Free radical-lipid interactions and their pathological consequences.

In this review we have tried to present the current thinking on the consequences for lipids of their interactions with free radicals and the pathological implications. In particular, atherosclerosis and cancer have been addressed. In the case of the former, it is not clear whether the initial oxidative event is an enzymic or free radical-mediated process as yet. However, the importance of the antioxidants in controlling LDL oxidation, macrophage uptake of oxidatively modified LDL and progression of atheroma in animal models certainly suggests an important propagative role for free radical-mediated events. With regard to cancer, oxidative modification of cell lipids has potential consequences for tumour cell proliferation. Whilst lipid hydroperoxides can serve as an origin of prostaglandins with tumour inhibitor (or immunosuppressive) properties, they may also influence cellular growth regulatory proteins normally dependent on membrane lipid integrity. Alternatively, they may function as a source of aldehydic breakdown products capable of 'down-regulating' cell proliferation through covalent modification of regulatory proteins. Oils rich in n-3 polyunsaturated fatty acids have toxic effects towards tumour cells. This toxicity is not mediated by prostaglandins but rather through the capacity of such agents to elevate the levels of lipid peroxides. This may be enhanced by active oxygen species released constitutively from tumour cells.

Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention?

alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells. (b) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all of the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused an increase in the level of alpha-T that was proportional to the level of inclusion of alpha-T in the medium. (c) Growth was stimulated by 7 and 14 mM alpha-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (d) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of alpha-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced.

t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress.

Erythrocytes were incubated with t-butyl hydroperoxide in the presence and absence of hemoglobin as a model system for oxidative stress and the alterations in the structure and integrity of the membranes were investigated. The results showed that in the presence of hemoglobin a significant modification in the membrane surface charge was induced but no such alteration was observed in peroxidized hemoglobin-free membranes. As increased hemoglobin oxidation occurred in the erythrocytes, membrane lipid peroxidation diminished, suggesting a protective role for methemoglobin in t-butyl hydroperoxide-induced lipid peroxidation. Electrophoresis on polyacrylamide gels showed modification of the cytoplasmic protein region but no high molecular weight aggregates formed at the concentrations of the hydroperoxide used in this work. The results suggest that the t-butyl hydroperoxide/normal erythrocyte system seems to be an instructive model for membrane perturbations characteristic of oxidative disorders.

Effects of N-methyl hexanoylhydroxamic acid (NMHH) and myoglobin on endothelial damage by hydrogen peroxide.

OBJECTIVE: The aim was to investigate the interaction of the novel antioxidant N-methyl hexanoylhydroxamic acid (NMHH) with myoglobin in protecting endothelial cells against H2O2 mediated damage. METHODS: Cultured bovine aortic endothelial cells were exposed to 50-100 microM H2O2 for 10-60 min with and without NMHH and/or myoglobin, and immediate or delayed damage was assessed by lactate dehydrogenase release, 3H adenine uptake, a tetrazolium reduction assay, and microscopy. RESULTS: Brief exposure to low concentrations of H2O2 caused cell damage, for which the tetrazolium reduction assay was the most sensitive assay, and inhibited subsequent cell division. NMHH in concentrations from 50 to 200 microM protected against damage provided it was present at the time of adding H2O2, and the effect was markedly potentiated by 10 microM oxymyoglobin, which had little protective effect alone. CONCLUSIONS: NMHH is an effective antioxidant which is markedly potentiated by low concentrations of oxymyoglobin. Oxymyoglobin may potentiate NMHH by scavenging H2O2 through the rapid formation of ferrylmyoglobin, which is then reduced by NMHH. This synergism may be particularly relevant to the protection of myoglobin-rich cells such as myocytes.

Novel monohydroxamate drugs attenuate myocardial reperfusion-induced arrhythmias.

