A unique antioxidant activity of phosphatidylserine on iron-induced lipid peroxidation of phospholipid bilayers

The relationship between the antioxidant effect of acidic phospholipids, phosphatidic acid (PA), phosphatidylglycerol (PG) and phosphatidylserine (PS), on iron-induced lipid peroxidation of phospholipid bilayers and their abilities to bind iron ion was examined in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC LUV). The effect of each acidic phospholipid added to the vesicles at 10 mol% was assessed by measuring phosphatidylcholine hydroperoxides (PC-OOH) and thiobarbituric acid-reactive substances. The addition of dipalmitoyl PS (DPPS) showed a significant inhibitory effect, although the other two acidic phospholipids, dipalmitoyl PA (DPPA) and dipalmitoyl PC (DPPG), did not exert the inhibition. Neither dipalmitoyl PC (DPPC) nor dipalmitoyl phophatidylethanolamine (DPPE) showed any remarkable inhibition on this system. None of the tested phospholipids affected the lipid peroxidation rate remarkably when the vesicles were exposed to a water-soluble radical generator. The iron-binding ability of each phospholipid was estimated on the basis of the amounts of iron recovered in the chloroform/methanol phase after separation of the vesicle solution to water/methanol and chloroform/methanol phases. EYPC LUV containing DPPS, DPPA, and DPPG had higher amounts of bound iron than those containing DPPC and DPPE, indicating that these three acidic phospholipids possess an iron-binding ability at a similar level. Nevertheless, only DPPS suppressed iron-dependent decomposition of PC-OOH significantly. Therefore, it is likely that these three acidic phospholipids possess a significant iron-binding ability, although this ability per se does not warrant them antioxidative activities. The ability to suppress the iron-dependent decomposition of PC-OOH may explain the unique antioxidant activity of PS.     

Effect of n−3 fatty acid supplementation on lipid peroxidation and protein aggregation in rat erythrocyte membranes

Human erythrocytes in the circulation undergo dynamic oxidative damage involving membrane lipid peroxidation and protein aggregation during aging. The present study was undertaken to determine the effect of n−3 fatty acid supplementation on lipid peroxidation and protein aggregation in the circulation and also the in vitro susceptibility of rat erythrocyte membranes to oxidative damage. Wistar male rats were fed a diet containing n−6 fatty acid-rich safflower oil or n−3 fatty acid-rich fish oil with an equal amount of vitamin E for 6 wk. n−3 Fatty acid content in erythrocyte membranes of rats fed fish oil was significantly higher than that of rats fed safflower oil. The degree of membrane lipid peroxidation and protein aggregation of rats fed fish oil was not significantly higher than that of rats fed safflower oil when the amounts of phospholipid hydroper-oxides, thiobarbituric acid-reactive substances, and detergent-insoluble protein aggregates were measured. When isolated erythrocytes were oxidized under aerobic conditions in the presence of Fe(III), the degree of membrane lipid peroxidation of erythrocytes from rats fed fish oil was increased to a greater extent than that of rats fed safflower oil, whereas the degree of membrane protein aggregation of both groups was increased in a similar extent. Hence, n−3 fatty acid supplementation did not affect lipid peroxidation and protein aggregation in membranes of circulating rat erythrocytes, and the supplementation increased the susceptibility of isolated erythrocytes to lipid peroxidation, but not to protein aggregation, under the aerobic conditions. If a sufficient amount of vitamin E is supplied, n−3 fatty acid supplementation may give no undesirable oxidative effects on rat erythrocytes in the circulation.     

