Aluminium salts accelerate peroxidation of membrane lipids stimulated by iron salts

Aluminium salts do not themselves stimulate peroxidation of ox-brain phospholipid liposomes, but they greatly accelerate the peroxidation induced by iron(II) salts at acidic pH values. This effect of Al(III) is not seen at pH 7.4, perhaps because Al(III) salts form insoluble complexes at this pH in aqueous solution. Peroxidation of liposomes in the presence of Al(III) and Fe(II) salts is inhibited by the chelating agent desferrioxamine, and by EDTA and diethylenetriaminepentaacetic acid at concentrations greater than those of Fe(II) salt. Aluminium salts slightly stimulate the peroxidation of peroxide-depleted linolenic acid micelles, but they do not accelerate the peroxidation induced by addition of iron(II) salts to the micelles at acidic pH. Aluminium salts accelerate the peroxidation observed when human erythrocytes are treated with hydrogen peroxide at pH 7.4. Desferrioxamine decreases the peroxidation. We suggest that Al(III) ions produce an alteration in membrane structure that facilitates lipid peroxidation, and that the increased formation of fluorescent age pigments in the nervous system of patients exposed to toxic amounts of Al(III) may be related to this phenomenon. The ability of desferal to bind both iron (III) and aluminium(III) salts and to inhibit lipid peroxidation makes it an especially useful chelating agent in the treatment of 'aluminium overload'.     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Relation of lipid peroxidation to loss of cations trapped in liposomes

Lipid peroxidation and alterations in cation loss have been induced in liposomes by ferrous ion, ascorbic acid, reduced and oxidized glutathione, and gamma radiation. Modifications of these effects by tocopherol and 2,6-di-tert-butyl-4-methylphenol (BHT) were studied when these antioxidants were either incorporated in the membrane or were added to already formed liposomes prior to the addition of the chemical agent or to irradiation. Lipid peroxidation, as indicated by the thiobarbituric acid test for malonic dialdehyde, did not correlate with alterations in cation loss. The largest amounts of lipid peroxidation induced by ascorbic acid and glutathione were associated with decreased cation loss. Inhibition of Fe(2+)- and radiation-induced lipid peroxidation by antioxidants did not inhibit the associated increase in cation loss. Tocopherol was a more effective antioxidant than BHT when it was incorporated in the membrane, whereas BHT was more effective when it was added to the liposomes after formation.     

Immunochemical study on the pathway of electron flow in reduced nicotinamide adenine dinucleotide-dependent microsomal lipid peroxidation.

NADH could support the lipid peroxidation of rat liver microsomes in the presence of ferric ions chelated by ADP(ADP-Fe). The reaction had a broad pH optimum (pH 5.8--7.4) and was more active in the acidic pH range. Antibodies to NADH-cytochrome b5 reductase [EC 1.6.2.2] and cytochrome b5 inhibited NADH-dependent lipid peroxidation in the presence of ADP-Fe, whereas the antibody against NADPH-cytochrome c reductase [EC 1.6.2.4] showed no inhibition. These oberservations suggest that the electron from NADH was supplied to the lipid peroxidation reaction via NADH-cytochrome b5 reductase and cytochrome b5. On the other hand, NADPH-supported lipid peroxidation was strongly inhibited by the antibody against NADPH-cytochrome c reductase, confirming the participation of this this flavoprotein in the NADPH-dependent reaction. In the presence of both ADP-Fe and ferric ions chelated by EDTA(EDTA-Fe), NADH-dependent lipid peroxidation was highly stimulated up to the level of the NADPH-dependent reaction. In this case, the antibody against cytochrome b5 could not inhibit the reaction, while the antibody against NADH-cytochrome b5 reductase did inhibit it, suggesting the direct transfer of electrons from NADH-cytochrome b5 reductase to EDTA-Fe complex.     

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (approximately threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.     

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.     

The role of iron in ferritin- and haemosiderin-mediated lipid peroxidation in liposomes.

Ferritin and haemosiderin were shown, by the measurement of malondialdehyde production and loss of polyunsaturated fatty acids, to stimulate lipid peroxidation in liposomes. At pH 7.4 ascorbate was additionally required to achieve peroxidation; however, peroxidation occurred at pH 4.5 in the presence of iron-proteins alone. The damage was completely inhibited by the incorporation of chain-breaking antioxidants (alpha-tocopherol and butylated hydroxytoluene) into the liposomes. Metal chelators (desferrioxamine and EDTA) also completely inhibited lipid peroxidation. These and further results indicate that, at pH 4.5, even in the absence of a reducing agent, iron is released from haemosiderin and can mediate oxidative damage to a lipid membrane.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.     