The novel monohydroxamates N-methyl hexanoylhydroxamic acid, N-methyl acetohydroxamic acid, and N-methyl butyrohydroxamic acid have antioxidant and iron chelating properties. They attenuated reperfusion-induced contractile dysfunction following long periods of ischaemia (50 min) in the isolated rat heart. Here we compare their effects and that of the trihydroxamate desferrioxamine on reperfusion-induced arrhythmias following short duration ischaemia (10 min). Isolated rat hearts were perfused by the Langendorff method, subjected to regional ischaemia and reperfusion. Arrhythmias induced during the first 5 min of reperfusion were quantified. Drugs (all at 150 microM) were introduced during the last 2 min of ischaemia and remained throughout reperfusion. Although the monohydroxamate- and desferrioxamine-treated hearts showed a reduction in the incidence of ventricular tachycardia and fibrillation, only the reduction in the incidence of sustained fibrillation ( > 3 min duration) in N-methyl acetohydroxamic acid--(27%), N-methyl hexanoylhydroxamic acid--(27%) and desferrioxamine-treated hearts (20%) was statistically significant (p < 0.05 vs control 73%; n = 15). There was a reduction in the severity of the arrhythmias, manifest as a significant increase in the duration of sinus rhythm in all the monohydroxamate-treated hearts, and a significant reduction (vs control 218 +/- 29 s; mean +/- SEM) in the duration of ventricular fibrillation in hearts treated with N-methyl acetohydroxamic acid (101 +/- 31 s) and desferrioxamine (112 +/- 30 s). This improvement was offset by an increase in the duration of ventricular tachycardia, in hearts treated with N-methyl acetohydroxamic acid, N-methyl butyrohydroxamic acid and desferrioxamine. These results suggest that these novel monohydroxamates, particularly N-methyl acetohydroxamic acid, attenuate reperfusion-induced arrhythmias in this model when introduced during the ischaemic period.

Ruptured erythrocytes inhibit the oxidation of membranes by 15-hydroperoxy-eicosatetraenoic acid.

In this study, the pro-oxidant effects of the hydroperoxide, 15-hydroperoxy-eicosatetraenoic acid (15-HPETE), on erythrocyte membranes and the modulation of the oxidation by haem proteins released from ruptured erythrocytes have been assessed. The results indicate that ruptured erythrocytes may act as an antioxidant in protecting membranes against oxidative stress induced by hydroperoxides and that it is the oxyhaemoglobin that is the active constituent of the protective mechanism. An important feature of the mechanism is the peroxidatic action of oxyhaemoglobin and its rate of reaction with 15-HPETE.

The identification of flavonoids as glycosides in human plasma.

This study describes evidence for the absorption of flavonoids and their presence in human plasma in the glycosylated form by HPLC analysis with photodiode array detection. Rutin and other quercetin glycosides, phloridzin, as well as an anthocyanin are detected simultaneously. In addition, a compound eluting with the spectral properties of the aurone family is identified. The results reveal that phloretin and quercetin are absorbed from the diet as glycosides. The polyphenols are detected in plasma from non-supplemented humans at individual levels in the range 0.5-1.6 microM.

Peroxynitrite oxidises catechols to o-quinones.

Nitration of phenolic compounds is a well-established mechanism on interaction with peroxynitrite. However, while nitration is the predominant reaction for monophenolic hydroxycinnamates, this does not take place with the catechol-containing hydroxycinnamate, caffeic acid. The aim of the present study was to investigate the mechanism of the chemical interaction of caffeic acid with peroxynitrite and to characterise the products formed. A novel compound was detected and characterised as the o-quinone of caffeic acid based on its reaction with nucleophilic thiol compounds, glutathione and L-cysteine. The same novel product was identified following the oxidation of caffeic acid in alkaline solutions confirming the identity of this species as a caffeic acid oxidation product.

Desferrioxamine as a lipid chain-breaking antioxidant in sickle erythrocyte membranes.

The mechanism of action of desferrioxamine in the inhibition of the catalysis of iron-induced oxidative damage has been ascribed to its ability to chelate available ferric ion (Kb = 10(31)). However, recent work has proposed that the trihydroxamate moiety of desferrioxamine can also be involved in electron transfer reactions involving the superoxide radical, peroxidase/hydrogen peroxide mixtures and ferryl myoglobin radicals. In this study we report evidence for the ability of desferrioxamine to inhibit peroxidative damage to pathological membranes with which non-haem iron is associated through a mechanism of action as a lipid chain breaking antioxidant, independently of its iron chelating properties.

Current status of antioxidant therapy.

There is evidence that free radical damage contributes to the aetiology of many chronic health problems such as emphysema, cardiovascular and inflammatory diseases, cataracts, and cancer. In this review we are not concerned with tissue damage in vivo induced directly by radicals from exogenous sources, such as air pollutants and tobacco smoke, high-pressure oxygen, irradiation, or through the metabolism of certain solvents, drugs, and pesticides. Rather, we address some of the disease states associated with increased oxidative stress from endogenous sources and the possible therapeutic advantage of the antioxidant treatment. This raises the question of the antioxidant status of individuals and its role in protection against amplification of certain disease processes. We have chosen to concentrate mainly on coronary heart disease, reperfusion injury, and organ storage for transplantation.

Modulation of the UVA activation of haem oxygenase, collagenase and cyclooxygenase gene expression by epigallocatechin in human skin cells.

