Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY(581/591).

A ratio-fluorescence assay was developed for on-line localization and quantification of lipid oxidation in living cells. The assay explores the oxidative sensitivity of C11-BODIPY(581/591). Upon oxidation, the fluorescence of this fluorophore shifts from red to green. The probe incorporates readily into cellular membranes and is about twice as sensitive to oxidation as arachidonic acid. Using confocal microscopy, the cumene hydroperoxide-induced oxidation of C11-BODIPY(581/591) was visualized at the sub-cellular level in rat-1 fibroblasts. Preloading of the cells with tocopherol retarded this oxidation. The data demonstrate that C11-BODIPY(581/591) is a valuable tool to quantify lipid oxidation and anti-oxidant efficacy in single cells.

Validation of the peroxidative indicators, cis-parinaric acid and parinaroyl-phospholipids, in a model system and cultured cardiac myocytes.

cis-Parinaric acid is increasingly being used in eukaryotic cells as a very sensitive marker for the initial stages of lipid peroxidation. Despite the increased application of this probe, no extensive validation, especially in cellular systems, has been performed. cis-Parinaric acid can either be inserted freely into biomembranes or incorporated (bio)synthetically into lipids (parinaroyl-lipid). Therefore, a direct comparison was made between the peroxidative behaviour of the two parinaroyl probes and the endogenous polyunsaturated fatty acids arachidonic and linoleic acid, in both an artificial lipidic system and in cultured neonatal rat heart cells. Three different radical generating systems were used, i.e., hydrogen peroxide, cumene hydroperoxide and the thermo-labile 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). The data demonstrate that the peroxidation rate of cis-parinaric acid is higher than that of the parinaroyl, arachidonoyl and linoleoyl lipids. The latter three displayed comparable peroxidation rates, showing that the peroxidative decay of parinaroyl-lipid is a good marker for the degradation of endogenous polyunsaturated fatty acids. Experimental results using the freely inserted cis-parinaric acid could potentially lead to an overestimation of the inflicted damage and should be interpreted with care. In addition, a comparison was made with the measurement of conjugated dienes and malon dialdehyde as thiobarbituric acid reactive substances. The results demonstrate that measurement of conjugated dienes and malon dialdehyde only provide information on peroxidative processes in vitro, but are not suitable for in-depth studies in cultured cells. In contrast, the use of the parinaroyl probes is a suitable, straightforward, sensitive and reproducible method for detecting the initial stages of lipid peroxidation in living cells.

Effect of tryptophan-N-formylated gramicidin on growth of Plasmodium berghei in mice.

The effect of tryptophan-N-formylated gramicidin (NFG) on the growth of Plasmodium berghei in mice was tested in three different experiments. NFG was shown to be capable of inhibiting the growth of the parasite in a dose-dependent way, although its action did not result in elimination of the parasite and was only temporary, preventing mice from early death, presumably due to cerebral malaria, but not from fatal generalized malaria. Intriguingly, a similar observation was made with two other drugs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, an inhibitor of viral and eukaryotic DNA polymerases, and the presumed topoisomerase II inhibitor, a bisquaternary quinolinium salt. A rise in the level of parasitemia after 8 days, despite continued treatment, was not due to parasite-induced reticulocytosis, as demonstrated in experiments in which this condition was induced artificially. NFG was added in the form of lipid vesicles in which the peptide had been incorporated. The inhibitory action of NFG was not modulated by the lipid composition of the vesicles. Control experiments did not demonstrate any toxicity of NFG when it was administered in lipid vesicles. The main observation is that NFG is able to inhibit the growth of a malaria parasite in vivo at concentrations that are well tolerated by the host.

Tryptophan-N-formylated gramicidin causes growth inhibition of Plasmodium falciparum by inducing potassium efflux from infected erythrocytes.

In a study of the supposed selective action of tryptophan-N-formylated gramicidin (NFG) on infected erythrocytes as well as the relationship between the ability of NFG to inhibit parasite growth and its capacity to induce potassium leakage from infected cells, a series of experiments was performed in which in vitro cultures of Plasmodium falciparum were incubated with NFG or gramicidin. Those cultures were subsequently assayed for intracellular sodium and potassium contents, cell lysis, and/or parasite viability. It is shown and discussed that although NFG can attack both infected and uninfected erythrocytes, resulting in potassium efflux from and sodium influx into these cells, the effects are much greater on infected erythrocytes than on uninfected ones. Furthermore, the results strongly suggest that NFG-mediated potassium efflux is the direct cause of parasite death.

