Cholesteryl Ester Hydroperoxides Research Articles

Inhibition of Mammalian 15-Lipoxygenase-Dependent Lipid Peroxidation in Low-Density Lipoprotein by Quercetin and Quercetin Monoglucosides

Lipoxygenase is suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation in the subendothelial space of the arterial wall. Since flavonoids such as quercetin are recognized as lipoxygenase inhibitors and they occur mainly in the glycoside form, we assessed the effect of quercetin and its glycosides (quercetin 3-O-β-glucopyranoside, Q3G; quercetin 4′-O-β-glucopyranoside, Q4′G; quercetin 7-O-β-glucopyranoside, Q7G) on rabbit reticulocyte 15-lipoxygenase (15-Lox)-induced human LDL lipid peroxidation and compared it with the inhibition obtained by ascorbic acid and α-tocopherol, the main water-soluble and lipid-soluble antioxidants in blood plasma, respectively. Quercetin inhibited the formation of cholesteryl ester hydroperoxides (CE-OOH) and endogenous α-tocopherol consumption effectively throughout the incubation period of 6 h. Ascorbic acid exhibited an effective inhibition only in the initial stage and LDL preloaded with fivefold α-tocopherol did not affect the formation of CE-OOH compared with the native LDL. CE-OOH formation was inhibited by both quercetin and quercetin monoglucosides in a concentration-dependent manner. Quercetin, Q3G, and Q7G exhibited a higher inhibitory effect than Q4′G (IC50: 0.3–0.5 μM for quercetin, Q3G, and Q7G and 1.2 μM for Q4′G). While endogenous α-tocopherol was completely depleted after 2 h of LDL oxidation, quercetin, Q7G, and Q3G prevented the consumption of α-tocopherol. Quercetin and its monoglucosides were also exhausted during the LDL oxidation. These results indicate that quercetin glycosides as well as its aglycone are capable of inhibiting lipoxygenase-induced LDL oxidation more efficiently than ascorbic acid and α-tocopherol.

Plasma LDL oxidation leads to its aggregation in the atherosclerotic apolipoprotein E-deficient mice.

Two major modifications of low density lipoprotein (LDL) that can lead to macrophage cholesterol accumulation and foam cell formation include its oxidation and aggregation. To find out whether these modifications can already occur in vivo in plasma and whether they are related to each other, the oxidation and aggregation states of plasma LDL were analyzed in the apolipoprotein E-deficient (E degree) transgenic mice during their aging (and the development of atherosclerosis), in comparison to plasma LDL from control mice. Plasma LDL from the E degree mice was already minimally oxidized at 1 month of age in comparison to control mice LDL, and it further oxidized with age in the E degree mice but not in the control mice. At 6 months of age, the contents of the E degree mice LDL-associated cholesteryl ester hydroperoxides, thiobarbituric acid reactive substances, and conjugated dienes were higher by two, three, and twofold, respectively, in comparison to LDL from the young, 1-month-old E degree mice. We also investigated the LDL aggregation state in E degree mice. In the young E degree mice, LDL oxidation was shown in comparison to control mice, but in both groups of young mice their LDL was not aggregated. In the E degree mice, however, the LDL aggregation state substantially increased with age, by as much as 125% at 6 months of age compared to the 1-month-old mice, whereas no significant aggregation could be detected in plasma LDL from control mice at the same age. To question the possible effect of LDL oxidation on its subsequent aggregation, LDL oxidation was induced by either copper ions, or by the free radical generator 2,2-azobis-2-amidinopropane hydrochloride, or by hypochlorite. All these oxidative systems led to LDL oxidation (to different degrees) and resulted in a similar, substantial LDL aggregation. These oxidation systems also enhanced the susceptibility of LDL to aggregation (induced by vortexing) by 23%, 28%, or 40%, respectively. To further analyze the relationships between the lipoprotein oxidation and its aggregation, LDL (0.1 mg of protein/mL) was incubated with 5 mumol/L CuSO4 at 37 degrees C in the absence or presence of the antioxidant, vitamin E (25 mumol/L). In the absence of vitamin E, a time-dependent increment in LDL oxidation was noted, which reached a plateau after 2 hours of incubation. LDL aggregation, however, only started at this time point and reached a plateau after only 5 hours of incubation. In the presence of vitamin E, both LDL oxidation and its aggregation were reduced at all time points studied. We extended the vitamin E study to the in vivo situation, and the effect of vitamin E supplementation to the E degree mice (50 mg.kg-1.d-1 for a 3-month period) on their plasma LDL oxidation and aggregation states was studied. Vitamin E supplementation to these mice resulted in a 35% reduction in the LDL oxidation state and in parallel, the LDL aggregation state was also reduced by 23%. These reductions in LDL oxidation and aggregation states were accompanied by a 33% reduction in the aortic lesion area, in comparison to nontreated E degree mice. We conclude that in E degree mice, LDL oxidation, which already took place in the plasma, can lead to the lipoprotein aggregation. These modified forms of LDL were shown to be taken up by macrophages at an enhanced rate, leading to foam cell formation. Thus, the use of an appropriate antioxidant can inhibit the formation of both atherogenic forms of LDL.

