Preferential hydrolysis of peroxidized phospholipid by lysosomal phospholipase C

The susceptibility of partially peroxidized liposomes of 2-[1-14C] linoleoylphosphatidylethanolamine ([14C]PE) to hydrolysis by cellular phospholipases was examined. [14C]PE was peroxidized by exposure to air at 37 degrees C, resulting in the formation of more polar derivatives, as determined by thin-layer chromatographic analysis. Hydrolysis of these partially peroxidized liposomes by lysosomal phospholipase C associated with cardiac sarcoplasmic reticulum, and by rat liver lysosomal phospholipase C, was greater than hydrolysis of non-peroxidized liposomes. By contrast, hydrolysis of liposomes by purified human synovial fluid phospholipase A2 or bacterial phospholipase C was almost completely inhibited by partial peroxidation of PE. Lysosomal phospholipase C preferentially hydrolyzed the peroxidized component of the lipid substrate which had accumulated during autoxidation. The major product recovered under these conditions was 2-monoacylglycerol, indicating sequential degradation by phospholipase C and diacylglycerol lipase. Liposomes peroxidized at pH 7.0 were more susceptible to hydrolysis by lysosomal phospholipases C than were liposomes peroxidized at pH 5.0, in spite of greater production of polar lipid after peroxidation at pH 5.0. Sodium bisulfite, an antioxidant and an inhibitor of lysosomal phospholipases, prevented: (1) lipid autoxidation, (2) hydrolysis of both non-peroxidized and peroxidized liposomes by sarcoplasmic reticulum and (3) loss of lipid phosphorus from endogenous lipids when sarcoplasmic reticulum was incubated at pH 5.0. These studies show that lipid peroxidation may modulate the susceptibility of phospholipid to attack by specific phospholipases, and may therefore be an important determinant in membrane dysfunction during injury. Preservation of membrane structural and functional integrity by antioxidants may result from inhibition of lipid peroxidation, which in turn may modulate cellular phospholipase activity.     

Cobaltous ion inhibition of lipid peroxidation in biological membranes.

The effect of cobalt on lipid peroxidation in biological membranes, phospholipid liposomes and fatty acid micelles was investigated. Cobaltous ion, at micromolar concentrations, inhibited iron-ascorbate induced lipid peroxidation in erythrocyte ghosts, microsomes and phosphatidylserine liposomes at pH 7.4. The pH seemed to be important for the anti-peroxidative effect of cobalt, because under slightly acidic conditions cobalt did not inhibit peroxidation. Cobalt was less effective in inhibiting peroxidation stimulated by organic hydroperoxides. Iron-ascorbate induced lipid peroxidation was also inhibited by EDTA. However, certain ratios of EDTA: cobalt in the reaction mixture stimulated peroxidation. Cobalt did not inhibit lipid peroxidation in linoleic acid micelles and phosphatidylethanolamine liposomes. The presence of phosphatidylserine, however, rendered these micelles and liposomes to cobalt inhibition. We conclude that the cobaltous ion is a potent inhibitor of lipid peroxidation in biological membranes and that the binding of cobalt to phosphatidylserine is necessary for the inhibitory effect of this metal ion.     

Inhibition of Lipid Peroxidation of Lecithin Liposomes Kept in a pH-Stat System Near Neutral pH

During 5 days of autoxidation of egg lecithin liposomes in nonbuffered saline pH dropped from an initial value of 7.4 to 4.5. A linear relationship between oxidation index and pH was obtained. Lipid peroxidation, monitored as conjugated diene and TBA-reactive products, was inhibited significantly by keep ing the samples under pH-controlled conditions (7.4 plusmn; 0.5), compared to controls. Obtained results indicate that the buffering capacity of Tris and Hepes buffers may play a role in their recently reported (D. Fiorentini et al. (1989) Free Radical Res. Commun., 6, 243) inhibitory action against lipid peroxidation of lecithin liposomes.     