We have investigated the modifying effects of epigallocatechin, a major polyphenolic constituent of green tea, on ultraviolet-A-activated gene expression in human fibroblasts and keratinocytes using the stress responsive enzymes: haem oxygenase-1, interstitial collagenase and cyclooxygenase-2. Although epigallocatechin strongly reduced ultraviolet-A-induced haem oxygenase-1 activation in skin-derived 'fibroblasts, the same compound activated collagenase and cyclooxygenase expression. In a keratinocyte cell line, ultraviolet-A-mediated haem oxygenase-1 over-expression was low and epigallocatechin failed to modulate it further. In contrast to the results with fibroblasts, ultraviolet-A activation of cyclooxygenase in keratinocytes was reduced by epigallocatechin. The results indicate that the effect of this green tea polyphenol on cellular stress responses is complex and may involve direct effects on signal transduction as well as changes that may be associated with its antioxidant activity.

Oxidised low density lipoproteins induce iron release from activated myoglobin.

Recent reports have detected the presence of iron in human atherosclerotic lesions [Biochem. J. 286 (1992) 901-905]. This study provides evidence for a biochemical mechanism whereby iron is released from myoglobin by low density lipoprotein (LDL) which has become oxidised by the ferryl myoglobin species. The haem destabilisation and iron release are inhibited by monohydroxamate compounds and desferrioxamine through their ability to inhibit the propagation of LDL oxidation. Thus, iron may derive from the myoglobin released from ruptured cells in the oxidising environment of the atherosclerotic lesion.

The interaction between ruptured erythrocytes and low-density lipoproteins.

Low-density lipoproteins (LDL) are oxidatively modified on interaction with haem proteins. The interaction of ruptured erythrocytes with LDL induces oxidative damage as detected by alterations in electrophoretic mobility and the peroxidation of the polyunsaturated fatty acyl chains. Difference spectroscopy reveals that the amplification of the oxidative process by the haem protein is related to the transition of the oxidation state of the haemoglobin in the erythrocyte lysate from the oxy [X-FeII-O2] to the ferryl [X-FeIV = O] form. The incorporation of the lipid-soluble antioxidant, butylated hydroxy toluene, at specific time points during the LDL-erythrocyte interaction prolongs the lag phase to oxidation and eliminates the oxy-to-ferryl conversion of the haemoglobin. The timescale of this haem conversion is related to the antioxidant status of the LDL.

The effects of pH on the oxidation of low-density lipoprotein by copper and metmyoglobin are different.

The amplification of low-density lipoprotein (LDL) peroxidation in vitro by copper and myoglobin are well-studied biochemical approaches for investigating the oxidative modification of LDL and its role in the pathogenesis of atherosclerosis. Since the acidity of the environment is increased in inflammatory sites, the aim of this study was to investigate the effects of acidic pH on the oxidisability of LDL mediated by the haem protein myoglobin in comparison with that of copper-mediated LDL oxidation. The results show that acidic pH enhances myoglobin-mediated LDL oxidation as measured by conjugated dienes, lipid hydroperoxides and electrophoretic mobility, whilst a retardation is observed with copper as pro-oxidant; the mechanism probably relates to the effects of pH on the decomposition and formation of lipid hydroperoxides and the relative influences of copper ions and of myoglobin under these conditions.

Interaction of peroxynitrite with carotenoids and tocopherols within low density lipoprotein.

Peroxynitrite is a powerful oxidising and nitrating agent generated in vivo by the combination of nitric oxide and superoxide. Previous studies have shown that on exposure to peroxynitrite, low density lipoprotein (LDL) is modified resulting in both a time- and concentration-dependent change to lipid and protein components. The present investigation highlights the reaction between carotenoids and tocopherols, present within the lipophilic phase of LDL, and peroxynitrite at varying concentrations. It was observed that the carotenoids were consumed by a significantly greater proportion than that of the tocopherols with lycopene (87.2 +/- 11%) being more reactive than beta-carotene (68.2 +/- 5.8%) when exposed to peroxynitrite (50 microM) for 1 min. Among the tocopherols, alpha-tocopherol (54.9 +/- 20.2%) was more extensively depleted than gamma-tocopherol (14.7 +/- 1.09%) at peroxynitrite concentration of 500 microM. It was also observed that peroxynitrite, unlike copper ions, does not lead to significant peroxidation of LDL as determined by the formation of conjugated dienes and thiobarbituric acid-reactive substances.

The antioxidant properties of theaflavins and their gallate esters--radical scavengers or metal chelators?