Antioxidant Activity of Vitamin C in Iron-overloaded Human Plasma

Vitamin C (ascorbic acid, AA) can act as an antioxidant or a pro-oxidant in vitro, depending on the absence or the presence, respectively, of redox-active metal ions. Some adults with iron-overload and some premature infants have potentially redox-active, bleomycin-detectable iron (BDI) in their plasma. Thus, it has been hypothesized that the combination of AA and BDI causes oxidative damage in vivo. We found that plasma of preterm infants contains high levels of AA and F2-isoprostanes, stable lipid peroxidation end products. However, F2-isoprostane levels were not different between those infants with BDI (138 +/- 51 pg/ml, n = 19) and those without (126 +/- 41 pg/ml, n = 10), and the same was true for protein carbonyls, a marker of protein oxidation (0.77 +/- 0.31 and 0.68 +/- 0.13 nmol/mg protein, respectively). Incubation of BDI-containing plasma from preterm infants did not result in detectable lipid hydroperoxide formation (</=10 nM cholesteryl ester hydroperoxides) as long as AA concentrations remained high. Furthermore, when excess iron was added to adult plasma, BDI became detectable, and endogenous AA was rapidly oxidized. Despite this apparent interaction between excess iron and endogenous AA, there was no detectable lipid peroxidation as long as AA was present at >10% of its initial concentration. Finally, when iron was added to plasma devoid of AA, lipid hydroperoxides were formed immediately, whereas endogenous and exogenous AA delayed the onset of iron-induced lipid peroxidation in a dose-dependent manner. These findings demonstrate that in iron-overloaded plasma, AA acts an antioxidant toward lipids. Furthermore, our data do not support the hypothesis that the combination of high plasma concentrations of AA and BDI, or BDI alone, causes oxidative damage to lipids and proteins in vivo.

Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection.

A method was developed for the simultaneous determination of phosphatidylcholine hydroperoxides (PCOOH) and cholesteryl ester hydroperoxides (CEOOH). Lipid hydroperoxides (LOOH) were quantitatively extracted from human plasma with a mixture of n-hexane and ethyl acetate, and separated by column-switching high-performance liquid chromatography using one aminopropyl column and two octyl columns followed by chemiluminescence detection. LOOHs could be completely separated from each other and detected at picomole levels. The results of method validation tests were satisfactory. This method was then applied to determine LOOH in normal human plasma; the levels of PCOOH and CEOOH found were 36.0+/-4.0 nM (mean+/-S.D., n=6) and 12.3+/-3.1 nM (mean+/-S.D., n=6), respectively.

Antioxidant protection of LDL by physiological concentrations of 17 beta-estradiol. Requirement for estradiol modification.

