Jeroen J.M. van den Berg

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.

Reinvestigation of the antioxidant properties of conjugated linoleic acid

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or-independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) or the water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 μM efficiently inhibited PLPC oxidation, as evident from a clear lagphase. In contrast, 0.75 μM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe2+ (500 μM/0.05–20 μM), both vitamin E (1 μM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 μM) efficiently inhibited PLPC oxidation. However, CLA (1–50 μM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.

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.

Membrane changes associated with lysis of red blood cells by hypochlorous acid

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidenced by ektacytometry, and an increase in K(+)-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, but complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.

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.

Correlation of membrane lipid peroxidation with oxidation of hemoglobin variants: possibly related to the rates of hemin release.

Experiments were performed to delineate the biochemical mechanism of hemoglobin (Hb)-catalyzed lipid peroxidation in human red blood cells (RBCs). Using a modified Langmuir trough lipid monolayer technique, we found that oxidized Hb induced an increase in lipid monolayer surface pressure, suggesting that oxidized Hb readily releases its heme moiety into the lipid monolayer. To confirm our interpretation that oxidized Hb readily releases its heme moiety, we monitored the fluorescence of Hb tryptophan upon oxidation of Hb. We found an increase in Hb fluorescence in the aqueous phase of our monolayer system after the addition of H2O2. The increase in fluorescence should reflect the departure of heme from globin due to a decrease in fluorescent quenching effect by the heme moiety. The rate of increase in lipid monolayer surface pressure upon Hb oxidation differed from Hb to Hb with an order of Hb E > F > S > A. The ability of various Hbs to affect lipid peroxidation in the RBC membrane, as monitored by the parinaric acid oxidation technique, followed this same order. In addition, hemin was shown to be a more potent catalyst of lipid peroxidation in RBC membrane than nonheme irons.

Differential reactivities of hypochlorous and hypobromous acids with purified Escherichia coli phospholipid: formation of haloamines and halohydrins

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong oxidants derived from myeloperoxidase and eosinophil peroxidase, the major antimicrobial enzymes of neutrophils and eosinophils, respectively. These oxidants are highly reactive with a wide range of biomolecules. At physiological pH, both HOCl and HOBr react readily with amines to form haloamines and with the unsaturated bonds of fatty acids to form halohydrins. We have investigated which of these reactions occur with phosphatidylethanolamine (PE), the predominant phospholipid of Escherichia coli. The formation of haloamines was determined by TLC and colorimetrically and the formation of halohydrins was determined by TLC and GC-MS. With HOCl, chloramines were much the preferred product and chlorohydrins were formed in substantial amounts only when HOCl was in excess of the amount required to convert the amine to the dichloramine. With HOBr at all concentrations, bromamines and bromohydrins were formed concurrently, indicating a greater relative reactivity with unsaturated fatty acids than with HOCl. The bromamine derivatives of PE, and other primary amines, were found to be more reactive than the equivalent chloramines, and were able to brominate the unsaturated bonds of fatty acids. Bromohydrins (formed directly or through the action of bromamines) may, therefore, be suitable biomarkers for the production of HOBr in vivo.

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.

Direct and continuous measurement of hydroperoxide-induced oxidative stress on the membrane of intact erythrocytes

Having minimized spectroscopic interference by hemoglobin (Hb), peroxidation processes in intact erythrocytes could be monitored in a continuous assay using the fluorescent polyunsaturated fatty acid, parinaric acid (PnA), as a peroxidation probe. Control experiments to establish the character of the method are described in detail. As a practical application, comparative studies were performed to monitor the response of normal and sickle Hb-containing human erythrocytes to oxidative stress in the PnA assay. After 10 min of incubation with 200 microM cumene hydroperoxide (cumOOH), peroxidation of PnA was found to be enhanced in erythrocytes from sickle cell disease patients (SS: 48 +/- 9% (n = 6) of initial amount had been peroxidized) compared to healthy controls (AA: 30 +/- 4% (n = 9)). PnA peroxidation in erythrocytes from sickle cell trait individuals (AS: 30 +/- 3% (n = 4)) was equal to that in control cells. The increased oxidation of PnA in sickle erythrocytes was accompanied by enhanced oxidation of Hb (metHb and hemichrome formation), indicating that sickle Hb mediates enhanced cumOOH-derived radical generation. It is concluded that PnA can be a useful tool in studying membrane peroxidation processes in intact normal and pathological erythrocytes.

MEASUREMENT OF REACTION PRODUCTS FROM HYPOCHLOROUS ACID AND UNSATURATED LIPIDS

Publisher Summary Neutrophils contain the enzyme myeloperoxidase (MPO) that catalyzes the conversion of hydrogen peroxide (H 2 O 2 ) and chloride (Cl - ) to hypochlorous acid (HOCl). HOCl is a highly reactive oxidant that is thought to be important in the antimicrobial action and the cytotoxic effects of activated neutrophils. Although proteins are known to be susceptible to HOCl-mediated oxidative modification, the oxidation by HOCl of unsaturated membrane lipids could be instrumental in destabilizing the membrane lipid matrix, leading to cell disruption. HOCl adds across double bonds to form chlorohydrins. The reaction with unsaturated fatty acids yields a mixture of fatty acid chlorohydrin positional isomers. Monounsaturated fatty acids form monochlorohydrins, whereas in polyunsaturated fatty acids all the double bonds are susceptible to modification. Low HOCl:fatty acid ratios predominantly give monochlorohydrins, and higher ratios increase the proportion of bis- and polychlorohydrin derivatives. This reaction occurs with unsaturated fatty acids in micelles and with sn-2 fatty acyl chains of phospholipids in vesicle membranes. Both reagent hypochlorite and the MPO system can generate these products. Equivalent reactions to form iodohydrins and bromohydrins are shown with various peroxidase systems.