Ferritin and haemosiderin in free radical generation, lipid peroxidation and protein damage

Iron storage proteins, ferritin and haemosiderin, release iron to a range of chelators and reducing agents, including citrate, acetate and ascorbate. Released iron promotes both hydroxyl radical formation in the presence of hydrogen peroxide and lipid peroxidation in liposomes. Ferritin protein is modified in such reactions, both by free radical cleavage and addition reactions with aldehyde products of lipid peroxidation.     

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+.     

A marked stimulation of Fe(3+)-dependent lipid peroxidation in phospholipid liposomes under acidic conditions

Lipid peroxidation in phosphatidylcholine liposomes induced by Fe(3+) alone, assessed by thiobarbituric acid-reactive substances (TBARS) production, was markedly enhanced as the solution pH was lowered from 7.4 to 5.5. On the other hand, at physiological pH, TBARS production by Fe(3+) was almost negligible. Results of the radical scavenger experiments with superoxide dismutase, catalase and hydroxyl radical ((&z.rad;)OH) scavengers (sodium benzoate, mannitol and dimethylthiourea), deoxyribose degradation and ESR spectrometry suggest that the stimulation of Fe(3+)-dependent lipid peroxidation under acidic conditions is involved in generation of superoxide anion (O(2)(&z.rad;-)), hydrogen peroxide (H(2)O(2)) and (&z.rad;)OH during the reaction. The stimulation of Fe(3+)-dependent TBARS production by increasing the [H(+)] completely disappeared by triphenylphosphine (TPP) treatment of the liposomes, but the reaction was reversible with either incorporation of cumen hydroperoxide (CumOOH) into the TPP-treated liposomes or the addition of CumOOH to the treated liposomes. Incubation of the CumOOH-incorporated TPP-treated liposomes with Fe(3+) at pH 5.5 also resulted in (&z.rad;)OH generation. Based on these results, a possible mechanism of stimulatory effect of Fe(3+) on lipid peroxidation under acidic conditions is discussed.     

O2- generation and lipid peroxidation during the oxidation of a glycated polypeptide, glycated polylysine, in the presence of iron-ADP

Oxidation of glycated polylysine, a model compound of glycated protein, caused O2- production even at physiological pH, which could be accelerated by Fe3(+)-ADP. An enediol structure in glycated polylysine and related compounds, which could be confirmed by I2 uptake, was related to their oxidizability. Glycated polylysine was easily coordinated with Fe3+ even in the presence of phosphate at pH 7.4 and the formation of the iron complex was prevented by desferrioxamine. The exposure of unsaturated phospholipid liposomes to glycated polylysine-Fe3(+)-ADP system caused the production of a thiobarbituric acid-reacting substance, which was completely inhibited by 5 microM alpha-tocopherol or 150 microM desferrioxamine and slightly by 0.5 microM SOD. Catalase (20 micrograms/ml) and 10 mM sodium-benzoate did not affect the iron-glycated polylysine-induced lipid peroxidation, indicating no participation of an OH. in this reaction. A ferrous ion-coordinated glycated polylysine may act as an initiator of phospholipid peroxidation in the presence of oxygen. A possible mechanism of the iron-glycated polylysine-induced lipid peroxidation was discussed.     

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'.     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.     

Importance of iron in lipid peroxidation in the tyrosinase/4-hydroxyanisole system: possible mechanism of killing of malignant melanoma cells by 4-hydroxyanisole.

Phospholipid peroxidation of unsaturated phospholipid liposomes in the tyrosinase(mushroom)-4-hydroxyanisole system was studied in both the presence and absence of Fe3+, as a model of melanocyte damage by this agent. Ferric ion is required for the lipid peroxidation, and maximal lipid peroxidation was achieved with a molar ratio of [Fe3+]/[4-hydroxyanisole] of about 1. The lipid peroxidation was significantly inhibited by ceruloplasmin (a ferroxidase), indicating that Fe3+, which would be coordinated with metabolites, catechols, should be reduced to express its oxidant property. Judging from the results obtained with inhibitors or scavengers of active oxygen species, O2-, H2O2, and .OH would not mainly involve in the lipid peroxidation.     