The use of cis-parinaric acid to measure lipid peroxidation in cardiomyocytes during ischemia and reperfusion.

cis-Parinaric acid (PnAc), a fluorescent, polyunsaturated fatty acid, was used to measure lipid peroxidation during simulated ischemia and reperfusion in cultured neonatal rat cardiomyocytes. PnAc was used both as free fatty acid, inserted in the membranes following cultivation of the cells, as well as constituent of the cellular complex lipids by metabolically integrating the fatty acid during growth. In the insertion experiments a pre-incubation with DL-aminocarnitine, an inhibitor of beta-oxidation, was necessary to prevent loss of fluorescent signal. Such a pre-incubation resulted in an enrichment of PnAc in the sarcolemma: In pre-treated cells 57 +/- 1.3% of total inserted PnAc is present in the sarcolemma compared to 27 +/- 5.7% in cells containing the integrated probe. Both methods to introduce PnAc into the cells were compared with respect to their sensitivity for an externally applied oxidative stress and thereafter lipid peroxidation during simulated ischemia and reperfusion was assayed. Going from normoxic to ischemic conditions lipid peroxidation did not increase and remained at a low level. When the ischemic cells were subsequently subjected to reperfusion (reintroduction of both oxygen and glucose), large scale lipid peroxidation was obvious. When, on the other hand, oxygen alone was reintroduced (reoxygenation) no increased lipid peroxidation was observed. These observations led to the conclusion that ischemia does not lead to an enhanced lipid peroxidation and that resumption of metabolic activity during reperfusion is necessary to induce lipid peroxidation.

Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes.

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptide-induced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.

Sarcolemmal phosphatidylethanolamine reorganization during simulated ischaemia and reperfusion: reversibility and ATP dependency.

Exposure of cultured neonatal rat heart cells to simulated ischaemia results in a cessation of the spontaneous contractile activity and changes at both the level of sarcolemmal phospholipid topology and the ultrastructural level. Reperfusion at a timepoint before irreversible cell damage develops leads to a recovery of contractile activity. Furthermore, the shift in transbilayer distribution of sarcolemmal phosphatidylethanolamine in favour of the outer membrane leaflet, due to the ischaemic period, is reversed during subsequent reperfusion. Also the morphological changes (mitochondrial oedema, reorganization of the mitochondrial cristae and the formation of extrusions at the sarcolemma) are reversible. At the same time total intracellular ATP levels are restored to 80% of control. The role of cellular ATP content on sarcolemmal phospholipid topology was further studied by the use of the calcium antagonist verapamil (10 microM), which preserved cellular ATP content by inhibiting cell contractility before the onset of ischaemia. After 120 min of ischaemia, cell ATP content was still 63% of control in the presence of verapamil, versus 20% of control in untreated cells. Verapamil treatment also prevented the loss of the asymmetrical distribution of phosphatidylethanolamine and sarcolemmal disruption, the latter occurring during 120 min of ischaemia in untreated cells. It is proposed that maintenance of phospholipid asymmetry of the sarcolemma of the myocytes depends on the cellular ATP concentrations, indicating the involvement of an ATP dependent aminophospholipid translocase.

The influence of a fish oil-enriched diet on the phospholipid fatty acid turnover in the rabbit red cell membrane in vivo.

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.

Comparison of the effects of dietary vitamin E on in vivo and in vitro parameters of lipid peroxidation in the rabbit.

