyme. Mazur2 first demonstrated the presence of an organophosphate-hydrolyzing enzyme in mammalian tissue, an observation that ultimately led to the identification of a human paraoxonase in serum in 1953.3 Paraoxonase—so named because of its ability to hydrolyze the toxic metabolite of parathion, paraoxon—was also shown early after its identification to manifest arylesterase activity, an effect that was underappreciated until the enzyme was found to play a role in modulating vascular oxidant stress many years later. Article see p 147 The paraoxonase story is a good example of the power of language to mislead. There is no teleological reason for mammals to have evolved an enzyme that can hydrolyze synthetic organophosphates; yet, paraoxonase was isolated in an effort to understand the endogenous metabolism of these exogenous neurotoxins. The enzymatic activity for which the enzyme is named is screened by using synthetic substrates without regard for the native substrate or its role in human (patho)biology. This focus on one aspect of the enzyme’s function, overemphasized by its denotation, delayed an appreciation of other potential—and perhaps more relevant—roles. In 1991, Mackness et al4 showed that paraoxonase …

Anti-peroxidation effects of vitamin E on low density lipoprotein and milk fat globule membrane of lactating goats: in vivo versus metal ion challenge in vitro

Ruminants are animals with mild oxidation risk considering characteristics of their plasma. The purpose of the present study was to determine if surplus vitamin E supplementation further improved their peroxidation status. Four lactating goats ( Capra ibex ibex) receiving a single intramuscular injection of 3000 IU d-α-tocopheryl acetate were monitored daily for a week. Plasma and milk levels of thiobarbituric acid reactive substances (TBARS) were measured to estimate the peroxidation status of overall body and mammary gland, respectively. Lipid hydroperoxide content of low density lipoprotein (LDL) and milk fat globule membrane (MFGM) were determined both immediately after isolation and after metal ion challenge to evaluate their spontaneous peroxidation in vivo and peroxidation susceptibility in vitro, respectively. The results showed that while plasma and milk levels of α-tocopherol peaked at day 2, the corresponding TBARS dropped to their lowest weekly levels. Content of preformed lipid hydroperoxides in LDL and MFGM remained unchanged ( P>0.05) during the study period, while peroxidation in LDL and MFGM on extended exposure to Cu 2+ was prevented by vitamin E enrichment. Therefore, surplus vitamin E was beneficial to the peroxidation-resistant mechanisms of the overall body and those specifically within mammary gland of lactating goats. Furthermore, peroxidation resistance of LDL and MFGM to copper challenge in vitro also benefited from vitamin E enrichment despite the fact their endogenous peroxidation seemed unresponsive. The hypothesis that peroxidation of LDL and MFGM of lactating goats in situ, which proceeds with metal ion-independent mechanisms, is minor was discussed.

Mechanisms by which cysteine can inhibit or promote the oxidation of low density lipoprotein by copper

Oxidised low density lipoprotein (LDL) may play a role in atherogenesis. We have investigated some of the mechanisms by which the thiol cysteine and the disulphide cystine can influence the oxidation of LDL by copper ions. Cysteine or cystine (100 μM) inhibited the oxidation of native LDL by copper in a simple phosphate buffer. One of the mechanisms by which cysteine (or more likely its oxidation products in the presence of copper) and cystine inhibited LDL oxidation was by decreasing the binding of copper to LDL (97% inhibition). Cysteine, but not cystine, rapidly reduced Cu 2+ to Cu +. This may help to explain the antioxidant effect of cysteine as it may limit the amount of Cu 2+ that is available to convert α-tocopherol in LDL into the prooxidant α-tocopherol radical. Cysteine (but not cystine) had a prooxidant effect, however, toward partially oxidised LDL in the presence of a low copper concentration, which may have been due to the rapid breakdown of lipid hydroperoxides in partially oxidised LDL by Cu + generated by cysteine. To prove that cysteine can cause the rapid breakdown of lipid hydroperoxides in LDL, we enriched LDL with lipid hydroperoxides using an azo initiator in the absence of copper. Cysteine, but not cystine, increased the rate of lipid hydroperoxide decomposition to thiobarbituric acid-reactive substances (TBARS) in the presence of copper.