Sulfite-induced lipid peroxidation

Sulfite initiated the peroxidation of linoleic acid and linolenic acid emulsions via a free radical mechanism. Peroxidation of these fatty acids required oxygen and sulfite and occurred with concomitant oxidation of sulfite to sulfate. In reaction mixtures containing linoleic acid, the formation of conjugated diene equaled the formation of hydroperoxide. In reaction mixtures containing linolenic acid emulsions, thiobarbituric acid reactive materials were also formed. Peroxidation was pH-dependent; peroxidation of linoleic acid proceeded between pH 4 and 7, but linolenic acid peroxidation was significant only if pH was below pH 6. The linoleic acid hydroperoxides thus formed were reduced and methylated to methyl hydroxystearate. Analysis of methyl hydroxystearate by gas chromatographymass spectrometry indicated that sulfite-induced peroxidation gave rise to the 9- and 13-hydroperoxy isomers. In addition to the hydroperoxides, sulfite adducts were detected. Hydroquinone, butylated hydroxytoluene and α-tocopherol effectively inhibited both sulfite oxidation and hydroperoxide formation. Conjugated diene formation also was inhibited by 4-thiouridine, suggesting that the reaction is mediated by the sulfite radical. No significant inhibition was observed with the addition of superoxide dismutase, catalase, or the hydroxyl radical scavengers, mannitol ort-butanol. A possible mechanism is presented to account for sulfite-induced peroxidation of linoleic acid.     

Microsomal lipid peroxidation: Effect of vitamin E and its functional interaction with phospholipid hydroperoxide glutathione peroxidase

The role of vitamin E in the protection against iron dependent lipid peroxidation was studied in rat liver microsomes and Triton-dispersed microsomal lipid micelles. In these systems, an antioxidant effect of vitamin E at a physiological ratio to phospholipids could be observed only in the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPX) and glutathione. The rationale of this cooperation is discussed on the basis of the hydroperoxyl radical scavenging capacity of vitamin E and the reduction of membrane hydroperoxides by PHGPX. The scavenging of lipid hydroperoxyl radicals by vitamin E, although inhibiting propagation of the peroxidative chain, produces lipid hydroperoxides from which ferrous iron generates alkoxyl radicals that react with vitamin E almost as fast as with fatty acids. Therefore, only if membrane hydroperoxides are continuously reduced by this specific peroxidase does the scavenging of hydroperoxyl radicals by vitamin E lead to an effective inhibition of lipid peroxidation.     

Measurement of lipid peroxidation in vivo: A comparison of different procedures

A study was undertaken to investigate whether some of the methods commonly used to detect lipid peroxidation of cellular membranes in vivo correlate with each other. The study was performed with the livers of bromobenzene-intoxicated mice, in which lipid peroxidation develops when the depletion of glutathione (GSH) reaches a threshold value. The methods tested and compared were the following: i) measurement of the malondialdehyde (MDA) content of the liver; ii) detection of diene conjugation absorption in liver phospholipids; iii) measurement of the loss of polyunsaturated fatty acids in liver phospholipids; and iv) determination of carbonyl functions formed in acyl residues of membrane phospholipids as a result of the peroxidative breakdown of phospholipid fatty acids. Correlations among the values obtained with these methods showed high statistical significances, indicating that the procedures measure lipid peroxidation in vivo with comparable reliability. Analogously, the four methods appeared also to correlate when applied to in vitro microsomal lipid peroxidation.     

Evaluation of a model system for the selective study of the lipid peroxidation process

The effect of molecular environment on the peroxidation of linoleic acid (LA), a polyunsaturated fatty acid (PUFA), initiated by ferrous ions was investigated in acidic and neutral pH conditions. Mixed nonionic surfactants TWEEN®‐20/LA micelles were established as a model system to obtain a surfactant‐in‐lipid aqueous system at high acidity level. The peroxidation of LA was induced by ferrous ions and the kinetics of the produced conjugated dienes was followed by UV measurements and the ferric thiocynate method. Ferrous ions were oxidized only by the preformed LA hydroperoxides, which under established conditions produced lipid alkoxyl and peroxyl radicals as the sole initiators of propagation. The results revealed the LA peroxidation process remained mainly unaffected within the 2.5<pH<5.5 range, while highly pH sensitive around pH 7. The propagation process prevailed at optimal concentrations of 500 µM of LA and 280 µM TWEEN®‐20, and at the ferrous ion concentration up to 75 µM, irrespective of the buffer used. Practical applications: A simple model system in water, suitable for the selective study of the lipid peroxidation propagation phase induced by ferrous ion is presented here. Fatty acids serve as model compounds susceptible to processes associated with oxidative radical initiated‐modifications of lipids. The obtained results contribute to a better understanding of the oxidative behavior of lipids, particularly those soluble in nonionic surfactant micelles in acidic medium. The oxidative stability of the PUFA in model systems containing TWEEN®‐20 and ferrous ion at low pH could be predicted and controlled by measuring the lipid hydroperoxide formation. The experimental conditions presented may also provide a suitable system for the study of the termination phase of lipid peroxidation.     