This study has investigated the effect of dietary vitamin E on markers of antioxidant status. Four groups of rabbits received diets containing 30 energy percent (en%) total fat (7.8 en% contributed by linoleic acid) for 12 weeks. D,1-alpha tocopheryl acetate was added to the diets to obtain a range of vitamin E concentrations (49, 114, 179, or 775 tocopherol equivalents per kg diet). Increased vitamin E concentrations were demonstrated in plasma lipoproteins and erythrocyte membranes following supplementation, and dietary effects on lipid peroxidation were investigated by (i) monitoring a fluorescent parinaric acid probe incorporated into erythrocyte membranes in vivo, (ii) determination of malondialdehyde and oxysterols in plasma, and (iii) investigation of the susceptibility of low density lipoprotein (LDL) to copper-induced conjugated diene formation in vitro. No effects of vitamin E were observed on parinaric acid oxidation in vivo or on the accumulation of lipid peroxidation products in plasma, but the resistance of LDL to oxidation in vitro increased significantly as vitamin E was supplemented to the diets. Our results demonstrate that under these dietary conditions (7.8 en% linoleic acid) increasing the vitamin E content of plasma and erythrocytes approximately two-fold does not reduce the level of lipid peroxidation in vivo, indicating sufficient antioxidant capacity on the lowest vitamin E diet. In contrast, LDL became more resistant to an extreme oxidative stress applied in vitro. The relevance of these assays to currently proposed mechanisms of atherosclerosis is discussed.

In vivo turnover of 1,2-dipalmitoylphosphatidylcholine and sphingomyelin in rabbit erythrocytes.

The in vivo turnover of both 1,2-dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) in rabbit erythrocytes was studied. DPPC, either 14C-labelled in the fatty acyl chain at the 2-position of the glycerol moiety or 3H-labelled in the choline's methyl group, and [N-methyl-14C]SM (bovine) were introduced into the membrane of freshly isolated rabbit erythrocytes by using phospholipid transfer proteins. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. Analysis of blood samples shows that both labels disappear from the circulating cells with the same rate, resulting in a half-time value of about 6.4-6.6 days. This result demonstrates that the loss of DPPC from the cells is due to transfer of intact molecules to the plasma and that a deacylation process is of no or minor importance as mechanism of renewal of DPPC. Labelled sphingomyelin, introduced into the rabbit erythrocyte membrane in a similar way, disappears from the circulating red cell with a half-time value of 15.5 days. This accounts for a daily replacement of the total SM pool by 3.2%.

Direct measurement of lipid peroxidation in submitochondrial particles.

The susceptibility of the polyunsaturated fatty acid parinaric acid (cis-PnA) to peroxidative damage with concomitant loss of its fluorescent character can be used to detect lipid peroxidation in a direct and sensitive way. The procedure, originally developed to measure peroxidation in lipid vesicles and erythrocyte membranes, has been adapted for the study of submitochondrial particles. Optimal conditions for the concentrations of cis-PnA (0.8 mol %), mitochondrial membrane (100 microM membrane phospholipid), and the radical generating system (50 microM NADH and 10 microM:1 mM Fe(III)-ADP) were established. In the absence of peroxidation inducing compounds, a stable fluorescent signal can be detected. Upon addition of NAD(P)H and ADP-Fe(III), lipid peroxidation starts, and the observed fluorescence decrease is a measure of peroxidation. Both NADH and NADPH were able to induce lipid peroxidation in submitochondrial particles in the presence of an iron chelate. The use of NADH resulted in higher rates of peroxidation compared with NADPH at the same concentration. Whereas the rate of NADH-induced lipid peroxidation was maximal at very low NADH concentrations (2.5 microM) and decreased when the concentration became higher, the NADPH-induced lipid peroxidation reaches saturation at 100 microM. NADH-induced lipid peroxidation in submitochondrial particles from different rat tissues (heart, skeletal muscle, and liver) resulted in a clear difference in peroxidation rates. The highest rates were observed in heart submitochondrial particles, while the lowest rates were obtained in submitochondrial particles derived from liver. Skeletal muscle submitochondrial particles showed intermediate rates of lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced Mg(2+)-ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity.

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg2+)-dependent adenosine triphosphatase (Mg(2+)-ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg(2+)-ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg(2+)-ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg(2+)-ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.

Stage-dependent effects of analogs of gramicidin A on the growth of Plasmodium falciparum in vitro.

Tryptophan-N-formylated gramicidin A, a nonhemolytic derivative of the toxic peptide antibiotic gramicidin A, has previously been shown to induce potassium leakage from Plasmodium falciparum-infected erythrocytes in vitro and to inhibit the growth of the parasite. In the present study the antimalarial activities of two other nonhemolytic derivatives of gramicidin A, viz., acylated gramicidin A and desformylated gramicidin A, were tested and compared with those of gramicidin A and tryptophan-N-formylated gramicidin A. The 50% growth-inhibitory concentrations (IC50 values) of the four compounds varied from 0.3 to 18.3 nM, and complete growth inhibition was detected within one parasitic growth cycle. Using highly synchronized cultures of P. falciparum, it was furthermore shown that the gramicidin analogs are inhibitory to all developmental stages of the parasite, although their efficiency in accomplishing growth inhibition was found, as expected, to be clearly stage-dependent and to increase with the age of the parasite.