The antioxidant properties of theaflavins and their gallate esters were studied by investigating their abilities to scavenge free radicals in the aqueous and lipophilic phases. The total relative antioxidant activities in the aqueous phase were assessed by measuring their direct ABTS.+ radical scavenging abilities, and by their efficacies in inhibiting the degradation of deoxyribose induced by iron. The propensities for enhancing the resistance of LDL to oxidation mediated by Cu2+ were also measured. The results show that the hierarchy of reactivity of these compounds as antioxidants is: theaflavin digallate > 3'-monogallate = 3-monogallate > theaflavin. Spectroscopic studies show that all the compounds chelate iron and copper; enhanced absorbance in the visible region is observed in the case of the iron-digallate complex, but not with copper.

Structure-antioxidant activity relationships of flavonoids and phenolic acids.

The recent explosion of interest in the bioactivity of the flavonoids of higher plants is due, at least in part, to the potential health benefits of these polyphenolic components of major dietary constituents. This review article discusses the biological properties of the flavonoids and focuses on the relationship between their antioxidant activity, as hydrogen donating free radical scavengers, and their chemical structures. This culminates in a proposed hierarchy of antioxidant activity in the aqueous phase. The cumulative findings concerning structure-antioxidant activity relationships in the lipophilic phase derive from studies on fatty acids, liposomes, and low-density lipoproteins; the factors underlying the influence of the different classes of polyphenols in enhancing their resistance to oxidation are discussed and support the contention that the partition coefficients of the flavonoids as well as their rates of reaction with the relevant radicals define the antioxidant activities in the lipophilic phase.

Urinary excretion of hydroxycinnamates and flavonoids after oral and intravenous administration.

The urinary recoveries of the hydroxycinnamates, ferulic acid (3-methoxy, 4-hydroxy cinnamic acid), and chlorogenic acid (the quinic acid ester of 3,4-dihydroxycinnamic acid), and three structurally related flavonoids were studied in the rat. For the latter, the aglycone quercetin was compared with its 3-glucoside (isoquercitrin) and 3-rhamnoglucoside (rutin). Doses of 50 mg/kg were administered via the oral and intravenous routes and urine collected over the subsequent 24-h period. Reverse phase HPLC with photo-diode array detection was used to analyze the unchanged compound and their metabolites excreted in the urine. Ferulic acid and isoquercitrin were orally absorbed (5.4 and 0.48% of administered dose, respectively) and are therefore bioavailable. In contrast, neither unchanged chlorogenic acid, rutin, quercetin, nor the conjugated metabolites in the form of glucuronide or sulphate were detected in the urine after oral dosing. All the flavonoids studied produced low total urinary recoveries after intravenous administration, 9.2% for quercetin-3-rhamnoglucoside, 6.7% for the 3-glucoside, and 2.4% for the aglycone, indicating that extensive metabolism to low molecular weight compounds or excretion via other routes may be occurring. Overall it can be stated that renal excretion is not a major pathway of elimination for intact flavonoids and hydroxycinnamates in the rat.

Bilirubin and ascorbate antioxidant activity in neonatal plasma.

Extremely low birth weight premature infants have been known for many years to have limited antioxidant protective capacity, especially with reference to those antioxidant components which do not cross the placenta until the third trimester of gestation. In this study the total antioxidant activity and the concentrations of individual antioxidants in plasma from premature neonates (27 +/- 2 weeks gestation) compared to term babies (38-41 weeks gestation) have been examined. The results show elevated levels of ascorbate at birth in the plasma of premature neonates compared with those of term babies, but the total plasma antioxidant status of the premature babies is significantly lower than that of term babies. At 5 days post-partum the ascorbate levels are within the normal adult range and plasma bilirubin levels are considerably enhanced in both groups, while the total plasma antioxidant status of the premature neonates has increased. Analysis of the relationship between the total plasma antioxidant activity and the bilirubin concentration show a direct, highly significant correlation for the term group, r2 = 0.774, consistent with significance of bilirubin as a plasma antioxidant.

Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein.

Oxidative stress is implicated in neuronal loss associated with neurodegeneration such as in Parkinson's disease, Alzheimer's disease and age-related cognitive decline. Recent reports indicate that the consumption of flavonoid-rich fruits partly reverses the age-related neuronal and cognitive decline. In this study, cultured striatal neurons were exposed to oxidized lipids in the form of low-density lipoprotein (oxLDL) as a model for the induction of oxidative injury, and the abilities of phenolic antioxidants, flavonoids and hydroxycinnamic acid derivatives, to attenuate this neuronal damage were examined. OxLDL was demonstrated to enter neuronal cells and to be capable of eliciting neurotoxicity in a dose- and time-dependent manner, inducing DNA fragmentation and cell lysis. Flavonoids exert protective effects, which appear to be related to specific structural characteristics, particularly relevant being those defining their reduction potentials and partition coefficients. In summary, these data suggest a possible role for flavonoids in reducing neurodegeneration associated with chronic disorders in which oxidative stress is implicated.