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: Nonspecificity of thiobarbituric acid-reactivity

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (∼20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/ mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced ∼40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA. TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result. Presented in part at the 78th American Oil Chemists' Society Annual Meeting, New Orleans, LA (May, 1987). Recipient of an Outstanding Paper Presentation Award (1).     

Lability of red blood cell membranes to lipid peroxidation: Application to humans fed polyunsaturated lipids

Red blood cell membranes (RBCM) were used to estimate human red blood cell lability to lipid peroxidationin vitro. RBCM were prepared from blood collected from humans fed diets with either 3 or 15% polyunsaturated fatty acids for 80 days. RBCM were isolated by centrifugation, and oxidative stress was induced byin vitro incubation with 0.1 or 0.5 mM tert-butyl hydroperoxide (t-BOOH) in the presence of 0.5 mg added hemoglobin. Lipid Peroxidation was evaluated by measurement of thiobarbituric acid-reactive substances (TBARS). Lipid peroxidation correlated with the protein content of RBCM in both noninduced and t-BOOH-induced lipid peroxidation systems. TBARS production was dependent on the amount of t-BOOH added to the RBCM. The production of TBARS by RBCM incubated with 0.5 mM t-BOOH was correlated with the arachidonic acid content in the red blood cells (RBC) from which RBCM were prepared. The methodology developed was useful for comparative estimations of the lability of RBCM to lipid peroxidation.     

The effect of beta blocking drugs on lipid peroxidation in rat heartin vitro

Beta-adrenergic receptor blocking drugs include a structurally related class of drugs that are employed clinically to treat a variety of cardiovascular disorders. Since these drugs exert additional nonspecific effects including membrane stabilization, representative samples including atenolol, dilevolol, labetolol, metoprolol and propranolol were studied to determine their influence on lipid peroxidation. Homogenates or liposomes of adult rat hearts were incubated in the presence of various concentrations of propranolol or equivalent concentrations of dilevolol, labetolol, metoprolol or atenolol. Lipid peroxidation was stimulated with 50 μM FeSO₄, 5 μMt-butyl hydroperoxide (homogenates) or 0.2 mM citrate FeSO₄ (liposomes) plus O₂. Lipid peroxidation, as assessed by both the thiobarbituric acid reaction and chemiluminescence, was reduced in a dose-dependent manner as the propranolol concentration was increased from 1 to 10 mM. The five beta-adrenergic receptor blocking drugs reduced lipid peroxidation both in crude homogenates and in liposomes; their effectiveness was related to their lipophilicity. Dilevolol, propranolol, labetolol and metoprolol at a concentration of 20 mM reduced lipid peroxidation by 45%, 37%, 35% and 28%, respectively. The hydrophilic blocker atenolol was ineffective in reducing lipid peroxidation event at elevated concentrations. Lipophilic beta-blocking drugs apparently are capable of exerting an antioxidant effect in protecting membrane lipids against peroxidation.     

Instability of malondialdehyde in the presence of H2O2: Implications for the thiobarbituric acid test

The content of thiobarbituric acid-reactive material (primarily malondialdehyde) is frequently used to estimate the extent of lipid peroxide formation. How-ever, malondialdehyde is unstable in the presence of millimolar concentrations of hydrogen peroxide. This observation considerably limits the applicability of the thiobarbituric acid test, as hydrogen peroxide is known to be formed in a number of lipid peroxidation-promoting systems. The instability of malondialdehyde in the presence of hydrogen peroxide seems to account for the inconsistent outcomes in studies relating the manipulations of intermediate H₂O₂ levels to the initiation of lipid peroxidation.     

