Aqueous Peroxyl Radicals Research Articles

Vitamin E in human low-density lipoprotein. When and how this antioxidant becomes a pro-oxidant.

Uptake of oxidatively modified low-density lipoprotein (LDL) by cells in the arterial wall is believed to be an important early event in the development of atherosclerosis. Because vitamin E is the major antioxidant present in human lipoproteins, it has received much attention as a suppressor of LDL lipid oxidation and as an epidemiological marker for ischaemic heart disease. However, a careful examination of lipid peroxidation in LDL induced by a steady flux of aqueous peroxyl radicals has demonstrated that, following consumption of endogenous ubiquinol-10, the rate of peroxidation (i) declines as vitamin E is consumed, (ii) is faster in the presence of vitamin E than following its complete consumption, (iii) is substantially accelerated by enrichment of the vitamin in LDL, either in vitro or by diet, and (iv) is virtually independent of the applied radical flux. We propose that perodixation is propagated within lipoprotein particles by reaction of the vitamin E radical (i.e. alpha-tocopheroxyl radical) with polyunsaturated fatty acid moieties in the lipid. This lipid peroxidation mechanism, which can readily be rationalized by the known chemistry of the alpha-tocopheroxyl radical and by the radical-isolating properties of fine emulsions such as LDL, explains how reagents which reduce the alpha-tocopheroxyl radical (i.e. vitamin C and ubiquinol-10) strongly inhibit lipid peroxidation in vitamin E-containing LDL.

High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors.

Analysis of untreated fresh blood plasma from healthy, fasting donors revealed that high density lipoprotein (HDL) particles carry most (approximately 85%) of the detectable oxidized core lipoprotein lipids. Low density lipoprotein (LDL) lipids are relatively peroxide-free. In vitro the mild oxidation of gel-filtered plasma from fasting donors with a low, steady flux of aqueous peroxyl radicals initially caused preferential oxidation of HDL rather than LDL lipids until most ubiquinol-10 present in LDL was consumed. Thereafter, LDL core lipids were oxidized more rapidly. Isolated lipoproteins behaved similarly. Preferential accumulation of lipid hydroperoxides in HDL reflects the lack of antioxidants in most HDL particles compared to LDL, which contained 8-12 alpha-tocopherol and 0.5-1.0 ubiquinol-10 molecules per particle. Cholesteryl ester hydroperoxides (CEOOHs) in HDL and LDL were stable when added to fresh plasma at 37 degrees C for up to 20 hr. Transfer of CEOOHs from HDL to LDL was too slow to have influenced the in vitro plasma oxidation data. Incubation of mildly oxidized LDL and HDL with cultured hepatocytes afforded a linear removal of CEOOHs from LDL (40% loss over 1 hr), whereas a fast-then-slow biphasic removal was observed for HDL. Our data show that HDL is the principal vehicle for circulating plasma lipid hydroperoxides and suggest that HDL lipids may be more rapidly oxidized than those in LDL in vivo. The rapid hepatic clearance of CEOOHs in HDL could imply a possible beneficial role of HDL by attenuating the build-up of oxidized lipids in LDL.

Antioxidant activity of xanthophylls on peroxyl radical-mediated phospholipid peroxidation

The ability of xanthophylls (canthaxanthin, zeaxanthin, and astaxanthin) as chain-breaking antioxidants was investigated in peroxyl radical-mediated peroxidation of phosphatidylcholine (PC) liposomes under atmospheric conditions using lipid-soluble and water-soluble radical generators. These xanthophylls retarded the chain propagation reaction of phosphatidylcholine hydroperoxides (PC-OOH) formation, although their activities to trap chain-carrying peroxyl radical were much less than that of alpha-tocopherol. In chick plasma studies, it was observed that endogenious xanthophylls participated in the antioxidant defenses against the attack of aqueous peroxyl radical. It was concluded that xanthophylls possess the ability to act as chain-breaking antioxidants in the peroxidation of membraneous phospholipids. Dietary xanthophylls may, therefore, be helpful in resisting membraneous phospholipids against oxidative damage in vivo.

Dietary supplementation with coenzyme Q 10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation

Ubiquinol-10 (CoQH 2, the reduced form of coenzyme Q 10) is a potent antioxidant present in human low-density lipoprotein (LDL). Supplementation of humans with ubiquinone-10 (CoQ, the oxidized coenzyme) increased the totaentrations of CoQH 2 in plasma acid and in all of its lipoproteins. Intake of a single oral dose of 100 or 200 mg CoQ increased the total plasma coenzyme content by 80 or 150%, respectively, within 6 h. Long-term supplementation (three times 100 mg CoQ/day) resulted in 4-fold enrichment of CoQH 2 in plasma and LDL with the latter containing 2.8 CoQH 2 molecules per LDL particle (on day 11). Approx. 80% of the coenzyme was present as CoQH 2 and the CoQH 2/CoQ ratio was unaffected by supplementation, indicating that the redox state of coenzyme Q 10 is tightly controlled in the blood. Oxidation of LDL containing various [CoQH 2] by a mild, steady flux of aqueous peroxyl radicals resulted immediately in very slow formation of lipid hydroperoxides. However, in each case the rate of lipid oxidation increased markedly with the disappearance of 80–90% CoQH 2. Moreover, the cumulative radical dose required to reach this ‘break point’ in lipid oxidation was proportional to the amount of CoQH 2 incorporated in vivo into the LDL. Thus, oral supplementation with CoQ increases CoQH 2 in the plasma and all lipoproteins thereby increasing the resistance of LDL to radical oxidation.

Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does alpha-tocopherol.