Effect of acetaminophen on the myeloperoxidase–hydrogen peroxide–nitrite mediated oxidation of LDL

Myeloperoxidase, in the presence of hydrogen peroxide and nitrite, promotes the lipid peroxidation of low density lipoprotein (LDL); the modified lipoprotein is then capable of being readily endocytosed by macrophages. Since acetaminophen has been shown to inhibit the leukocyte myeloperoxidase antimicrobial system and is, under certain experimental conditions, an antioxidant, the effect of acetaminophen on the myeloperoxidase–hydrogen peroxide–nitrite mediated oxidation of LDL was examined. The content of LDL lipid hydroperoxides after incubation with 50 nM myeloperoxidase, 100 μM nitrite and a hydrogen peroxide generating system for 6 h was reduced by approx. 80% in the presence of 25–250 μM acetaminophen. The production of thiobarbituric acid-reactive substances was also inhibited by acetaminophen to a similar extent. Acetylsalicylic acid (25–100 μM) did not inhibit LDL lipid peroxidation mediated by the myeloperoxidase enzyme system. LDL, treated with myeloperoxidase, hydrogen peroxide and nitrite for 14 h, was metabolized by macrophages to a much greater extent than native LDL. The presence of acetaminophen prevented the modification of LDL; the lipoprotein was metabolized by macrophages to the same extent as was native LDL. These results demonstrate that acetaminophen is a potent inhibitor of the myeloperoxidase–hydrogen peroxide–nitrite mediated modification of LDL.

Ascorbic acid protects against peroxidative modification of low-density lipoprotein, maintaining its recognition by LDL receptors.

Peroxidatively modified low-density lipoprotein (LDL) may contribute to atherosclerotic processes; therefore, protecting LDL against peroxidation may thus reduce or retard the progression of atherosclerosis. We have evaluated the protective effects of ascorbic acid on copper-catalyzed LDL peroxidative modification. The protective effects of ascorbic acid on copper-catalyzed LDL peroxidative modification were examined by measurement of concentration of lipid hydroperoxides in LDL and by the provision of LDL cholesterol to lymphocytes via LDL receptor-mediated pathway. The measurement of concentration of lipid hydroperoxides in LDL showed that ascorbic acid inhibited peroxidative modification of LDL. Also, ascorbic acid preserved the ability of LDL to be recognized by LDL receptors in peripheral blood lymphocytes to the same extent as native LDL. These findings indicate that ascorbic acid may protect LDL against peroxidative modification, maintaining its ability to act as a ligand for LDL receptors in vivo.

Macrophages Can Decrease the Level of Cholesteryl Ester Hydroperoxides in Low Density Lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.

Potent antioxidant activity of a dithiocarbamate-related compound from a marine hydroid

Recently, we discovered a novel class of natural products, named the tridentatols, in a marine hydroid. Close examination of their molecular structures suggested that they may have antioxidant activity. This observation prompted us to evaluate in vitro the capacity of one of these tridentatols, viz. tridentatol A, to inhibit lipid peroxidation using human low density lipoprotein (LDL) as an experimental model. LDL was incubated with 5 μM cupric chloride (Cu 2+) in the absence and presence of tridentatol A or a reference antioxidant standard, i.e. vitamin E. The onset of rapid formation of conjugated lipid hydroperoxides was delayed in a concentration-dependent manner by tridentatol A. More specifically, LDL incubated with Cu 2+ had a lag-phase time (the elapsed time before the onset of rapid formation of conjugated lipid hydroperoxides) of 150 min. However, when 0.5 μM tridentatol A was present during incubation, the lag phase time was extended to 225 min. With 1 μM tridentatol A, the lag phase time was 300 min. The same concentrations of vitamin E produced noticeably lower lag phase times. Thus, compared with vitamin E, tridentatol A better protected against the formation of conjugated lipid hydroperoxides in LDL. Direct colorimetric measurements of both lipid hydroperoxides and thiobarbituric acid-reactive substances confirmed the greater potency of tridentatol A relative to vitamin E. Furthermore, tridentatol A negated the Cu 2+-induced increase in electrophoretic mobility of LDL to a greater extent than vitamin E. In conclusion, tridentatol A is a powerful antioxidant against lipid peroxidation of LDL and is significantly more potent than vitamin E in this regard.

