Preformed Lipid Hydroperoxides Research Articles

Biphasic Effect of Curcuminoids on Oxidation of Postprandial Chylomicrons.

Chylomicrons are large, triglyceride-rich lipoproteins that transport dietary lipids and lipophilic micronutrients through the lymphatic system and into the venous circulation. They therefore represent a valuable means of investigating the bioavailability of a range of bioactive molecules. Curcuminoids have a complex reputation, with most in vitro studies suggesting a beneficial effect (e.g., as an antioxidant), while in vivo work is often, at best, equivocal. We have prepared a curcuminoid extract from fresh turmeric and investigated the transfer of curcuminoids to chylomicrons by rapid ultracentrifugation. We subsequently characterized the chylomicrons in terms of their susceptibility to oxidation. There was a dose-dependent relationship between the curcuminoid level in chylomicrons and decreased levels of preformed lipid hydroperoxides (P < .05), which was broadly mirrored by paraoxonase arylesterase activity. At lower dosages (1000-3000 mg), curcuminoids protected chylomicrons against copper(II)-mediated oxidation, but at higher levels (4000-5000 mg), a pro-oxidant effect was observed. In conclusion, we report a clear biphasic (hormetic) effect of curcuminoids on lipid oxidation, which supports the theory that low dosages of bioactive compounds can have beneficial effects, while higher dosages may have an equivocal or negative impact on the disease.

The state of systemic oxidative stress during Parkinson’s disease

The development of oxidative stress is considered as one of the key molecular mechanisms that lead to the death of dopamine-producing neurons in Parkinson’s disease (PD). Here, we characterized the oxidative status (OS) of patients depending on the severity of the disease, the time course of the disease, and treatment with antiparkinsonian drugs. The OS was evaluated using Fe2+-induced chemiluminescence of serum lipoproteins (LPs) on the basis of informative parameters, including preformed lipid hydroperoxides, the maximum intensity of lipid peroxidation (LPO) and LP resistance to oxidation, due to the activity of the endogenous antioxidant system. In all patients, regardless of the disease stage (second, third, or fourth), we observed a comparable unidirectional increase in the level of lipid hydroperoxides and maximum intensity of (LPO). However, the activity of the endogenous antioxidant system decreased proportionally to the disease severity: second stage > third stage > fourth stage. In patients at the second and third stages of the disease who were treated with antiparkinsonian drugs we found a disruption of the OS in comparison with untreated patients of the same stage. In general, the deterioration of the OS in patients at the second and third stages of the disease, which included an increase in the level of lipid hydroperoxides and the maximum intensity of LPO, was detected in both patients receiving levodopa at a daily dose of more than 300 mg and in patients treated with drugs that did not contain levodopa. The decrease in the activity of the endogenous antioxidant system in these patients did not depend on the method of treatment and the dose of levodopa. The duration of treatment (up to 5 years) did not influence the OS of patients at the second and third stages of the disease. These data confirm the concept of the significance of oxidative stress in the pathogenesis of PD and indicate the advisability of using antioxidant drugs in complex therapy of PD.

Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varying histidine content and hemin affinity.

The compound 4-hydroxy-2-nonenal (HNE) dissolved in water was examined to remove potential effects of using ethanol to solubilize the aldehyde such as altering protein structure or redox properties of myoglobin (Mb). HNE became covalently bound to sperm whale Mb at up to five sites based on ESI-MS analysis. Adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Alkylation of P88H/Q152HMb with HNE accelerated metMb formation and subsequent lipid oxidation. P88H/Q152HMb exposed to HNE enhanced lipid oxidation compared to wild-type Mb exposed to HNE. Results using H97A Mb suggested that the combination of HNE and low hemin affinity facilitated rapid decomposition of preformed lipid hydroperoxides to secondary lipid oxidation products. HNE and HHE (4-hydroxy-2-hexenal) facilitated Mb-mediated lipid oxidation similarly. The potential mechanisms by which Mb binding of α,β-unsaturated aldehydes affect Mb oxidation and the onset of lipid oxidation are discussed.

