Jos A.F. Op den Kamp

C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (Micro)spectroscopic characterization and validation of methodology

C11-BODIPY(581/591) is a fluorescent radio-probe for indexing lipid peroxidation and antioxidant efficacy in model membrane systems and living cells, with excellent characteristics: (i) emission in the visible range of the electromagnetic spectrum, with good spectral separation of the nonoxidized (595 nm) and oxidized (520 nm) forms; (ii) has a high quantum yield and because of this, low labeling concentrations can be used, ensuring minimal perturbation of the membrane whilst retaining favorable signal to noise ratios; (iii) has a good photo-stability and displays very few fluorescence artifacts; (iv) is virtually insensitive to environmental changes, i.e., pH or solvent polarity; (v) is lipophilic and as such easily enters membranes; (vi) once oxidized, C11-BODIPY(581/591) remains lipophilic and does not spontaneously leave the lipid bilayer; (vii) C11-BODIPY(581/591) localizes in two distinct pools within the lipid bilayer, a shallow pool at 18 A and a deep pool at < 7.5 A from the center of the bilayer; (viii) is not cytotoxic to rat-1 fibroblasts up to 50 microM; (ix) is sensitive to a variety of oxy-radicals and peroxynitrite, but not to superoxide, nitric oxide, transition metal ions, and hydroperoxides per se; (x) its sensitivity to oxidation is comparable to that of endogenous fatty acyl moieties.

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non‐specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6‐times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP‐depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.

Kinetics and site specificity of hydroperoxide-induced oxidative damage in red blood cells

To provide a detailed description of the time course and the site specificity of hydroperoxide-induced oxidative stress in red blood cells (RBCs), we have characterized the action of a membrane-soluble (cumene hydroperoxide [cumOOH]) and a water-soluble (hydrogen peroxide [H2O2]) oxidant. The fluorescent polyunsaturated fatty acid (PUFA) parinaric acid (PnA) was used to probe peroxidation processes in the membrane, and oxidation of hemoglobin (Hb) was measured spectrophotometrically as an indicator of cytosolic oxidative stress. The observed degradation patterns of PnA and Hb were clearly distinct for each oxidant. At comparable oxidant concentrations, the cumulative oxidative stress on the RBC membrane was always much higher with cumOOH, whereas much more Hb oxidation was measured with H2O2. The kinetics of Hb oxidation as well as the nature of the products formed were different for each oxidant. The main Hb oxidation product generated gradually by cumOOH was metHb, whereas H2O2 caused the rapid formation of ferrylHb. CumOOH caused more oxidation of endogenous PUFAs and of vitamin E, while the degradation pattern of vitamin E closely resembled that of PnA. At high oxidant concentrations, extensive cell lysis was observed after prolonged incubation. Butylated hydroxytoluene (BHT) completely prevented oxidation of endogenous PUFAs but did not completely prevent hemolysis, indicating that factors other than lipid peroxidation are also important in causing lysis of RBCs. The action of cumOOH is characterized by a gradual reaction with Hb, generating radicals that produce an oxidative stress primarily directed at the membrane, which increases in time to a maximum and then gradually decreases. In contrast, H2O2 crosses the RBC membrane and reacts rapidly with Hb, generating a very reactive radical species that has Hb, not the membrane, as a prime target. H2O2-induced oxidative stress is at a maximum immediately after addition of this oxidant and decreases rapidly to zero in a short time. These findings provide further insight into the mode of action of hydroperoxides and the mechanism of compartmentalization of RBC oxidative damage.

Flip-flop rates of individual molecular species of phosphatidylcholine in the human red cell membrane

Trace amounts of four different, well-defined species of phosphatidyl[N-methyl-14C]choline ([14C]PC), differing in their fatty acyl constituents, were introduced exclusively into the outer membrane leaflet of the intact erythrocyte by using a PC-specific phospholipid transfer protein. The rate of transbilayer equilibration of these probe molecules was calculated from the time-dependent decay in specific radioactivity of the PC pool in the outer monolayer, which was discriminated from that in the inner leaflet by treating the intact cells with phospholipase A2 in the presence of sphingomyelinase C. At 37 degrees C, 1,2-dipalmitoyl-, 1,2-dioleoyl-, 1-palmitoyl-2-linoleoyl- and 1-palmitoyl-2-arachidonoyl-PC revealed halftime values for the rate of their transbilayer equilibration of 26.3 +/- 4.4, 14.4 +/- 3.5, 2.9 +/- 1.7 and 9.7 +/- 1.6 h, respectively.

Increased n-3 polyunsaturated fatty acid content of red blood cells from fish oil-fed rabbits increases in vitro lipid peroxidation, but decreases hemolysis

In view of a possible relationship between fish oil, lipid peroxidation, and atherosclerosis, the in vitro lipid peroxidation susceptibility of red blood cells (RBCs) from rabbits on conventional (-FO) and fish oil-enriched diets (+FO) was investigated. The diet caused substantial increases in the RBC concentrations of n-3 polyunsaturated fatty acids (PUFAs), in combination with decreases in the concentration of oleic acid (18:1) and linoleic acid (18:2). Cumene hydroperoxide-induced oxidative stress led to increased overall fatty acid peroxidation in +FO RBCs compared with with -FO RBCs, as quantitated by GLC fatty acid analysis. However, the increased overall susceptibility to lipid peroxidation of +FO RBCs was not reflected in increased peroxidation of every individual fatty acid. This was observed for endogenous arachidonic acid (20:4) as well as, in separate experiments, for exogenously added parinaric acid (PnA). The increased cumene hydroperoxide-induced PUFA oxidation in +FO RBCs was accompanied by a lesser extent of hemolysis. To account for these observations, it is proposed that the increased n-3 PUFA content of +FO RBCs serves as an oxidizable buffer. The present data suggest that oxidation of fatty acids can occur until a critically low level of intact phospholipid in the RBC membrane is reached, after which the membrane destabilizes and hemolysis occurs. At the same time, the PUFA buffer in +FO RBCs could also prevent oxidative damage to specific membrane proteins, which could also help prevent cell lysis.