In vivo turnover of phospholipids in rabbit erythrocytes.

The rate of phospholipid turnover in erythrocyte membranes in vivo has been studied using a recently developed procedure (Kuypers, F.A., Easton, E.W., van den Hoven, R., Wensing, T., Roelofsen, B., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1985) Biochim. Biophys. Acta 819, 170-178). The technique is based on the application of phospholipid transfer proteins in order to introduce trace amounts of radiolabelled phospholipids in the membrane of isolated erythrocytes, followed by re-injection of the erythrocytes into the bloodstream of the animal. The most abundant species of the phosphatidylcholine (PC) class, 1-palmitoyl,2-linoleoyl PC, has, on the basis of loss of the radioactivity in its fatty acyl part, a relatively high turnover with a half-time value of 1.5 days. Other PC species studied exhibit more moderate turnover rates of about 5 days for 1-palmitoyl,2-oleoyl PC and 1-stearoyl,2-arachidonoyl PC. Dipalmitoyl PC, labelled in the polar headgroup, turns over at a slow rate with a half-time value of 9 days. From these data and the relative abundance of the various species, it can be calculated that, on a daily basis in vivo, about one third of the total PC pool in rabbit erythrocyte membranes is replaced and/or modified by de-/reacylation. The only phosphatidylethanolamine (PE) species studied so far, 1-palmitoyl,2-arachidonoyl PE, appeared to be renewed at a relatively low rate with a half-time value of 12 days. The data demonstrate that the in vivo turnover values of phospholipids in the erythrocyte membrane may depend on their polar head group structure, their localization in the membrane and, to a large extent, on their fatty acid composition.

Testosterone-induced changes in phosphatidylcholine molecular species composition of Plasmodium chabaudi-infected erythrocytes.

This study is concerned with the influence of testosterone on the phospholipid class and the phosphatidylcholine molecular species composition of various fractions obtained from the blood of Plasmodium chabaudi-infected mice. Blood plasma, infected erythrocytes, isolated parasites and erythrocyte membranes isolated from both non-infected and infected erythrocytes in the form of ghosts were analysed. In general, the phospholipid classes remained unaffected, while the phosphatidylcholine (PC) molecular species composition showed differences after testosterone treatment. In infected erythrocytes, there was a decrease in 16:0/20:4-PC and 18:0/20:4-PC and an increase in 16:0/18:2(16:0/20:3)-PC. The decrease of 16:0/20:4-PC was exclusively confined to parasites. The rise in 16:0/18:2(16:0/20:3)-PC and the diminution of 18:0/20:4-PC occurred in the erythrocyte membrane of both infected ghosts and non-infected ghosts as well as in the blood plasma. It is suggested that these changes occur primarily in the plasma thereby influencing the erythrocyte membranes. The decrease in 16:0/20:4-PC supports the view of the independence of the parasite from the biosynthetic lipid pathways of its host cell.

Loss of asymmetric distribution of sarcolemmal phosphatidylethanolamine during simulated ischemia in the isolated neonatal rat cardiomyocyte.

In the present study we have investigated the reorganization of the sarcolemmal phospholipids during the first 60 minutes of simulated ischemia ("ischemia") as induced by anoxia, volume restriction, and nutrient deprivation. Experiments were carried out on [3H]acetate-labeled neonatal rat cardiomyocytes and isolated (nonradiolabeled) sarcolemmal membranes obtained from the same culture system. After 60 minutes of "ischemia," cellular high-energy phosphate (ATP) levels had decreased to approximately 40% of the control values, but no significant phospholipid hydrolysis was detected. Labeling experiments using the nonpermeant (primary amine-containing phospholipid) probe trinitrobenzenesulfonic acid and nonlytic treatment with (different) exogenous phospholipases A2 were both indicative of a shifted transbilayer distribution of the hexagonalII phase-preferring and fusion-promoting sarcolemmal phosphatidylethanolamine in favor of the outer membrane leaflet. This specific change in sarcolemmal phospholipid asymmetry preceded the loss of integrity of the sarcolemma, monitored by the release of lactate dehydrogenase as well as by scanning electron microscopy. It is proposed that, in addition to the previously reported lateral phospholipid reorganization, uncontrolled transbilayer movement of the non-bilayer-preferring phosphatidylethanolamine from the inner to the outer leaflet of the sarcolemma is an additional factor in destabilizing the lipid bilayer, eventually leading to the irreversible membrane damage seen after a prolonged period of ischemia.