Mechanism for the enhanced peroxidation of linoleic acid by a titanium dioxide/hypochlorite system

Nanosized titanium dioxide (TiO2) is a common component of sunscreen preparations and cosmetics as it reflects UV and visible light in accordance to Rayleigh’s law. However, in aqueous environments, TiO2 is an efficient photocatalyst, producing superoxide (O2-) and hydroxyl (HO) radicals, which are highly damaging to biomolecules. We investigated the role of TiO2 in promoting the peroxidation of linoleic acid (LA) alone and in the presence of hypochlorous acid (HOCl). TiO2 significantly enhanced peroxidation of LA, which was further enhanced in the presence of HOCl. This latter finding involved the formation of singlet molecular oxygen in a Russell-type mechanism appearing to involve preformed lipid hydroperoxides (LOOH). In addition to lipid peroxidation, HOCl also mediated formation of 18:1 monochlorohydrins, which in the presence of TiO2 appeared to decompose to kinetic products which supplemented peroxidation of linoleic acid. We present a theoretical mechanism which fits the available experimental data and may partially explain the dichotomy associated with HOCls role in lipid modification.

Redox reactions of myoglobin.

Failure to maintain myoglobin (Mb) in the reduced state causes the formation of metMb, ferryl Mb species, and cross-linked Mb. Dissociation of ferriprotoporphyrin IX from the globin and release of iron atoms can also occur as oxidized Mb accumulates. These modifications may contribute to various oxidative pathologies in muscle and muscle foods. The mechanism of ferryl Mb-mediated oxidative damage to nearby structures has been partially elucidated. Dissociation of ferriprotoporphyrin IX from metMb occurs more readily at acidic pH values. The dissociated ferriprotoporphyrin IX (also called hemin) readily decomposes preformed lipid hydroperoxides to reactive oxygen species. Heme oxygenase as well as lipophilic free radicals can degrade the protoporphyrin IX moiety, which results in the formation of free iron. The multiple pathways by which Mb can incur toxicity create difficulties in determining the major cause of oxidative damage in a particular system. Peroxides and low pH activate each of the oxidative Mb forms, ferriprotoporphyrin IX, and released iron. Determining the relative concentration of these species is technically difficult, but essential to a complete understanding of oxidative pathology in muscle tissue. Improved methods to assess the different pathways of Mb toxicity are needed. Although significant advances have been made in the understanding of Mb interactions with other biomolecules, further investigation is needed to understand the physical and chemical nature of these interactions.

In vitro inhibition of linoleic acid peroxidation by primary S-nitrosothiols

Nitric oxide (•NO) is an effective chain-breaking antioxidant in the inhibition of lipid peroxidation and circulates in vivo mainly as primary S-nitrosothiols (RSNOs). In this work, the in vitro peroxidation of linoleic acid-SDS comicelles (LA-SDS) catalyzed by soybean lipoxygenase (SLO) and FeII ions was monitored in the presence and absence of three primary RSNOs: S-nitrosocysteine, S-nitroso-N-acetylcysteyne and S-nitrosoglutathione. Kinetic measurements based on the formation of conjugated double bonds and fluorescent oxidized LA-lysine adducts, showed that RSNOs are more potent antioxidants than their corresponding free thiols (RSHs) in equimolar conditions. These results are consistent with the blocking of LA-SDS peroxidation by RSNOs through the inactivation of peroxyl/alkoxyl (LOO•/LO•) radicals, leading to nitrogen-containing products of oxidized LA, which release free •NO. These results indicate that endogenous RSNOs may play a major role in the blocking of lipid peroxidation in vivo, through the primary inactivation of alkoxyl/peroxyl radicals and also of preformed lipid hydroperoxides.

Efficiency of Hemoglobin from Rainbow Trout, Cod, and Herring in Promotion of Hydroperoxide-Derived Free Radicals

Hemoglobin (Hb) from rainbow trout (Oncorhynchus mykiss), Atlantic cod (Gadus morhua), and herring (Clupea harengus) were evaluated in terms of capacity to generate hydroperoxide-derived free radicals in aqueous solution at pH 6.8. Cumene hydroperoxide (CumOOH) was used as a model for preformed lipid hydroperoxides, and free radicals were monitored by stabilizing with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and further detection by electron spin resonance spectroscopy. The generation of hydroperoxide-derived free radicals was found to be Hb concentration-dependent, and intermediate Hb concentrations corresponding to hydroperoxide/Hb molar ratios of roughly 200 were found most active. The results indicated the following relative activity in the formation of hydroperoxide-derived free radicals under low and medium hydroperoxide levels (hydroperoxide/Hb molar ratios < or =5): cod Hb approximately herring Hb > trout Hb. This free radical generating-activity was not affected by heating fish hemoglobins at 70 degrees C for 10 and 45 min in aqueous solution. Cod and herring Hb also exhibited faster autoxidation rates and stronger redox instability in the presence of hydroperoxides compared to trout Hb. According to their ability to generate hydroperoxide-derived free radicals, hemoglobin from cod and herring showed more activity than trout Hb as catalysts of lipid oxidation in lecithin liposomes. These data emphasize the central contribution of the free radicals formed through hydroperoxide-decomposition on the hemoglobin-mediated lipid oxidation.