Glutathione and antioxidants protect microsomes against lipid peroxidation and enzyme inactivation

The study investigated the relationship between lipid peroxidation and enzyme inactivation in rat hepatic microsomes and whether prior inactivation of aldehyde dehydrogenase (ALDH) exacerbated inactivation of other enzymes. In microsomes incubated with 2.5 μM iron as ferric sulfate and 50 μM ascorbate, ALDH, glucose-6-phosphate (G6Pase) and cytochrome P450 (Cyt-P450) levels decreased rapidly and concurrently with increased levels of thiobarbituric acid-reactive substances. Microsomal glutathioneS-transferase and nicotinamide adenine dinucleotide phosphate-cytochromec reductase were little affected during 1 hr of incubation. Addition of reduced glutathione partially protected, andN,N′-diphenyl-p-phenylenediamine and butylated hydroxytoluene completely protected microsomes against inactivation of ALDH, G6Pase and Cyt-P450, as well as lipid peroxidation induced by iron and ascorbate. ALDH was more susceptible than G6Pase to inactivation by iron and ascorbate, and was thus an excellent marker for oxidative stress. Inhibition of ALDH by cyanamide injection of rats exacerbated the inactivation of G6Pase in microsomes incubated with 0.1 mM, but not 25 μM 4-hydroxynonenal (4-HN). 4-HN did not stimulate lipid peroxidation. Thus, 4-HN may play a minor role in microsomal enzyme inactivation. In contrast, lipid, peroxyl radicals play an important role in microsomal enzyme inactivation, as evidenced by the prevention of both lipid peroxidation and enzyme inactivation by chain-breaking antioxidants.     

Effect of dietary menhaden oil and vitamin E onin vivo lipid peroxidation induced by iron

Weanling rats were fed diets containing 10% menhaden oil (MO) or 10% corn oil-lard (1∶1, COL) with low (≤5 IU/kg) or supplementary (35 IU/kg) vitamin E for six weeks. The rats were killed 30 min after injection with 24 mg iron/kg as ferrous chloride because thiobarbituric acid-reactive substances (TBARS) in liver homogenates were highest at 30 min after injection of iron into rats fed a standard diet. Tissue homogenates were used either without incubation (zero-time) or after incubation at 37°C for 1 hr. In addition to TBARS and conjugated dienes, headspace hexanal and total volatiles (TOV) determined by capillary gas chromatography were useful indices of lipid peroxidation since they were decreased by vitamin E supplementation and were increased with increasing iron dose. Regardless of the dietary lipid used, vitamin E supplementation decreased headspace hexanal, TOV, TBARS and conjugated dienes in both zero-time and incubated homogenates of liver and kidney. Dietary MO increased TBARS in both zero-time and incubated homogenates of tissue from rats injected with iron. In contrast, dietary MO decreased hexanal and TOV in incubated tissue homogenates. The study demonstrated the usefulness and limitations of using hexanal and TOV as indices of lipid peroxidation.     

Singlet oxygen in copper-catalyzed lipid peroxidation in erythrocyte membranes

Lipid hydroperoxide was generated in human erythrocyte membranes by irradiation with near ultraviolet (UV) light in the presence of a photosensitizer, hematoporphyrin, but no production of 2-thiobarbituric acid-reactive materials (malonaldehyde and its precursors) was detected. Incubation of the irradiated membranes with CuSO₄ led to increased levels of hydroperoxide and formation of malonaldehyde. Hydroperoxides were essential for initiating the Cu(II)-catalyzed peroxidation as no significant activity was observed with nonirradiated membranes and Cu(II) unless an organic peroxide, either t-butyl hydroperoxide or cumene hydroperoxide, was added. Catalytic activity was also found with Fe(II), but not with other metal ions tested. The peroxidation catalyzed with Cu(II) was partially inhibited by several singlet oxygen quenchers but was not affected by superoxide dismutase, catalase or OH^• radical scavengers. The possible involvement of singlet oxygen in the Cu(II)-catalyzed peroxidation reaction was further supported by a 3-fold enhancement of malonaldehyde production in D₂O.     