Transbilayer movement of various phosphatidylcholine species in intact human erythrocytes

Phosphatidylcholine specific phospholipid exchange protein was used to introduce (14C)‐labeled phosphatidylcholine of different fatty acyl compositions into the intact human erythrocyte. Hydrolysis by a combination of phospholipase A2 and sphingomyelinase was applied to prove that originally all newly introduced phosphatidylcholine resided in the outer monolayer. Subsequently the erythrocytes were reincubated at 37°C, and redistribution of the introduced (14C)phosphatidylcholine was monitored by applying the combination of phospholipases after different times of incubation. In the situation where 20% of the native erythrocyte phosphatidylcholine had been replaced by phosphatidylcholine from (14C)choline‐labeled rat liver microsomal membranes, a slow translocation of the (14C)microsomal phosphatidylcholine was found, with a half‐time of transbilayer equilibration of 10.8 hr. Furthermore, the transbilayer movement of probe amounts of (14C)dipalmitoyl‐phosphatidylcholine, (14C)egg phosphatidylcholine and (14C)soybean phosphatidylcholine was studied under conditions whereby the fatty acyl composition of the bulk erythrocyte phosphatidylcholine remained unchanged. In correlation to the increasing unsaturation of the probe, half‐times for the transbilayer equilibration were calculated to be 26.9, 12.8, and 8.1 hr, respectively.

The cooperative action of vitamins E and C in the protection against peroxidation of parinaric acid in human erythrocyte membranes

The influence of vitamins E and C on the initial stages of lipid peroxidation in human erythrocyte membranes was assessed with the fluorescent polyunsaturated fatty acid, parinaric acid, as probe molecule. Cumene hydroperoxide was used as initiator with either haemin-Fe3+ or Cu2+ as metal ion cofactor. The effect of vitamin C (pro- or antioxidant) appeared to be determined by the localisation of the metal ions, either in the water phase or in the membrane. Vitamin C is only able to reduce metal ions in the water phase, which results in acceleration of radical generation and subsequent enhancement of parinaric acid peroxidation. Thus, interaction of vitamin C with Cu2+ in the water phase led to drastically enhanced peroxidation of parinaric acid. In contrast, when only membrane-associated haemin-Fe3+ was present, vitamin C functioned as an antioxidant at all concentrations tested (0-10 microM). In a system with haemin-Fe3+ equilibrated between the water phase and the membranes, less than 5 microM vitamin C produced an overall prooxidant, and greater than 15 microM vitamin C an overall antioxidant effect. At vitamin C concentrations of 5-15 microM, continuous measurement of parinaric acid fluorescence revealed a shift in the vitamin C effect from antioxidant to prooxidant within the time-course of an assay. Vitamin E exhibited a protective effect on peroxidation initiated by cumene (per)oxyl radicals with haemin-Fe3+ as cofactor, by inducing a concentration-dependent extension of the lag-phase in parinaric acid peroxidation. Vitamin E appeared to be much more effective compared with vitamin C in scavenging radicals in this system. This indicates that vitamin C has only a limited ability to react with cumene (per)oxyl radicals in the membrane. The combination of vitamins E and C produced a protective effect on parinaric acid peroxidation exceeding the sum of their individual contributions. Moreover, the rate of vitamin E consumption was drastically lowered in the presence of vitamin C, whereas the rate of vitamin C consumption hardly decreased in the presence of vitamin E. The results are discussed in terms of a reaction scheme where the relative contributions of a number of reactions are considered to determine the total effect of added vitamin C or E. Vitamin E radicals constitute an additional substrate for vitamin C, resulting in a more than additive shift in the overall effect to the antioxidant side.

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.

Complete exchange of phosphatidylcholine from intact erythrocytes after protein crosslinking

Treatment of human erythrocytes with tetrathionate or diamide resulted in extensive crosslinking of membranous and cytoskeletal proteins. Such treatment was followed by an incubation with phosphatidylcholine specific exchange protein to investigate the rate and extent of exchange of phosphatidylcholine between the erythrocytes and 14C-labeled phosphatidylcholine containing microsomal membranes or vesicles. Exchange profiles showed that the exchange of phosphatidylcholine is facilitated in treated cells when compared to control erythrocytes and, more importantly, that all of the phosphatidylcholine is exchangeable after protein crosslinking whereas in control cells only the phosphatidylcholine pool located in the outer layer of the membrane is exchangeable. These observations demonstrate that crosslinking of cytoskeletal and membraneous proteins enhances the rate of transbilayer movement of phosphatidylcholine considerably.

Structural and dynamic aspects of phosphatidylcholine in the human erythrocyte membrane

Abstract A protein responsible for the phosphatidylcholine-specific transfer of phospholipid across membranes provides a useful tool for retailoring the composition of the phosphatidylcholine species in the erythrocyte membrane. Using the same protein, the transbilayer mobility of this phospholipid in normal and abnormal erythrocytes can be determined.