Heme-Mediated Production of Free Radicals via Preformed Lipid Hydroperoxide Fragmentation

Electron spin resonance (ESR) spectroscopy and the spin-trapping technique were used to investigate the capacity of several hemoglobin (Hb) forms of rainbow trout (oxyHb and metHb), free hemin (oxidized form of heme group), and hemin complexed with bovine serum albumin (BSA) to promote formation of free radicals via fragmentation of preformed lipid hydroperoxides. Cumene hydroperoxide (CumOOH) was used as a model for lipid hydroperoxide, and free radicals were monitored by stabilizing with the spin traps alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Two different types of free radicals, hydroxyl and carbon-centered radicals, were identified as a result of the interaction of the heme-containing systems and CumOOH. Carbon-centered radicals were found to be mainly heme-mediated because the addition of the iron-chelating agent EDTA did not affect the formation of POBN/carbon-centered adducts. Hemin alone was the best promoter for the production of POBN/carbon-centered radicals in the presence of low hydroperoxide concentrations (below equimolar condition over heme group), whereas hemin/BSA and oxyHb were more active in generating radicals at high hydroperoxide concentrations or after successive interactions with hydroperoxides. This finding can be explained by the coexistence of two different facts: (i) the interaction between hemin and lipid hydroperoxides seems to be more efficient in the case of free hemin compared to heme-protein complexes and (ii) a faster degradation of hemin is produced without the presence of a protein fraction, globin or albumin. The comparison of oxyHb and metHb also suggested that both Hb redox states have similar capacities to generate oxidative stress via cleavage of preformed lipid hydroperoxides.

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.

EPR spin-trapping evidence for the direct, one-electron reduction of tert-butylhydroperoxide to the tert-butoxyl radical by copper(II): paradigm for a previously overlooked reaction in the initiation of lipid peroxidation.

Lipid peroxidation is often initiated using Cu(II) ions. It is widely assumed that Cu(II) oxidizes preformed lipid hydroperoxides to peroxyl radicals, which propagate oxidation of the parent fatty acid via hydrogen atom abstraction. However, the oxidation of alkyl hydroperoxides by Cu(II) is thermodynamically unfavorable. An alternative means by which Cu(II) ions could initiate lipid peroxidation is by their one-electron reduction of lipid hydroperoxides to alkoxyl radicals, which would be accompanied by the generation of Cu(III). We have investigated by EPR spectroscopy, in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the reactions of various Cu(II) chelates with tert-butylhydroperoxide. Spectra contained signals from the tert-butoxyl, methyl, and methoxyl radical adducts. In many previous studies, the signal from the methoxyl adduct has been assigned incorrectly to the tert-butylperoxyl adduct, which is now known to be unstable, releasing the tert-butoxyl radical upon decomposition. This either is trapped by 5,5-dimethyl-1-pyrroline N-oxide or undergoes beta-scission to the methyl radical, which either is trapped or reacts with molecular oxygen to give, ultimately, the methoxyl radical adduct. By using metal chelates that are known to be specific in either their oxidation or reduction of tert-butylhydroperoxide (the Cu(II) complex of bathocuproine disulfonic acid and the Fe(II) complex of diethylenetriaminepentaacetic acid, respectively) for comparison, we have been able to deduce, from the relative concentrations of the three radical adducts, that the Cu(II) complexes tested each reduce tert-butylhydroperoxide directly to the tert-butoxyl radical. These findings suggest that a previously overlooked reaction, namely the direct reduction of preformed lipid hydroperoxides to alkoxyl radicals by Cu(II), may be responsible for the initiation of lipid peroxidation by Cu(II) ions.

Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate

In crude synaptosomal fractions from rat brain exposed to iron and ascorbate, enhanced lipid peroxidation (more than 3-fold compared to control), loss of protein thiols up to the extent of 40% compared to control, increased incorporation of carbonyl groups into proteins (more than 4.5-fold compared to control) and non-disulphide covalent cross-linking of membrane proteins have been observed. The phenomena are not inhibited by catalase or hydroxyl radical scavengers like mannitol or dimethyl sulphoxide. However, chain breaking antioxidants like α-tocopherol and butylated hydroxytoluene prevent both lipid peroxidation and accompanying protein oxidation. It is suggested that in this system lipid peroxidation propagated by the decomposition of preformed lipid hydroperoxides by iron and ascorbate is the primary event and products of the peroxidation process cause secondary protein damage. In view of high ascorbate content of brain and availability of several transition metals, such ascorbate mediated oxidative damage may be relevant in the aetiopathogenesis of several neurodegenerative disorders as well as ageing of brain.