Rapid headspace gas chromatography of hexanal as a measure of lipid peroxidation in biological samples

A rapid, sensitive and convenient capillary gas chromatographic-headspace method was developed to determine hexanal as an important volatile decomposition product of hydroperoxides formed from n−6 polyunsaturated fatty acids in rat liver samples. Total volatiles were also determined as a measure of overall lipid peroxidation. Samples of headspace taken from sealed serum bottles incubated at 37°C were injected into a gas chromatograph. It was possible to make 15 determinations per hour. This method is convenient because no special sample manipulations are necessary. The addition of 0.5 mM ascorbic acid prior to gas chromatographic, analysis significantly increased hexanal production. The applicability of the method was demonstrated in studies of the effect of iron in the presence or absence of hydroperoxides of methyl linoleate and methyl linolenate andtert-butyl hydroperoxide on rat liver homogenates, slices and microsomes. A rapid silica cartridge chromatographic procedure was used to purify hydroperoxides from autoxidized methyl linoleate and methyl linolenate, and hydroperoxy epidioxides (cyclic peroxides) from autoxidized methyl linolenate in 20–40 mg quantities. The hydroperoxides and hydroperoxy epidioxides of methyl linolenate were, effective inducers n−6 polyunsaturated fatty acid peroxidation in liver homogenates. Hexanal and thiobarbituric acid-reacting substances were signficantly correlated in liver homogenates and microsomes but not in slices. This specific method for hexanal, a known product of peroxidation of n−6 polyunsaturated fatty acids, can be used as a good measure of lipid peroxidation. Presented at the joint meeting of the Amerian Society for Cell Biology and the American Society for Biochemistry and Molecular Biology, San Francisco, CA January 29–February 2, 1989.     

Time dependent changes occurring in rat liver microsomes upon lipid peroxidation

Steady-state fluorescence anisotropy of diphenylhexatriene and n-(9-anthroyloxy)stearic acids (n=2,12) in rat liver microsomes showed a marked increase in the early stages of enzymatically or non-enzymatically induced lipid peroxidation. The changes in fluorescence anisotropy occurred in parallel with the formation of thiobarbituric acid-reactive substances (TBA-RS). Parallel to these changes, the fluorescence emitted from peroxidized microsomes increased markedly in the early stages of lipid peroxidation. In contrast to the changes in the fluorescence anisotropy and in the formation of TBA-RS, the fluorescence showed a continuing increase over the three hr period of lipid peroxidation. Glucose-6-phosphatase was inactivated in the early stages of lipid peroxidation, whereas NADH-cytochrome b₅ reductase underwent a slow deactivation over three hr. The apparently slow deactivation of the peripheral protein may be explained by the formation of fluorescent substances.     

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 μM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 μM) or the combination of ferric ion (10 μM) and ascorbic acid (500 μM), in the presence of reduced glutathione (GSH, 800 μM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 μM). ATG similarly reversed an inhibition of phosphatidyl-choline hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classifical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 μM ATG. ATG, at concentrations up to 250 μM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%). These data demonstrate the potential of ATG to increase toxicity of lipid peroxidative stimuli by inhibition of microsomal and cytosolic defense mechanisms. Although ATG did not inhibit Triton-enhanced PHGPX activity, overall evidence points toward inhibition of this enzyme as the mechanism for ATG-augmented lipid peroxidation and supports the conclusion that PHGPX plays a major role in the cellular defense mechanism.     

Rapid isolation of microsomes for studies of lipid peroxidation

Conventional isolation of microsomes by high-speed centrifugation from isotonic sucrose requires exposure to air for several hours, leading to the formation of low levels of lipid peroxidation products. Sucrose interferes in protein and malondialdehyde assays and provides no protection against lipid peroxidation during workup. A new procedure for the purification of microsomes from rat liver substitutes mannitol (a hydroxyl radical scavenger) for sucrose and takes advantage of the properties of morpholinopropane sulfonic acid (MOPS) buffer and triethylenetetramine to provide protection against lipid peroxidation during the rapid (less than one hour) workup and subsequent low-temperature storage. The microsomal fractions prepared by the proposed method are free of detectable mitochondrial contamination and at least as pure overall as those prepared by the conventional method, but they have higher glucose-6-phosphatase and laurate hydroxylase activities and significantly less malondialdehyde than conventional microsomes at the time isolation is complete. Laurate hydroxylase activity is more stable during frozen storage in mannitol medium. The kinetics of lipid peroxidation in vitro are quite different for microsomes prepared by the two methods.     