A phenolic antioxidant from the freshwater orchid, Habenaria repens

Recently, an unusual compound named habenariol was isolated from the freshwater orchid, Habenaria repens. Its phenolic structure suggested that habenariol should have substantial antioxidant activity. This possibility was investigated by evaluating the capacity of habenariol to inhibit copper-induced lipid peroxidation of human low density lipoprotein (LDL), a popular experimental model. LDL was incubated with 5 μM cupric chloride in the presence and absence of habenariol or a positive control, viz., α-tocopherol. Both kinetic and end-point spectrophotometric assays were used to determine extent of lipid peroxidation of LDL. In the kinetic assay, the time elapsing before the onset of rapid formation of conjugated lipid hydroperoxides in LDL (marked by a sharp increase in UV absorbance) was prolonged by habenariol, indicative of an antioxidant effect. In the end-point assay, direct colorimetric measurement confirmed habenariol's ability to inhibit formation of lipid hydroperoxides. However, in both assays, habenariol was less potent than α-tocopherol in inhibiting lipid peroxidation of LDL.

The effect of selective low-density lipoprotein apheresis on plasma lipoperoxides and antioxidant vitamins in familial hypercholesterolemic patients.

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder characterized by a lifelong elevation in the concentration of low-density lipoprotein (LDL) bound cholesterol in blood by cholesterol deposits and by early coronary artery disease. The LDL apheresis technique has been introduced with the goal of reducing LDL cholesterol levels, thereby preventing the development of atherosclerosis. The literature on LDL apheresis reports 2 different facets, the therapeutic aspect associated with the lessening of LDL concentration and the initiation of a peroxidation process associated with the biocompatibility of the artificial membrane. Lipid and protein peroxidation gives rise to toxic and atherogenic hydroperoxide, mostly lipid hydroperoxides, and derivative compounds, which may offset the benefit of the procedure. In this paper, plasma hydroperoxide levels are determined along with the elevation of the serum and LDL antioxidant status in hypercholesterolemic patients before and following repeated LDL apheresis sessions. Hydroperoxide concentration has been expressed both in terms of plasma volume and LDL concentration. A highly significant increase in LDL lipid hydroperoxides is demonstrated when expressed in terms of LDL concentration and is associated with the LDL apheresis procedure. The usefulness of antioxidant supplementation in LDL apheresis is discussed.

Alpha-tocopherol protects the peroxidative modification of LDL to be recognized by LDL receptors.

Peroxidatively modified low-density lipoprotein (LDL) may contribute to the atherosclerotic process; therefore, protecting LDL against peroxidation may reduce or retard the progression of atherosclerosis. We evaluated the effect of alpha-tocopherol on copper-catalyzed LDL peroxidative modification. The protective effects of alpha-tocopherol on copper-catalyzed LDL peroxidative modification were examined by measurement of the concentration of lipid hydroperoxides in LDL and by the provision of LDL cholesterol to lymphocytes via the LDL receptor-mediated pathway. The measurement of concentration of lipid hydroperoxides in LDL showed that alpha-tocopherol inhibited the peroxidative modification of LDL. Also, alpha-tocopherol preserved the ability of LDL to be recognized by LDL receptors in peripheral blood lymphocytes to the same extent as native LDL. These findings indicate that alpha-tocopherol may protect LDL against peroxidative modification, maintaining its ability to act as a ligand for LDL receptors in vivo.

Endothelial cells prevent accumulation of lipid hydroperoxides in low-density lipoprotein.

A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35 +/- 0.08 versus 0.23 +/- 0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8 +/- 4.4 versus 0.49 +/- 0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu2+ (0 to 4 mumol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu2+ exceeded 0.75 mumol/L. At 1.5 and 4 mumol/L Cu2+, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu(2+)-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (< 22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs (approximately equal to 33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.

Free radical-mediated oxidation of lipids induced by hemoglobin in aqueous dispersions

In order to elucidate the effects of hemoglobin on the oxidative damage, the oxidations of soybean phosphatidylcholine (PC) liposomes and low density lipoprotein (LDL) in aqueous dispersions induced by ruptured erythrocytes (hemolysate) and methemoglobin were studied. The rate of oxidation of soybean PC liposomes by hemolysate was much faster than that induced by the same concentration of ferric sufate or cupric chloride and increased with the increase in the amount of hemolysate. When soybean PC was treated beforehand with ebselen, which decomposes hydroperoxides but can not scavenge free radicals, the oxidation did not proceed. Furthermore, the oxidation was suppressed by the addition of pentamethylchromanol in liposomal membrane. The reaction of hemoglobin with methyl linoleate hydroperoxide in aqueous dispersions was also studied. It was found that hemolysate and methemoglobin decomposed the hydroperoxides much faster than cupric chloride and ferric sulfate. The electron spin resonance study strongly suggested that both hemolysate and methemoglobin decomposed hydroperoxides rapidly to form alkoxyl and peroxyl radicals. It was also found out by spectrophotometrical study and by using carbon monoxide and potassium cyanide that hemolysate was oxidized by organic hydroperoxide to form methemoglobin and ferryhemoglobin. The rate of oxidation of LDL induced by hemolysate was much slower than that of soybean PC lyposomes, while LDL was oxidized fater than soybean PC liposomes by cupric chloride under the same conditions. However, in the presence of tert-butyl hydroperoxide, the oxidation of LDL induced by hemolysate proceeded rapidly. These results were interpreted by both the chemical reactivity and the accessibility of metal catalyst to the lipid hydroperoxides in LDL and liposomes. It was suggested that hemoglobin released by hemorrhage might play a crucial role in the initiation of oxidative damage in vivo.

Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation.

Oxidative modification of low density lipoprotein (LDL) has been suggested to play a casual role in human atherosclerosis, and prevention of LDL oxidation may be an effective strategy to prevent or slow the progression of this disease. It is important, therefore, to identify the factors that determine LDL's susceptibility to oxidation. We have analyzed 62 human LDL samples for content of antioxidants, preformed lipid hydroperoxides, and cholesterol. To investigate their oxidative susceptibility, the LDL samples were exposed to either a metal ion-dependent (Cu2+) or -independent (aqueous peroxyl radicals) oxidizing system; the length of the lag phase of inhibited lipid peroxidation was measured, as well as the rate of lipid peroxidation during the lag and ensuing propagation phases. The susceptibility of LDL to metal ion-dependent oxidation was not related to its susceptibility to metal ion-independent oxidation. A strong predictor of an increased susceptibility of LDL to metal ion-dependent oxidation was a decreased vitamin E-to-cholesterol ratio, in contrast to the vitamin E-to-protein ratio. Elevated levels of performed lipid hydroperoxides in LDL and an increased cholesterol content were also associated with an increased susceptibility of the lipoprotein to Cu(2+)-induced oxidation. Remarkably, a strong predictor of an increased susceptibility of LDL to metal ion-independent oxidation was an increased, rather than decreased, vitamin E content relative to protein. An increased cholesterol content also was associated with an increased oxidative susceptibility of LDL to aqueous peroxyl radicals, while preformed lipid hydroperoxides showed no significant correlation. Ubiquinol-10, beta-carotene, and lycopene, whether quantitated relative to cholesterol or protein, did not show significant protective effects against both metal ion-dependent and -independent oxidation of LDL. Our data suggest that a high lipid content of LDL, relative to its protein content, renders the lipoprotein more susceptible to oxidative modification, while vitamin E may have either a protective or promoting effect on LDL oxidation, depending on the oxidative stress conditions. Other known antioxidants in LDL do not appear to play a significant role in protecting LDL against oxidative modification.

Inhibition of LDL oxidation by antioxidants.

Low density lipoprotein (LDL) consists of about 3000 fatty acids (50% polyunsaturated) and a single molecule apolipoprotein B (500 kDa). The endogenous antioxidants of LDL consist mainly of tocopherols and few carotenoids, which protect the PUFAS against oxidation. That native LDL contains traces of oxidation products has not been proved yet. Oxidatively modified LDL (oLDL) exhibits cytotoxic and chemotactic activities, furthermore it leads to foam cell formation, a critical step in atherogenesis. The oxidation of LDL is a free radical process and leads to various aldehydic products. The oxidation of LDL is initiated by cells as well as by transition metals like Cu2+. In both cases the oxidation goes through three consecutive phases. The lag-phase is characterized by minimal degradation of PUFAs but a loss of the antioxidants. Thereafter the PUFAs are oxidized to lipid hydroperoxides, which are only intermediates (propagation-phase). These intermediates will decompose to aldehydic products, accompanied by several additional changes in the LDL particle (decomposition-phase). For increased macrophage uptake oLDL must reach the late decomposition-phase; the presence of lipid hydroperoxides in LDL is not sufficient. It is suggested that binding of aldehydes to free amino groups of Apo B is the reason for macrophage uptake. This is supported by the finding that antibodies against aldehyde-modified LDL are able to recognize oxidized LDL in atherosclerotic lesions. Antioxidants like alpha-tocopherol are able to protect LDL against oxidation. The duration of the lag-phase shows a linear relationship with the content of alpha-tocopherol in LDL. Yet the efficiency of alpha-tocopherol to protect LDL shows strong individual variation.