The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids.

Myeloperoxidase (MPO), an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases including atherosclerosis. The major oxidant produced by MPO, hypochlorous acid (HOCl), is able to modify a great variety of biomolecules by chlorination and/or oxidation. In this paper the reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with either reagent HOCl or HOCl generated by the MPO-hydrogen peroxide-chloride system are reviewed. One of the major issues has been whether the reaction of HOCl with lipids of low density lipoprotein (LDL) yields predominantly chlorohydrins or lipid hydroperoxides. Electrospray mass spectrometry provided direct evidence that chlorohydrins rather than peroxides are the major products of HOCl- or MPO-treated LDL phosphatidylcholines. Nevertheless lipid peroxidation is a possible alternative reaction of HOCl with polyunsaturated fatty acids if an additional radical source such as pre-formed lipid hydroperoxides is available. In phospholipids carrying a primary amino group such as phosphatidylethanolamine chloramines are the preferred products compared to chlorohydrins. Cholesterol can be converted by HOCl to great variety of oxysterols besides three isomers of chlorohydrins. For the situation in vivo it appears that the type of reaction occurring between HOCl and lipids would very much depend on the circumstances, e.g. the pH and the presence of radical initiators. The biological effects of lipid chlorohydrins are not yet well understood. It has been shown that chlorohydrins of both unsaturated fatty acids as well as of cholesterol may cause lysis of target cells, possibly by disruption of membrane structures.

Effect of pH on lipid oxidation using trout hemolysate as a catalyst: a possible role for deoxyhemoglobin.

Hemoglobin-mediated lipid oxidation was studied by adding hemolysate to washed cod muscle. Three pH values were examined (pH 7.6, 7.2, and 6.0). The lag time prior to rancidity and thiobarbituric acid reactive substance development decreased greatly as the pH was reduced (p < 0.01). Formation of methemoglobin due to autoxidation of the heme pigment was found to occur more rapidly at reduced pH. Also, the level of deoxyhemoglobin was found to sharply increase with pH reduction in the range of pH 7.6-6.0. This suggested a potential role for deoxyhemoglobin as a catalyst. ATP lowered hemoglobin oxygenation at pH 7.2. Peroxidation of linoleic acid by oxy/deoxyhemoglobin and methemoglobin was investigated at two levels of preformed lipid hydroperoxides. At a reduced level of preformed lipid hydroperoxides, oxy/deoxyhemoglobin stimulated peroxidation of linoleic acid, whereas methemoglobin did not. At the higher level of preformed lipid hydroperoxides, both oxy/deoxyhemoglobin and methemoglobin were active. This investigation suggests that reduced hemoglobins played an important role in lipid oxidation processes.

Troglitazone protects human erythrocytes from oxidant damage.

The antidiabetic drug troglitazone contains the active chromanol ring of alpha-tocopherol, which should give it antioxidant properties within cells. In these studies, the antioxidant effects of troglitazone were tested in human erythrocytes and in their ghosts. Troglitazone bound to erythrocyte ghosts in a linear manner and was retained even after centrifugation washes. In response to an oxidant stress generated by a water-soluble free radical initiator, troglitazone that was bound to erythrocyte ghosts was oxidized, but induced a lag-phase in the disappearance of endogenous alpha-tocopherol and in the appearance of lipid hydroperoxides. Troglitazone also delayed loss of endogenous alpha-tocopherol and hemolysis in washed intact erythrocytes in response to free radical-induced extracellular oxidant stress. To mimic exposure of erythrocytes to lipid hydroperoxides in vivo, erythrocytes were incubated with phospholipid liposomes that contained small amounts of preformed lipid hydroperoxides. This induced an oxidant stress in both the liposomes and cells. Troglitazone in concentrations above 4 microM almost completely prevented further appearance of lipid hydroperoxides in the liposomes, and also completely preserved alpha-tocopherol in the erythrocytes. The present results suggest that troglitazone will help to prevent peroxidative damage to erythrocytes in areas of excessive oxidant stress in the vascular bed.