Exposure to estrogens reduces the risk for coronary artery disease and associated clinical events; however, the mechanisms responsible for these observations are not clear. Supraphysiological levels of estrogens act as antioxidants in vitro, limiting oxidation of low-density lipoprotein (LDL), an event implicated in atherogenesis. We investigated the conditions under which physiological concentrations of 17 beta-estradiol (E2) inhibit oxidative modification of LDL. Plasma incubated with E2 (0.1 to 100 nmol/L) for 4 hours yielded LDL that demonstrated a dose-related increase in resistance to oxidation by Cu2+ as measured by conjugated diene formation. This effect was dependent on plasma, because incubation of isolated LDL with E2 at these concentrations in buffered saline produced no effect on Cu(2+)-mediated oxidation. Incubation of plasma with E2 had no effect on LDL alpha-tocopherol content or cholesteryl ester hydroperoxide formation during the 4-hour incubation. Plasma incubation with [3H]E2 was associated with dose-dependent association of 3H with LDL. High-performance liquid chromatographic analysis of LDL derived from plasma incubated with [3H]E2 indicated that the majority of the associated species were not detectable as authentic E2 but as nonpolar forms of E2 that were susceptible to base hydrolysis consistent with fatty acid esterification of E2. Plasma-mediated association of E2 and subsequent antioxidant protection was inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), an inhibitor of plasma acyltransferase activity. Exposure of LDL to physiological levels of E2 in a plasma milieu is associated with enhanced resistance to Cu(2+)-mediated oxidation and incorporation of E2 derivatives into LDL. This antioxidant capacity may be another means by which E2 limits coronary artery disease in women.

Increased Levels Of Plasma Cholesteryl Ester Hydroperoxides In Patients With Subarachnoid Hemorrhage

The pathophysiology of subarachnoid hemorrhage (SAH) may involve free radical production and lipid peroxidation. We examined plasma levels of cholesteryl ester hydroperoxides (CEOOH) and antioxidants in 25 patients with SAH, and 10 neurologic controls with lacunar stroke. Patients with SAH had significantly increased plasma levels of CEOOH, which peaked on day 5 after the ictus. Concentrations of CEOOH were significantly increased, and ascorbic acid concentrations were significantly decreased in patients who developed vasospasm compared with patients without vasospasm. Increased levels of CEOOH were associated with increased mortality and correlated with clinical outcome scales. These results implicate oxidative stress in the pathogenesis of SAH and suggest that measurements of CEOOH in plasma may be useful both prognostically as well as in monitoring therapeutic interventions.

Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells.

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.

Inhibition of oxidation of low density lipoprotein by troglitazone

The effect of a new oral hypoglycemic agent troglitazone, (±)-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl-methoxy)benzyl]-2,4-thiazolidinedione as an antioxidant against the free radical-mediated oxidation of low density lipoprotein (LDL) was studied. The oxidation of LDL gives cholesteryl ester hydroperoxide and phosphatidylcholine hydroperoxide as major primary products. Troglitazone incorporated exogenously into LDL inhibited the oxidations of LDL induced by either aqueous or lipophilic peroxyl radicals and suppressed the formation of lipid hydroperoxides efficiently. Ascorbic acid added into the aqueous phase spared both endogenous α-tocopherol and troglitazone in LDL. It was also found by absorption spectroscopic and electron spin resonance (ESR) studies that troglitazone reacted rapidly with a galvinoxyl radical to give a chromanoxyl radical which gives the same ESR spectrum as α-tocopherol. This ESR spectrum disappeared rapidly when ascorbic acid was added into the system. These results show that troglitazone acts as a potent antioxidant and protects LDL from oxidative modification.