Role of cytochrome P-450 in ochratoxin A-stimulated lipid peroxidation.

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe3+, ethylenediaminetetraacetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe3+. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

The effect of zinc on iron-induced lipid peroxidation in different lipid systems including liposomes and micelles

The effect of zinc on FeSO4/ascorbic acid-induced lipid peroxidation was measured by the thiobarbituric acid assay in various lipid systems including small unilamellar liposomes prepared from egg phosphatidylcholine (EPC), ionic micelles prepared from arachidonic acid (C20:4), non-ionic monocomponent micelles prepared from EPC-derived, methylated fatty acids, and an eicosatetrene emulsion. With the exception of C20:4 micelles, zinc inhibited lipid peroxidation in each of the above systems in a similar dose-related fashion, with 0.5 mM zinc having maximal effect. Gas-chromatographic fatty acid analysis too indicated a protective effect of zinc against FeCl3-induced lipid peroxidation in soybean PC vesicles, which do not contain C20:4 moieties. These findings, in particular the inhibition of lipid peroxidation in eicosatetrene emulsion, suggest that the presence of uncharged polar head groups, or packing of lipid molecules into ordered self-assemblages (membranes and micelles) have no critical influence on the antioxidant effect of zinc. The results with Fe2+ are compatible with the concept that zinc interferes with the formation of Fe2+-oxygen-enoic complexes. This mechanism, however, cannot account for the inhibition by zinc of the Fe#+-induced lipid peroxidation, suggesting the involvement of other types of zinc effects in these systems.     

Role of cytochrome P-450 in ochratoxin a-stimulated lipid peroxidation

The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe³⁺, ethylenediaminetetra-acetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe³⁺. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.     

Membrane lipid peroxidation by ultrasound: Mechanism and implications

Ultrasonic radiation produced a dose-dependent linear increase in lipid peroxidation in the liposomes membrane as reflected in the measurement of conjugated dienes, lipid hydroperoxides and malondialdehydes. Ultrasound induced malondialdehyde production could not be inhibited by any significant degree by superoxide dismutase or histidine or dimethyl furan but was very significantly inhibited by butylated hydroxytoluene, cholesterol, sodium benzoate, dimethyl sulphoxide, sodium formate and EDTA. The scavenger studies indicated the functional role of hydroxyl radicals in the initiation of ultrasound induced lipid peroxidation.     

Oxidation of ferrous iron during peroxidation of lipid substrates

Oxidation of Fe2+ in solution was dependent upon medium composition and the presence of lipid. The complete oxidation of Fe2+ in 0.9% saline was markedly accelerated in the presence of phosphate or EDTA and the ferrous oxidation product formed was readily recoverable as Fe2+ by ascorbate reduction. In contrast, in the presence of either brain synaptosomal membranes, phospholipid liposomes, fatty acid micelles or H2O2, less than 50% of the Fe2+ oxidized during an incubation could be recovered as Fe2+ via reduction with ascorbate. In the presence of unsaturated lipid, oxidation of Fe2+ was associated with peroxidation of lipid, as assessed by the uptake of O2 and formation of thiobarbituric acid-reactive products during incubations. Although relatively little Fe2+ oxidation or lipid peroxidation occurred in saline with synaptosomes or linoleic acid micelles during an incubation with Fe2+ alone, significant Fe2+ oxidation and lipid peroxidation occurred in incubations containing a 1:1 ratio of Fe2+ and Fe3+. Extensive Fe2+ oxidation and lipid peroxidation also occurred with Fe2+ alone in saline incubations with either linolenic or arachidonic acid acid micelles or liposomes prepared from dilinoleoylphosphatidylcholine. While a 1:1 ratio of Fe2+ and Fe3+ enhanced thiobarbituric acid-reactive product formation in incubations containing linolenic or arachidonic micelles, it reduced the rate of O2 consumption as compared with Fe2+ alone. The results demonstrate that oxidation of Fe2+ in incubations containing lipid substrates is linked to and accelerated by peroxidation of those substrates. Furthermore, the results suggest that oxidation of Fe2+ in the presence of lipid or H2O2 creates forms of iron which differ from those formed during simple Fe2+ autoxidation.     

Site-specific mechanisms of initiation by chelated iron and inhibition by alpha-tocopherol of lipid peroxide-dependent lipid peroxidation in charged micelles.