Lipid Oxidation in Human Low-Density Lipoprotein Induced by Metmyoglobin/H2O2: Involvement of α-Tocopheroxyl and Phosphatidylcholine Alkoxyl Radicals

Metmyoglobin (metMb) and H(2)O(2) can oxidize low-density lipoprotein (LDL) in vitro, and oxidized LDL may be atherogenic. The role of alpha-tocopherol (alpha-TOH) in LDL oxidation by peroxidases such as metMb is unclear. Herein, we show that during metMb/H(2)O(2)-induced oxidation of native LDL, alpha-tocopheroxyl radical (alpha-TO(*)) and hydroperoxides and alcohols of cholesteryl esters [CE-O(O)H] and phosphatidylcholine [PC-O(O)H] accumulate concomitantly with alpha-TOH consumption. The ratio of accumulating CE-O(O)H to PC-O(O)H remains constant as long as alpha-TOH is present. Accumulation of CE-O(O)H is dependent on, and correlates with, LDL's alpha-TOH content, yet does not require preformed lipid hydroperoxides or H(2)O(2). This indicates that in native LDL alpha-TOH can act as a phase-transfer agent and alpha-TO(*) as a chain-transfer agent propagating LDL lipid peroxidation via tocopherol-mediated peroxidation (TMP). After alpha-TOH depletion, CE-O(O)H continues to accumulate, albeit at a slower rate than in the presence of alpha-TOH. This second phase of LDL oxidation is accompanied by depletion of PC-OOH, a rapid increase in the CE-O(O)H/PC-O(O)H ratio, formation of lipid-derived alkoxyl radicals and phosphatidylcholine hydroxides (PC-OH), and accumulation of a second organic radical, characterized by a broad singlet EPR signal. The latter persists for several hours at 37 degrees C. We conclude that metMb/H(2)O(2)-induced peroxidation of LDL lipids occurs initially via TMP. After alpha-TOH depletion, cholesteryl esters peroxidize at higher fractional rates than surface phospholipids, and this appears to be mediated at least in part via reactions involving alkoxyl radicals derived from the peroxidatic activity of metMb on PC-OOH.

Antioxidant defenses in rat intestine and mesenteric lymph

Dietary oxysterols can reach the circulation and this may contribute to atherosclerosis, where lipid oxidation is thought to be important. There is also evidence that, in rats,peroxidized lipids are absorbed and transported into lymph [Aw TY, Williams MW, Gray L. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am J Physiol 1992; 262: G99–G106], although the method used to detect lipid peroxides lacked specificity. We tested whether intragastric administration of vegetable oils containing triglyceride hydroperoxides (TG-OOH) to rats resulted in detectable lipid hydroperoxides in mesenteric lymph. Using sensitive HPLC with postcolumn chemiluminescence detection, we were unable to detect hydroperoxides of triglycerides, cholesterylesters or phospholipids during the course of lipid absorption, and lymph levels of ascorbate, urate, α-tocopherol and ubiquinol-9 did not change significantly. By contrast, we observed a striking reducing activity judged by the efficient reduction of administered ubiquinones-9 and -10 to the corresponding ubiquinols. Exposure of rat lymph and isolated chylomicrons to aqueous peroxyl radicals revealed patterns of antioxidant consumption and lipid hydroperoxide formation similar to those described previously for human extravascular fluids and isolated lipoproteins, respectively. In particular, rates of TG-OOH formation in lymph and chylomicrons were very low to undetectable as long as ascorbate and/or ubiquinols were present, but subsequently proceeded in a chain reaction despite the presence of α-tocopherol. These studies demonstrate that rat intestine and mesenteric lymph possess efficient antioxidant defenses against preformed lipid hydroperoxides and (peroxyl) radical mediated lipid oxidation. We conclude that dietary lipid hydroperoxides or postprandial oxidation of lipids are not likely to contribute to these particular forms of oxidized lipids in circulation and aortic tissue.