Phospholipase A 2 activity in non-glycated and glycated low density lipoproteins

The oxidation of low density lipoprotein (LDL) is believed to be an important risk factor for atherosclerosis. We have previously reported that glycation of LDL enhances LDL oxidation. Incubation of LDL in the presence of 200 mM glucose resulted in the enhanced formation of phosphatidylcholine hydroperoxide (PC-OOH) and cholesteryl ester hydroperoxide (CE-OOH). Fe 3+-ADP accelerated the formation of hydroperoxides. The concentration of PC-OOH was much smaller than that of CE-OOH. In addition, we found a PC-OOH decomposing activity in LDL by following linoleic acid hydroperoxide (18:2-OOH) formation from 1-stearoyl-2-[13′-( S)-hydroperoxy-9′,11′-octadecadienoyl]- sn-glycero-3-phosphocholine (SLPC-OOH). Hydrolysis was similar between LDL and glycated LDL. Pretreatment by para-bromophenacylbromide, histidine modifier and phospholipase A 2 inhibitor, retarded the formation of 18:2-OOH only by 30%. The addition of equimolar platelet activating factor (PAF) reduced hydrolysis by 50%, indicating PAF acetylhydrolase may be responsible for the hydrolysis of PC-OOH.

Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of α-Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation

There is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D-alpha-tocopherol, alpha-TOH) or dietary supplementation with D-alpha-TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of alpha-TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal-containing Ham's F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.5 +/- 2 molecules of alpha-TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ10H2, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Ham's F-10 medium resulted in a time-dependent loss of alpha-TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of alpha-TOH, and the rate of this oxidation decreased on alpha-TOH depletion. In vitro enrichment of LDL with alpha-TOH resulted in an LDL particle containing sixfold to sevenfold more alpha-TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo alpha-TOH-deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Ham's F-10-initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patient's LDL. Oral supplementation of healthy individuals for 5 days with either alpha-TOH or coenzyme Q increased the LDL levels of alpha-TOH and CoQ10H2 by two to three or three to four times, respectively. alpha-TOH-supplemented LDL was significantly more prone to oxidation, whereas CoQ10H2-enriched LDL was more resistant to oxidation initiation by Ham's F-10 medium than native LDL. Cosupplementation with both alpha-TOH and coenzyme Q resulted in LDL with increased levels of alpha-TOH and CoQ10H2, and such LDL was markedly more resistant to initiation of oxidation than native or alpha-TOH-enriched LDL. These results demonstrate that oral supplementation with alpha-TOH alone results in LDL that is more prone to oxidation initiation, whereas cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.

Reaction of Phosphatidylcholine Hydroperoxide in Human Plasma: The Role of Peroxidase and Lecithin:Cholesterol Acyltransferase

In order to elucidate the reason why phosphatidylcholine hydroperoxide is unstable in human plasma, 1-palmitoyl-2-linoleoylphosphatidylcholine hydroperoxide (PLPC-OOH) was incubated aerobically in human plasma at 37°C, and its decomposition products were measured. The major product was the corresponding alcohol (PLPC-OH) and this reduction probably occurred by an enzymatic process since no acceleration in ascorbate depletion and no significant decrease in other plasma antioxidants were observed upon addition of PLPC-OOH. Cholesteryl linoleate hydroperoxide and its alcohol (Ch18:2-OH) were also detected as minor products. Similarly, 1-stearoyl-2-arachidonoylphosphatidylcholine hydroperoxide gave its alcohol (SAPC-OH) as a major product and cholesteryl arachidonate hydroperoxide and its hydroxide (Ch20:4-OH) as minor products. These oxidized cholesteryl esters are likely to be produced by the action of lecithin:cholesterol acyltransferase (LCAT) present in high-density lipoprotein (HDL) since (a) incubation of PLPC-OH and SAPC-OH in human plasma gave Ch18:2-OH and Ch20:4-OH, respectively, (b) isolated human HDL converted PLPC-OH to Ch18:2-OH and SAPC-OH to Ch20:4-OH, while isolated human low-density lipoprotein was inactive for this conversion, and (c) formation of oxidized cholesteryl esters in plasma and HDL was inhibited by the LCAT inhibitor 5,5′-dithiobis(2-nitrobenzoic acid). A possible beneficial role of LCAT for converting phosphatidylcholine hydroperoxide to cholesteryl ester hydroperoxide is also discussed.

Rapid reduction and removal of HDL- but not LDL-associated cholesteryl ester hydroperoxides by rat liver perfused in situ.