To obtain information on the role of iron-catalyzed lipid peroxidation in the presence of the small amount of lipid peroxide in deterioration of biological membranes, we examined factors affecting peroxidation of fatty acids in charged micelles. Peroxidation of linoleic acid (LA) was catalyzed by Fe2+ via reductive cleavage of linoleic acid hydroperoxide (LOOH) in negatively charged sodium dodecyl sulfate micelles, but not in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles. However, this Fe2(+)-induced, LOOH-dependent lipid peroxidation could be induced in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). The linoleic acid alkoxy radical (LO.) generated by the LOOH-dependent Fenton reaction was also trapped by N-t-butyl-alpha-phenylnitrone at the surface of TTAB micelles in the presence of NTA, but not in its absence. The degradation rates of two spin probes, N-oxyl-4,4'-dimethyloxazolidine derivatives of stearic acid (5-NS and 16-NS), were investigated to determine the site of production of radicals formed during LOOH-dependent lipid peroxidation. The rate of consumption of 16-NS during the LOOH-dependent Fenton-like reaction was higher in TTAB micelles containing LA than in those containing lauric acid (LauA), although the rates of formation of LO. in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same and were as low as that of 16-NS consumption in LauA micelles. 16-NS was more inhibitory than 5-NS of LOOH-dependent lipid peroxidation, and this inhibition was associated with its higher consumption of 16-NS than of 5-NS. alpha-Tocopherol inhibited NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation in TTAB micelles, and was oxidized during this inhibition process. The rate and amount of alpha-tocopherol oxidized by the LOOH-dependent Fenton reaction were higher in LA micelles than in LauA micelles. alpha-Tocopherol inhibited the consumption of 16-NS during NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation more effectively than that of 5-NS. The distribution of the chromanol moiety of alpha-tocopherol was studied by the fluorescence quenching method. There was no difference between Stern-Volmer plots of the quenchings of alpha-tocopherol fluorescence by 5-NS and 16-NS. From these results, we discuss the mechanism of induction of LOOH-dependent peroxidation of LA and the mechanism of the antioxidant effects of alpha-tocopherol on it from the viewpoint of site-specific reaction.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Peroxide-dependent and -independent lipid peroxidations catalyzed by chelated iron.

Oxidation of linoleic acid (LA) in tetradecyltrimethylammonium bromide micelles was induced by ferrous- and ferric-chelates in the presence of linoleic acid hydroperoxide (LOOH). Ferrous-chelates also induced lipid peroxidation in the presence of H2O2, but ferric-chelates did not, thought they could generate OH-radicals in the presence of H2O2, resulting in deoxyribose degradation. Of the chelators tested, nitrilotriacetic acid (NTA) chelated with iron showed the highest activity for induction of H2O2- and LOOH-dependent lipid peroxidations and H2O2-dependent deoxyribose degradation. NTA with ferrous ion, but not with ferric ion, also initiated oxidation of LA after a short lag period in the absence of peroxides such as H2O2 and LOOH, but other chelators with ferrous ion did not. The peroxide-independent lipid peroxidation and associated oxidation of ferrous-NTA to ferric-NTA progressed in two steps: an induction step in a lag period and then a propagation step. Ferrous ion complexed with NTA was autoxidized pH-dependently and synchronously with oxygen uptake. The rates of both reactions increased with increase of pH, but were not related to the length of the lag period, which was also dependent on pH, and was shortest at pH 4.2. The EPR spectrum of the ferric-NTA complex prepared directly from ferric salt was different from that of the complex prepared from ferrous salt, confirming that some ferric-type active oxygen participated in induction of peroxide-independent lipid peroxidation. From these results, we propose a possible mechanism of lipid peroxidation induced by ferrous-NTA without peroxides. The finding that iron-NTA had the highest activity for induction of the oxidations of LA and deoxyribose is discussed in relation to the carcinogenic and nephrotoxic effects of this chelating agent.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

The influence of various lipids on the activity of bovine milk galactosyltransferase

Purified bovine milk galactosyltransferase was combined with liposomes of different lipid composition. The activity was markedly affected by the nature of the lipid used. Thus phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol stimulated the activity, while phosphatidic acid and phosphatidylserine inhibited the activity of the transferase. Phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid had identical fatty acid compositions, yet phosphatidylcholine and phosphatidylglycerol stimulated the activity while phosphatidic acid inhibited the activity. The effect on the enzyme was probably related to the nature of the head group since the inhibition by phosphatidic acid could be converted to stimulation by methylating the phosphatidic acid. The properties of several of the head groups is discussed. The physical state of the lipid was shown to affect the activity markedly. When the enzyme was combined with dimyristylphosphatidylcholine the activity was markedly stimulated when the lipid was in the liquid-crystalline state i.e., above the phase transition.     

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.