The relationship between fatty acid peroxidation and α-tocopherol consumption in isolated normal and transformed hepatocytes

The response of normal and transformed rat hepatocytes to oxidative stress was investigated. Isolated normal rat hepatocytes and differentiated hepatoma cells (the Fao cell line was derived from the Reuber H 35 rat hepatoma) in suspension were incubated with the ADP/Fe³⁺ chelate for 30 min at 37°C. Membrane lipid oxidation was assessed by measuring (i) free malondialdehyde (MDA) production by a high-performance liquid chromatography (HPLC) procedure, (ii) membrane fatty acid disappearance as judged by capillary gas chromatography, and (iii) α-tocopherol oxidation as determined by HPLC and electrochemical detection. The addition of iron led to increased MDA production in normal as well as in transformed cells, and to simultaneous consumption of polyunsaturated fatty acids (PUFA) and α-tocopherol. In addition, in Fao cells more α-tocopherol was consumed during lipid peroxidation while less PUFA was oxidized. Lipid peroxidation was lower in tumoral hepatocytes than in normal cells. This could be due to a difference in membrane lipid composition because of a lower PUFA content and a higher α-tocopherol level in Fao cells. During oxidation, Fao cells produced 1.5 to 2 times less MDA than normal cells, while in the tumoral cells the amount of oxidized PUFA having 3 or more double bonds was 7 to 8 times lower. Therefore, measuring MDA alone as an index of lipid peroxidation did not allow for proper comparison of the membrane lipid oxidizability of transformed cellsvs. the membrane lipid oxidizability of normal cells.     

Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate

The purpose of this study was to test the hypothesis that multiple antioxygenic nutrients provide increased protection against lipid peroxidative damage to rat liver. Rats were fed diets (i) deficient in vitamin E and selenium (Diet 1), (ii) supplemented with vitamin E and selenium (Diet 2), (iii) supplemented with (ii) and in addition trolox C,N-acetylcysteine, coenzyme Q₀, and (+)-catechin (Diet 3), or (iv) supplemented with (iii) and in addition β-carotene, ascorbic acid palmitate, canthaxanthin, and coenzyme Q₁₀ (Diet 4). Liver homogenates were obtained from three rats fed each of the diets for six weeks and were incubated at 37°C up to two hours with and without exogenous tertiary-butyl hydroperoxide (TBHP) or Cu²⁺. Lipid peroxidation was determined by measurement of thiobarbituric acid substances. Diets 2 and 3 significantly protected againstin vivo hepatic lipid peroxidation, and this protection was augmented by Diet 4. Diets 2, 3, and 4 were protective against mild oxidation induced by TBHP or Cu²⁺. During incubations with exogenous TBHP and Cu²⁺, there were only small differences between diets supplemented with antioxidants in inhibition of lipid peroxidation, indicating that diets supplemented with vitamin E and selenium (Diet 2) may have provided the maximal protection for liver. The possible mechanisms of protection provided by multiple antioxidants in diets were discussed. Protection by multiple antioxidants against lipid peroxidation may translate to prevention of peroxidative damage to human tissue, a factor in human disease.     

Susceptibility to peroxidation of the major mycelial lipids of Cunninghamella echinulata

The distribution of hydroperoxides among lipid fractions (namely neutral lipids, glycolipids, sphingolipids, and phospholipids) of the oleaginous fungus Cunninghamella echinulata was studied during the growth cycle. The lipid hydroperoxide content of total lipids increased during growth. Neutral lipids contained a small amount of hydroperoxides (0.78×10^–4–5×10^–4 µmol/mg), glycolipids plus sphingolipids contained large amounts of hydroperoxides (14×10^–4–17.6×10^–4 µmol/mg), while the hydroperoxide content of phospholipids increased greatly during growth (from 4.6×10^–4 to 38×10^–4 µmol/mg). In addition, the distribution of lipid hydroperoxides among the major phospholipid classes indicated that the neutral phospholipids, namely phosphatidylcholine and phosphatidylethanolamine, were more susceptible to peroxidation than the anionic ones (i.e. phosphatidylinositol and phosphatidylserine). A novel parameter, namely “specific lipid peroxidizability”, which is able to evaluate the susceptibility to peroxidation (peroxidizability) of each lipid fraction/class due to the nature of the lipid itself, was introduced. By using this parameter it was found that peroxidizability depended on the nature of the individual lipid rather than on its polyunsaturated fatty acid content.