Tumor necrosis factor-induced permeability increase of negatively charged phospholipid vesicles

The effect of human tumor necrosis factor (TNF) on the permeability properties of liposomes containing phosphatidylserine at pH 5-6, as demonstrated by the calcein efflux. However, it did not induce any permeability change in such liposomes at neutral pH. The TNF-induced calcein efflux was also observed when an other acidic lipid was used as a component of the liposomes, i.e., phosphatidic acid or dicetyl phosphate. On the other hand, liposomes composed of neutral phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin showed little increases in permeability when incubated with TNF above pH 5.0. The TNF-induced permeability change was inhibited by the addition of polyaspartic acid, while it was not affected by the presence of 0.5 mM calcium ions. These data suggest that the negative charges on the liposomal surface trigger the interaction between TNF and liposomes. However, when the pH of the reaction mixture was decreased to 4.5, TNF-induced calcein efflux was observed even from neutral liposomes. When TNF was incubated with 8-anilinonaphthalene-1-sulfonic acid, the fluorescence intensity of this fluorophore increased with a decrease in the pH of the solution from 7 to 5, and a drastic increase in fluorescence was observed at pH 4.5. These data suggest that the hydrophobic region of TNF is also important for liposomal damage. Furthermore, the potencies of TNF and its derivative as to the induction of the permeability change paralleled their cytotoxic effects on mouse L929 cells, suggesting that the effect of TNF on liposomal membranes is related to its biological action.     

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+.     

The mode of action of polyacetylene and thiophene photosensitizers on liposome permeability to glucose

The mode of action of the two photosensitizers 1-phenylhepta-1,3,5-triyne and alpha-terthienyl on membrane permeability was investigated using liposomes entrapped with glucose as a model membrane system. Upon exposure to UV-A light, alpha-terthienyl, and to a much lesser extent phenylheptatriyne, induced leakage of glucose via a photodynamic mechanism in liposomes which had a high degree of unsaturated fatty acid side chains. Enhanced permeability to glucose in these liposomes due to the action of alpha-terthienyl and phenylheptatriyne involved lipid peroxidation, but neither of the two assays used to monitor lipid peroxidation (malondialdehyde and peroxide formation) was directly correlated with the increase in liposome permeability. In liposomes with highly ordered lipid where the fatty acid side chains are saturated, alpha-terthienyl had no effect on glucose permeability. In contrast, phenylheptatriyne was very effective in increasing glucose permeability in these liposomes via a photodynamic mechanism. Addition of lysophosphatidylcholine, which perturbs the order of lipid packing, to these liposomes, completely inhibited the effect of phenylheptatriyne. Conversely, incorporation of cholesterol which increases lipid order, into egg PC liposomes, enhanced the action of phenylheptatriyne. These data suggest that under UV-A irradiation (a) alpha-terthienyl and phenylheptatriyne enhance permeability in liposomes with a high degree of unsaturation involving lipid peroxidation and (b) phenylheptatriyne enhances membrane permeability through some other mechanism when present in a bilayer with a highly ordered lipid environment.     

Reconstitution studies on the involvement of radiation-induced lipid peroxidation in damage to membrane enzymes

The effect of radiation on the drug-metabolizing enzyme system of microsomes, reconstituted with liposomes of microsomal phospholipids, NADPH-cytochrome P-450 reductase and cytochrome P-450, was examined to elucidate the role of lipid peroxidation of membranes in radiation-induced damage to membrane-bound enzymes. The reconstituted system of non-irradiated enzymes with irradiated liposomes showed a low activity of hexobarbital hydroxylation, whereas irradiated enzymes combined with non-irradiated liposomes exhibited an activity equal to that of unirradiated controls. Irradiation of liposomes caused a decrease in cytochrome P-450 content by destruction of the haem of cytochrome P-450 and also inhibited the binding capacity of cytochrome P-450 for hexobarbital. The relationship between radiation-induced lipid peroxidation and membrane-bound enzymes is discussed.     