To test whether high-density lipoproteins (HDL) could aid in the removal in vivo of potentially atherogenic oxidized lipids, we perfused rat liver in situ with buffer supplemented with isolated human HDL containing small amounts of cholesteryl linoleate hydro(pero)xides [CH18:2-O(O)H]. Perfusion resulted in the rapid removal of Ch18:2-O(O)H from HDL with a half-life (t1/2)of 11.4 min., faster than that of unoxidized cholesteryl linoleate, and dependent of the presence of the liver. In addition, the liver enhanced the reduction of Ch18:2-OOH associated with HDL remaining in the perfusate buffer. Perfusion resulted in the release of a hepatic activity that enhanced the reduction of HDL-associated CH18:2-OOH and was resistant to heat treatment. In contrast with the situation with HDL, low-density lipoprotein (LDL)-associated CH18:2-O(O)H were neither removed nor reduced by perfused rat liver within the time course studied, in support of a possible role for HDL in the detoxification of circulating lipid hydroperoxides in vivo.

Oxidation of Human Plasma by Water- and Lipid-soluble Radical Initiators.

The formation of phosphatidylcholine hydroperoxide (PC-OOH), phosphatidylcholine hydroxide (PC-OH), cholesteryl ester hydroperoxide (CE-OOH), cholesteryl ester hydroxide (CE-OH), free fatty acid hydroperoxide (FFA-OOH), and free fatty acid hydroxide (FFA-OH), and changes in the levels of plasma antioxidants such as urate, ascorbate, bilirubin, ubiquinol-10, and α-tocopherol were measured during the oxidation of human plasma initiated with water- and lipid-soluble initiators in an aerobic condition at 37°C to assess the oxidation of plasma lipids and its inhibition by antioxidants. Rapid decrease in ascorbate level was induced by a water-soluble initiator and that in ubiquinol-10 and α-tocopherol levels by a lipid-soluble initiator indicate that the former initiator is active in water-phase while the latter in the lipid-phase. In addition to PC-OOH and CE-OOH, considerable amount of PC-OH was formed during plasma oxidation. PC-OH is likely to be produced by the action of peroxidase since the addition of PC-OOH to human plasma gave PC-OH as the major product. Considerable amount of CE-OH was also formed during plasma oxidation. Since CE-OOH was relatively stable in human plasma, CH-OH is likely to be derived from PC-OH by the action of lecithin : cholesterol acyltransferase. Little formation of FFA-OOH and FFA-OH suggests that phospholipase A 2 like activity and platelet-activating factor acetylhydrolase are not significant factors in the decomposition of PC-OOH and PC-DH. The differences between the initiators in plasma oxidation and between the oxidation of high and low density lipoproteins are discussed.

Coulometric detection in high-performance liquid chromatographic analysis of cholesteryl ester hydroperoxides

A highly sensitive and simple method for the determination of cholesteryl ester hydroperoxides (CHE-OOH) was developed using high-performance liquid chromatography (HPLC) with coulometric electrochemical detection. The lowest detectable level by this technique was 2 pmol for cholesteryl linoleate hydroperoxides at the signal-to-noise ratio of 3. This method was applied to the determination of ChE-OOH presumably present in human plasma. Although ChE-OOH could not be detected, the ChE-OOH level in the fluid was estimated to be less than 27 nM. It was found that the extraction efficiency of an internal standard, cholesteryl nervonate, was decreased by lowering its amount spiked to the plasma. The concentration of ChE-OOH in human plasma and plasma lipoprotein, which were peroxidized with a radical initiator in vitro, could be determined by use of this standard. HPLC-coulometric technique is, therefore, useful to measure the peroxidation of plasma lipids in vitro.