A Microassay to Assess the Oxidative Resistance of Low-Density Lipoproteins

Oxidative modification of LDL is implicated in the pathogenesis of atherosclerosis (1)(2). Susceptibility of LDL to oxidative modification is suggested to be an independent risk factor for coronary atherosclerosis, and recent epidemiological studies revealed protective effects of antioxidants on development and progression of atherosclerosis (3)(4)(5). We have previously demonstrated that hemin readily intercalates into LDL particles and rapidly oxidizes LDL in vitro (6). Hemin-catalyzed oxidation of LDL can be accelerated by activated inflammatory cells, small amounts of hydrogen peroxide, or preformed lipid hydroperoxides within the LDL. That such hemin-induced oxidative modification of LDL may be involved in atherogenesis is supported by the finding that hemin-sensitive genes in endothelium (7)(8) are up-regulated in atherosclerotic lesions (9). The aim of the present study was to establish a clinical laboratory microassay, based on the time kinetics of hemin-catalyzed lipid peroxidation of LDL, for assessing LDL resistance to oxidative modification, and to determine the optimal conditions and reproducibility of the assay. Plasma LDL was isolated from 1 g/L Na2EDTA-anticoagulated venous blood after a 2000 g centrifugation for 20 min at 4 °C, and the density of plasma was adjusted to 1210 g/L with KBr. After a two-layer gradient was made in a 5.1-mL Quick-Seal polyallomer tube (Beckman Instruments) by layering normal saline containing 100 mg/L of Na2EDTA on 1.5 mL of density-adjusted plasma, a single spin gradient ultracentrifugation at 228 000 g at 4 °C for 90 min (VTi 65.2 rotor, Beckman Instruments) was performed to isolate LDL. The LDL fraction proved pure …

Troglitazone Reduces LDL Oxidation and Lowers Plasma E-selectin Concentration in NIDDM Patients

Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule E-selectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 +/- 0.10 nmol/l (vs. 5.32 +/- 0.10 nmol/l in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 +/- 0.03 to 1.19 +/- 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 +/- 2.33 to 43.7 +/- 1.77 microg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.

Antioxidant activity of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) is of great interest for epidemiological and clinical diagnostic reasons, as well as for basic scientific research, because it is thought to precede the development of atherosclerotic lesions (). Measurement of individual antioxidants such as α-tocopherol, is, of course, a primary route to the study of LDL oxidation. However, such measurements are not always possible and do not take into account the potential contribution to the antioxidant activity of LDL from a wide variety of different antioxidants whose exact nature may not be known. Oxidized LDL can itself be quantitatively measured () as well as LDL oxidizability (, , ). However, the measurement of LDL oxidizability involves a prolonged and variable incubation period and the results obtained from the studies previously quoted are, in a number of respects, contradictory. LDL oxidizability measurements clearly reflect a number of factors other than the antioxidant content of LDL (for example, the presence of pre-formed lipid hydroperoxides and the ratio of saturated to unsaturated fatty acids) (), which may, of course, be a desirable feature because oxidized LDL itself is the end product of a multi-factorial process.

α-Tocopherol as a Reductant for Cu(II) in Human Lipoproteins: TRIGGERING ROLE IN THE INITIATION OF LIPOPROTEIN OXIDATION

Initiation of lipid peroxidation by Cu(II) requires reduction of Cu(II) to Cu(I) as a first step. It is unclear, however, whether this reaction occurs in the course of lipoprotein oxidation. It is also unknown which reductant, if any, can drive the reduction of Cu(II) in this case. We found that Cu(II) was rapidly reduced to Cu(I) by all major human lipoproteins (high, low, and very low density lipoproteins (HDL, LDL, and VLDL), and chylomicrons). Cu(II)-reducing activity was associated with a lipid moiety of the lipoproteins. The rates of Cu(II) reduction by different lipoproteins were similar when the lipoproteins were adjusted to similar alpha-tocopherol concentrations. Enriching lipoproteins with alpha-tocopherol considerably increased the rate of CU(II) reduction. CU(II) reduction by alpha-tocopherol-deficient LDL isolated from a patient with familial inherited vitamin E deficiency was found to occur much slower in comparison with LDL isolated from a donor with a normal plasma level of alpha-tocopherol. Initial rate of CU(II) reduction by alpha-tocopherol-deficient LDL was found to be zero. Enriching LDL with ubiquinol-10 to concentrations close to those of alpha-tocopherol did not influence the reaction rate. When LDL was treated with ebselen to eliminate preformed lipid hydroperoxides, the reaction rate was also not changed significantly. CU(II) reduction was accompanied by a consumption of lipoprotein alpha-tocopherol and accumulation of conjugated dienes in the samples. Increasing alpha-tocopherol content in lipoproteins slightly decreased the rate of conjugated diene accumulation in LDL and HDL and considerably increased it in VLDL. The results suggest that alpha-tocopherol plays a triggering role in the lipoprotein oxidation by CU(II), providing its initial step as follows: alpha TocH + CU(II) --> alpha Toc. + Cu(I) + H+. This reaction appears to diminish or totally eliminate the antioxidative activity of alpha-tocopherol in the course of lipoprotein oxidation.