The temporal disappearance of natural antioxidants associated with human low density lipoprotein (LDL) in relation to the appearance of various classes of lipid hydroperoxides was investigated under three types of oxidizing conditions. Freshly isolated LDL from plasma of healthy subjects was free of detectable amounts of lipid hydroperoxides as measured by HPLC postcolumn chemiluminescence detection. Exposure of such LDL to a mild, constant flux of aqueous peroxyl radicals led to rapid and complete oxidation of ubiquinol-10, followed by slower partial depletion of lycopene, beta-carotene, and alpha-tocopherol. After an initial lag period of complete inhibition of detectable lipid peroxidation, formation of hydroperoxides of cholesterol esters, triglycerides, and phospholipids was observed. The onset of detectable lipid peroxidation corresponded closely with the completion of ubiquinol-10 consumption. However, small amounts of ascorbate, present as a contaminant in the LDL preparation, rather than ubiquinol-10 itself were responsible for the initial lag period. Thus, complete consumption of ubiquinol-10 was preceded by that of ascorbate, and exposure of ascorbate-free LDL to aqueous peroxyl radicals resulted in immediate formation of detectable amounts of lipid hydroperoxides. The rate of radical-mediated formation of lipid hydroperoxides in ascorbate-free LDL was low as long as ubiquinol-10 was present, but increased rapidly after its consumption, even though more than 80% and 95% of endogenous carotenoids and alpha-tocopherol, respectively, were still present. Qualitatively similar results were obtained when peroxyl radicals were generated within LDL or when the lipoprotein was exposed to oxidants produced by activated human polymorphonuclear leukocytes. LDL oxidation was reduced significantly by supplementing the lipoprotein preparation with physiological amounts of either ascorbate or ubiquinol-10. Our data show that ubiquinol-10 is much more efficient in inhibiting LDL oxidation than either lycopene, beta-carotene, or alpha-tocopherol.

Ascorbate is an outstanding antioxidant in human blood plasma.

We have shown recently that the temporal order of antioxidant consumption in human blood plasma exposed to a constant flux of aqueous peroxyl radicals is ascorbate = protein thiols greater than bilirubin greater than urate greater than alpha-tocopherol and that detectable lipid peroxidation starts only after ascorbate has been consumed completely. In this paper, we show that it is indeed ascorbate that completely protects plasma lipids against detectable peroxidative damage induced by aqueous peroxyl radicals and that ascorbate is the only plasma antioxidant that can do so. Plasma devoid of ascorbate, but no other endogenous antioxidant, is extremely vulnerable to oxidant stress and susceptible to peroxidative damage to lipids. The plasma proteins' thiols, although they become oxidized immediately upon exposure to aqueous peroxyl radicals, are inefficient radical scavengers and appear to be consumed mainly by autoxidation. Our data demonstrate that ascorbate is the most effective aqueous-phase antioxidant in human blood plasma and suggest that in humans ascorbate is a physiological antioxidant of major importance for protection against diseases and degenerative processes caused by oxidant stress.

Antioxidant defenses and lipid peroxidation in human blood plasma.

The temporal disappearance in human blood plasma of endogenous antioxidants in relation to the appearance of various classes of lipid hydroperoxides measured by HPLC postcolumn chemiluminescence detection has been investigated under two types of oxidizing conditions. Exposure of plasma to aqueous peroxyl radicals generated at a constant rate leads immediately to oxidation of endogenous ascorbate and sulfhydryl groups, followed by sequential depletion of bilirubin, urate, and alpha-tocopherol. Stimulating polymorphonuclear leukocytes in plasma initiates very rapid oxidation of ascorbate, followed by partial depletion of urate. Once ascorbate is consumed completely, micromolar concentrations of hydroperoxides of plasma phospholipids, triglycerides, and cholesterol esters appear simultaneously, even though sulfhydryl groups, bilirubin, urate, and alpha-tocopherol are still present at high concentrations. Nonesterified fatty acids, the only lipid class in plasma not transported in lipoproteins but bound to albumin, are preserved from peroxidative damage even after complete oxidation of ascorbate, most likely due to site-specific antioxidant protection by albumin-bound bilirubin and possibly by albumin itself. Thus, in plasma ascorbate and, in a site-specific manner, bilirubin appear to be much more effective in protecting lipids from peroxidative damage by aqueous oxidants than all the other endogenous antioxidants. Hydroperoxides of linoleic acid, phosphatidylcholine, and cholesterol added to plasma in the absence of added reducing substrates are degraded, in contrast to hydroperoxides of trilinolein and cholesterol linoleate. These findings indicate the presence of a selective peroxidase activity operative under physiological conditions. Our data suggest that in states of leukocyte activation and other types of acute or chronic oxidative stress such a simple regimen as controlled ascorbate supplementation could prove helpful in preventing formation of lipid hydroperoxides, some of which cannot be detoxified by endogenous plasma activities and thus might cause damage to critical targets.

The antioxidant role of vitamin C

Abstract Ascorbate has been demonstrated to be an effective antioxidant. It can act both directly, by reaction with aqueous peroxyl radicals, and indirectly, by restoring the antioxidant properties of fat-soluble vitamin E. The overall consequence of these antioxidant activities is the beneficial control of lipid peroxidation of cellular membranes including those surrounding as well as within intracellular organelles. Intracellular free radical attack on non-lipid nuclear material may also be diminished. In addition to reviewing the chemical basis of the antioxidant function of vitamin C, this report will focus on the importance of vitamin C as an antioxidant component of plasma as well as the extracellular fluids surrounding the lung, lens and retina. The protection by vitamin C of phagocytic cells involved in the defense against pathogen invasion will also be discussed. This review presents evidence which supports the importance of vitamin C as a component of the overall antioxidant protective mechanisms found in cells and tissues. The data are consistent and form a strong consensus for investigating the importance of the antioxidant function of vitamin C in the maintenance of human health.