Supplementation with Carotenoids Inhibits Singlet Oxygen-Mediated Oxidation of Human Plasma Low-Density Lipoprotein

To study the role of carotenoids in the antioxidant defense against oxidative modification of low-density lipoprotein (LDL) particles, human LDL rich in β-carotene and lycopene was prepared from a healthy volunteer following long-term supplementation with tomato juice. This carotenoid-supplemented LDL accumulated cholesteryl ester hydroperoxides (CE-OOH) more slowly than the LDL prepared before supplementation when the suspensions containing these LDL were subjected to a singlet oxygen-generating system. However, there was no significant difference in the rate of CE-OOH accumulation between the two suspensions when they were exposed to a water-soluble radical generator. Therefore, it is strongly suggested that supplementation of LDL with carotenoids mainly improves the antioxidant defense against the attack of singlet oxygen. Keywords: Carotenoids; singlet oxygen; low-density lipoprotein; atherosclerosis

Oxidative modification and antioxidant protection of human low density lipoprotein at high and low oxygen partial pressures

Oxidative modification of low density lipoprotein (LDL) in the subendothelial space of the arterial wall has been implicated as an initial process in atherosclerosis. In vitro studies of LDL oxidation are usually done at ambient oxygen partial pressure (pO2; approximately 160 torr, or 21% O2), which is considerably higher than arterial tissue pO2 (30-70 torr, and as low as 20 torr, or 2.5% O2, in atherosclerotic lesions). In addition, beta-carotene acts as an efficient free radical scavenger only at low pO2. Therefore, we investigated the effects of high (20%) and low (2%) pO2 on the kinetics of LDL oxidation, and the effectiveness of beta-carotene compared to other physiological antioxidants in preventing LDL oxidation. At low pO2, the rate of Cu(2+)-induced oxidative modification of LDL was lower than at high pO2. Furthermore, at high pO2 there was a distinct lag phase preceding the propagation phase of lipid peroxidation in Cu(2+)-exposed LDL, as measured by cholesteryl ester hydroperoxide formation; in contrast, there appeared to be no distinct lipid peroxidation lag phase in LDL incubated with Cu2+ at low pO2. Elevating alpha-tocopherol levels in LDL about 5-fold resulted in significant antioxidant protection: the lipid peroxidation lag phase at high pO2 increased by 45% (from 58 +/- 11 to 84 +/- 3 min, P < 0.05), and the initial rate (0-1 h) of lipid hydroperoxide formation at low pO2 was reduced by 52% (from 11.6 +/- 1.9 to 5.6 +/- 1.0 nmol/mg LDL protein/h, P < 0.01). In contrast, increasing LDL beta-carotene levels about 6-fold did not inhibit LDL oxidation at either pO2. Most remarkably, low concentrations of ascorbic acid (30 microM) drastically reduced LDL oxidation, regardless of pO2: the lipid peroxidation lag phase at high pO2 increased more than 7-fold (from 46 +/- 11 min to > 360 min, P < 0.001), and at low pO2 no lipid hydroperoxides could be detected for at least 6 h of incubation. These results show that at low physiological pO2, Cu(2+)-induced LDL oxidation occurs at a significantly lower rate than at ambient pO2. At both high and low pO2, beta-carotene cannot inhibit LDL oxidation, whereas alpha-tocopherol has a moderate protective effect, and low physiological concentrations of ascorbic acid very strongly suppress LDL oxidation.

Lipoperoxides in LDL incubated with fibroblasts that overexpress 15-lipoxygenase.

Oxidative modification of LDL plays an important role in early atherogenesis but the mechanisms, nonenzymatic and/or enzymatic, by which LDL is oxidized in vivo remain to be established. Several lines of evidence suggest that cellular 15-lipoxygenase (arachidonate 15-oxidoreductase, EC.1.13.11.13) (15-LO) may contribute to oxidative modification of LDL, including recent studies demonstrating that murine fibroblasts overexpressing 15-LO have an enhanced capacity to oxidize LDL in the medium. The present studies were undertaken to better understand the mechanisms by which cells expressing 15-LO bring about oxidative modification of LDL. LDL incubated 1-2 h with the 15-LO-enriched cells showed a much higher lipoperoxide (LOOH) content than did LDL incubated with control cells. By far the largest absolute increase occurred in cholesteryl ester hydroperoxide (CE-OOH), a much lesser increase in free fatty acid hydroperoxides (FFA-OOH), and only a very small increase in phospholipid hydroperoxides (PL-OOH). Addition of EDTA to the medium abolished these increases in LDL lipid hydroperoxides. Enrichment of LDL with probucol or vitamin E also prevented CE-OOH accumulation. Incubation of LDL with linoleic acid hydroperoxide in the absence of cells also caused a significant increase in CE-OOH and this was markedly inhibited by EDTA. These findings provide further evidence for the potential of 15-LO to participate in LDL oxidation by way of a mechanism involving introduction of LOOH into the LDL particle followed by metal-catalyzed propagation.