Iron release from haemosiderin and ferritin by therapeutic and physiological chelators.

Iron release from both human and horse spleen haemosiderin to desferrioxamine was substantially less than that released from ferritin samples. This finding contradicts a previous report [Kontoghiorges, Chambers & Hoffbrand (1987) Biochem. J. 241, 87-92]. Differences in phosphate content of cores and in core size between haemosiderin and ferritin did not account for the different iron-release rates. Iron released to acetate was found to stimulate lipid peroxidation in liposomes, whereas that released to stronger chelators such as citrate and desferal did not. Absorption spectra and gel-filtration studies suggest that the acetate-solubilized iron was in the form of low-molecular-mass (less than 5 kDa) ferrihydrite fragments.     

The effect of concentration on the antioxidant effectiveness of alpha-tocopherol in lipid peroxidation induced by superoxide free radicals

Egg yolk phosphatidylcholine liposomes were rapidly oxidized in the presence of chelated iron and a superoxide-generating system. alpha-Tocopherol incorporated in the bilayer was oxidized at the same time. No lipid or alpha-tocopherol oxidation occurred in liposomes composed of dimyristoyl phosphatidylcholine. The antioxidant did not inhibit lipid peroxidation until its concentration reached a critical level, which depended on the effectiveness of the oxidative stress. Beyond this level, peroxidation was inhibited completely and, simultaneously, the rate of oxidation of tocopherol was lowered. The results suggest that the antioxidant efficiency of alpha-tocopherol depends on its ability to react mainly with the chain-initiating or chain-propagating lipid radicals. This, in turn, is closely tied to the tocopherol content of the membrane. Ascorbate inhibited the consumption of alpha-tocopherol, possibly by regenerating its reduced form.     

Lipoxygenase may be involved in cationic liposome-induced macrophage apoptosis

The purpose of this study was to determine the source of reactive oxygen species (ROS) generation and the contribution of ROS to the apoptosis of RAW264.7 cells induced by cationic liposomes. Cationic liposome-induced apoptosis was inhibited by lipoxygenase inhibitors, but not inhibitors of NADPH-oxidase, xanthine oxidase or cyclooxygenase. ROS generation induced by cationic liposomes was also inhibited by the lipoxygenase inhibitor NDGA. Furthermore, lipid peroxidation was observed following liposome treatment, but the apoptosis was not inhibited by the antioxidant alpha-tocopherol. These findings suggested that lipoxygenase is responsible for ROS generation, and ROS but not lipid peroxidation acts as a key mediator in the progress of apoptosis induced by cationic liposomes.     

Reaction of xanthine oxidase-derived oxidants with lipid and protein of human plasma

Xanthine oxidase and purines have recently been detected in the circulation during acute viral infection and following hepatotoxicity and shock. Reactions of xanthine oxidase-generated oxidants with human plasma or bovine serum albumin (BSA) and egg phosphatidylcholine (PC) liposomes have been studied by measuring protein sulfhydryl oxidation and two markers of free radical-mediated lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and conjugated dienes. Plasma incubated with 5 mU/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (Hx) for 2 h at 37 degrees C had 25-53% oxidation of sulfhydryl groups, with greater than 80% of the oxidation occurring during the first 20 min of the reaction. Concentrations of BSA similar to those present in serum, when exposed to XO/Hx-mediated oxidative stress, showed an even greater decrease in sulfhydryl concentration than that of plasma. No significant increase in plasma TBARS and conjugated dienes was observed during the 2-h incubation period in the presence of XO. Egg PC liposomes, suspended to a plasma phospholipid-equivalent concentration, showed a minor increase in TBARS and conjugated dienes under similar XO/Hx incubation conditions. In the presence of 0.23 mM BSA, lipid peroxidation was completely inhibited. A similar inhibition of lipid peroxidation was induced by cysteine but not by uric acid. Electrophoretic and arsenite-mediated sulfur reduction analysis revealed that BSA was oxidized beyond the disulfide form, with sulfenic acid formed during the initial period of oxidation. Protein sulfhydryls served as sacrificial antioxidants, preventing plasma lipid peroxidation, as well as being targets for oxidative damage. Plasma protein thiol oxidation was determined to be a more sensitive and specific indication of oxidant stress to the vascular compartment than assessment of lipid oxidation byproducts.     