Conformational changes in oxidized phospholipids and their preferential hydrolysis by phospholipase A2: a monolayer study.

Cleavage of oxidized fatty acids by phospholipase A2 has been implicated as the first step in the repair mechanism for oxidative damage to membrane phospholipids. However, the mechanism by which this enzyme preferentially hydrolyzes oxidized fatty acyl chains is poorly understood. Using a lipid monolayer technique, we found that the molecular surface areas of 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)-phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) were increased by as much as 50% relative to the parent nonoxidized 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC). These experimental data directly indicate a drastically changed molecular conformation of oxidized phospholipids in which the hydroperoxy or hydroxy group in the sn-2 fatty acid is close to the lipid-water interface. Phospholipases A2 from porcine pancreas and from bee venom were shown to break down PLPC-OOH and PLPC-OH monolayers much faster than PLPC monolayers. In all cases, the presence of serum albumin in the subphase enhanced monolayer breakdown by extracting hydrolysis products from the monolayer, but monolayer breakdown was always much faster for oxidized than for nonoxidized PLPC. This did not appear to be due to change in the extent of monolayer penetration by phospholipase A2, since enzyme-monolayer interaction studies revealed essentially identical penetration behavior of bee venom phospholipase A2 with PLPC, PLPC-OOH, and PLPC-OH monolayers. We propose that the altered molecular conformation of oxidized phospholipids facilitates access to the sn-2 ester bond, thereby ensuring their preferential hydrolysis in the presence of a phospholipase A2.

Molecular species of membrane phospholipids containing arachidonic acid and linoleic acid contribute to the interindividual variability of red blood cell Na(+)-Li+ countertransport: in vivo and in vitro evidence.

Previous studies indicate a particular sensitivity of red blood cell Na(+)-Li+ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na(+)-Li+ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na(+)-Li+ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl-PC and in diacyl-PE the species 16:0/20:4 and 16:0/18:2 were, respectively, positively and negatively related to the apparent maximal velocity of Na(+)-Li+ countertransport. The sum of all species with 20:4 at sn2 of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 18:2 a negative correlation (r = -0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na+. To evaluate whether the associations between molecular species of membrane phospholipids and Na(+)-Li+ countertransport activity were indicative of a causal relationship, the species 16:0/20:4-PC and 16:0/18:2-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 16:0/18:2-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 16:0/20:4-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes.

cis-Parinaric acid (PnA) was used as a fluorescent probe to study lipid peroxidation in nonparasitized and Plasmodium falciparum-parasitized erythrocytes, upon challenge by cumene hydroperoxide and tert-butyl hydroperoxide. Parasitized erythrocytes were less susceptible toward lipid peroxidation than nonparasitized erythrocytes with which they had been cultured. Furthermore, nonparasitized erythrocytes cultured together with parasitized cells, and thereafter isolated on a Percoll gradient, were less susceptible toward lipid peroxidation than erythrocytes kept under the same experimental conditions but in the absence of parasitized cells. We concluded, therefore, that the intracellular development of the parasite leads to an increase in the resistance against oxidative stress, not only of the host cell membrane of the parasitized erythrocyte, but also in the plasma membrane of the neighboring cells. The erythrocyte cytosol of parasitized cells and/or the intraerythrocytic parasite was required for the increased protection of the host cell membrane, since ghosts prepared from parasitized erythrocytes were more susceptible to lipid peroxidation than those prepared from nonparasitized ones. Vitamin E content of parasitized erythrocytes was lower than that of nonparasitized cells. However, parasitized erythrocytes promoted extracellular reduction of ferricyanide at higher rates, which might be indicative of a larger cytosolic reductive capacity. It is suggested that the improved response of intact erythrocytes is due to an increased reduction potential of the host-erythrocyte cytosol. The role of vitamin C as a mediator of this process is discussed.