HDL, its enzymes and its potential to influence lipid peroxidation

In seeking an explanation of the inverse relationship between serum high density lipoprotein (HDL) concentration and coronary heart disease (CHD) incidence, most investigations have been directed at its role in reverse cholesterol transport. However, recently it has become clear that HDL has the potential to limit oxidative modification of low density lipoprotein (LDL) whether induced by transition metals or by cells in tissue culture. In view of the current theory that oxidative modification of LDL is an important element in atherogenesis, this suggests another potential mechanism by which HDL might impede the development of CHD. HDL is the major carrier of cholesteryl ester hydroperoxides, but more than this it appears to have the prolonged capacity to decrease the total amount of lipid peroxides generated on LDL during oxidation while the quantity accumulating on HDL itself reaches an early plateau. These effects are not explained by chain-breaking antioxidants present in HDL and are likely to involve an enzymic mechanism. Several enzymes are present on HDL: paraoxonase, lecithin:cholesterol acyl transferase, platelet activating factor acetylhydrolase, phospholipase D and protease. Apolipoproteins, such as apolipoprotein AI, could also have enzymic activity. Evidence that some of these might act to metabolise lipid peroxidation products, such as oxidised phospholipids and lyso-phosphatidylcholine, is discussed in this review.

Cholesterylester hydroperoxide reducing activity associated with high density lipoproteins

Cholesterylester hydroperoxide reducing activity associated with high density lipoproteins

Cholesterylester hydroperoxide reducing activity associated with isolated high- and low-density lipoproteins

Exposure of isolated high-(HDL) and low-density lipoproteins (LDL) to aqueous peroxyl radicals generated from a thermo-labile azo-compound resulted in immediate formation of cholesteryllinoleate hydroxide (Ch18:2-OH) in addition to hydroperoxides of cholesteryllinoleate (Ch 18: 2- OOH) and phospholipids. Ch 18: 2- OH was also formed in peroxyl radical-oxidizing human plasma devoid of ascorbate or low molecular weight compounds or isolated lipoproteins in the presence of desferrioxamine. In contrast, peroxyl radical-mediated oxidation of HDL or LDL lipid extracts or detergent-solubilized lipoproteins resulted in the formation of Ch 18:2-OOH without concomitant formation of Ch 18:2-OH. Heat treatment of the isolated lipoproteins prior to oxidation greatly reduced Ch18:2-OH formation. Compared to the concentrations of Ch18:2-OOH accumulating, formation of Ch18:2-OH was more pronounced in oxidizing HDL than LDL isolated from the same blood donor. The levels of Ch18: 2-OH detected after prolonged oxidation periods were independent of the radical flux to which the lipoproteins were exposed. In the absence of peroxyl radical generator, [ 3H]Chl8: 2-OOH associated with HDL was converted readily and in a biphasic manner into [ 3H]Chl8: 2-OH upon incubation at 37 but not 4°C. LDL-associated [ 3H]Ch18: 2-OOH were also reduced, albeit with an initial reaction rate -10 times slower than that observed with labelled HDL. Together, the results show that cholesterylester hydroxides are formed during (peroxyl) radical-mediated oxidation of isolated intact HDL and LDL under transition metal-free conditions. The findings suggest the presence of a hydroperoxide reducing activity in isolated human lipoproteins, particularly HDL.