Ascorbic Acid Inhibits Lipid Peroxidation but Enhances DNA Damage in Rat Liver Nuclei Incubated with Iron Ions

In this report we studied DNA damage and lipid peroxidation in rat liver nuclei incubated with iron ions for up to 2 hrs in order to examine whether nuclear DNA damage was dependent on membrane lipid peroxidation. Lipid peroxidation was measured as thio-barbituric acid-reactive substances (TBARS) and DNA damage was measured as 8-OH-deoxyguanosine (8-OH-dG). We showed that Fe(II) induced nuclear lipid peroxidation dose-dependently but only the highest concentration (1.0 mM) used induced appreciable 8-OH-dG. Fe(II1) up to 1 mM induced minimal lipid peroxidation and negligible amounts of 8-OH-dG. Ascorbic acid enhanced Fe(II)-induced lipid peroxidation at a ratio to Fe(II) of 1:l but strongly inhibited peroxidation at ratios of 2.5:l and 5:l. By contrast, ascorbate markedly enhanced DNA damage at all ratios tested and in a concentration-dependent manner. The nuclear DNA damage induced by 1 niM FeSO₄/5 mM ascorbic acid was largely inhibited by iron chelators and by dimethylsulphoxide and manni-tol, indicating the involvement of OH. Hydrogen peroxide and superoxide anions were also involved, as DNA damage was partially inhibited by catalase and, to a lesser extent, by superoxide dismutase. The chain-breaking antioxidants butylated hydroxytoluene and diphenylamine (an alkoxyl radical scavenger) did not inhibit DNA damage. Hence, this study demonstrated that ascorbic acid enhanced Fe(II)-induced DNA base modification which was not dependent on lipid peroxidation in rat liver nuclei.     

The mode of action of polyacetylene and thiophene photosensitizers on liposome permeability to glucose

The mode of action of the two photosensitizers 1-phenylhepta-1,3,5-triyne and α-terthienyl on membrane permeability was investigated using liposomes entrapped with glucose as a model membrane system. Upon exposure to UV-A light, α-terthienyl, and to a much lesser extent phenylheptatriyne, induced leakage of glucose via a photodynamic mechanism in liposomes which had a high degree of unsaturated fatty acid side chains. Enhanced permeability to glucose in these liposomes due to the action of α-terthienyl and phenylheptatriyne involved lipid peroxidation, but neither of the two assays used to monitor lipid peroxidation (malondialdehyde and peroxide formation) was directly correlated with the increase in liposome permeability. In liposomes with highly ordered lipid where the fatty acid side chains are saturated, α-terthienyl had no effect on glucose permeability. In contrast, phenylheptatriyne was very effective in increasing glucose permeability in these liposomes via a photodynamic mechanism. Addition of lysophosphatidylcholine, which perturbs the order of lipid packing, to these liposomes, completely inhibited the effect of phenylheptatriyne. Conversely, incorporation of cholesterol which increases lipid order, into egg PC liposomes, enhanced the action of phenylheptatriyne. These data suggest that under UV-A irradiation (a) α-terthienyl and phenylheptatriyne enhance permeability in liposomes with a high degree of unsaturation involving lipid peroxidation and (b) phenylheptatriyne enhances membrane permeability through some other mechanism when present in a bilayer with a highly ordered lipid environment.