hr. J. Biochem. Vol. 24, No. I, pp. 121-128, 1992 Printed in Great Britain. All rights reserved
Copyright
0
0020-7 11X/92 $5.00 + 0.00 1991 Pergamon Press plc
POSSIBLE INVOLVEMENT OF MYELOPEROXIDASE LIPID PEROXIDATION
IN
TRRRSASTRLMASZYI+SKA,~* ELISABETRKUKOVETZ,’ G. EGGRR~ and R. J. SCHADR’ ‘Institute of Biochemistry, University of Graz Schubertstrasse 1, A-8010 Graz, Austria and *Institute of Functional Pathology, University of Graz, Mozartgasse 14/D, A-8010 Graz, Austria (Received 28 January 1991) Abstract-l. Exposure of liposomes to the MPO-H,O,-Clsystem results in oxidation of lipids. Malondialdehyde and 4-hydroxynonenal are formed. 2. Oxidation of liposomes by stimulated rat neutrophils, assessed by malondialdehyde formation, is inhibited by KCN. This indicates involvement of MPO in the process. 3. The MPO-H,O, system oxidizes mildly LDL but in the presence of chloride a propagation phase, with a rapid increase of conjugated diene formation, was observed.
INTRODUCTION Myeloperoxidase (MPO)-an enzyme of neutrophilic granulocytes is released from the azurophilic granules after stimulation of the cells. The list of the activities of the enzyme is long: it peroxidizes classical peroxidase substrates such as phenols and aromatic amines;
in combination with H,02 and chloride ions it produces HOC1 which chlorinates many substrates such as amines, amino acids, proteins and other biologically active substances (for review see Zgliczynski and Stelmaszynska, 1988). This enzyme also exerts oxidase-peroxidase activity towards such substrates as NADH (Odajima, 1971), some thiols (Svensson, 1988) and acetoacetate (Harrison and Seed, 1981). MPO may develop its activity in uivo after stimulation of neutrophils, which triggers the respiratory burst producing hydrogen peroxide among other oxygen radicals, i.e. superoxide anion and probably hydroxyl radicals (for review see Winterbourn, 1990). Considerable interest is focused on the role of the neutrophil respiratory burst products in tumor cell cytotoxicity. The mechanisms of cytotoxic action of neutrophils are various and their effectiveness depends in great part on the target cell resistance. The cytotoxic action of neutrophils is partly based on the MPO-halogenating system, at least towards some cell lines (Slivka et al., 1980; Clark and Klebanoff, 1975; Weiss and Slivka, 1982). The question arises, as to what extent this cytotoxicity is dependent on membrane lipid peroxidation and formation of its secondary cytotoxic products. There is some information concerning the contribution of activated neutrophils to lipid peroxidation (Stossel et al., 1974; Claster et al., 1984; Carlin, 1985; Thomas et al., 1986). Recently it was found that in a model of acute inflammation activated neutrophils
*On leave from the Institute of Medical Biochemistry, Medical Academy, ul. Kopernika Poland. Bc 24,1--H
7, 31-034 Krakow,
form 4-hydroxynonenal (HNE )-one of the most cytotoxic products of lipid peroxidation (Schaur, 1989). Few investigations focus on MPO involvement in lipid pet-oxidation. It has been found that cofactors such as acetoacetate (Harrison et al., 1981) or chelated iron and iodide (Carlin and Djursiiter, 1988) are needed for MPO-mediated lipid peroxidation, assessed by thiobarbituric acid-reactive substance test (TBARS). Sepe and Clark (1985) have shown that the MPO-chlorinating system can lyse phospholipid liposomes. The aim of this study was to ascertain whether the MPO-H,O,-Clsystem acts on phospholipid liposomes and is responsible for the production of cytotoxic secondary products of lipid peroxidation (HNE). Also, this work was performed because of its possible relevance to atherosclerosis, which is now connected with oxidative modification of low density lipoprotein (Esterbauer et al., 1989). It is known that stimulated phagocytes of blood-neutrophils and monocytes oxidize low density lipoprotein making it cytotoxic to vascular cells (Cathcart et al., 1985). Therefore an attempt was made to show that the MPO-H,O, system and predominantly the MPOHrO&lsystem is involved in peroxidation of LDL.
MATERIALS AND METHODS Asolectin (phosphatidylcholine from soybean) containing 55% linoleid-acid and 8.2% linolenic acid (Kagawa and Racker, 1966). taurine and butvlated hvdroxvtoluene (BHT) were obtained from Sigma Chemical-Company (St iouis; U.S.A.). 2,2’azino-di-(3-ethyl-benzothiazohne-(6)-sulfonic acid (ABTS) and thiobarbituric acid (TBA) were from Serva (Fed. Rep. Germany). Myeloperoxidase (MPO) was a generous gift from Professor Inge Olsson (Lund University, Sweden). The enzyme activity was measured with ABTS as a substrate (Piitter and Becker, 1986). H,O, solutions were prepared from 30% H,O, (Merck, Fed. Rep. Germany) and standardized manganometrically. Polystyrene particles (0.8-l pm in diameter) were prepared in the Institute of
122
TERESA STELMA~ZY~SKA
Catalysis and Surface Chemistry, PAN, Krakow, Poland. The particles were washed thoroughly with water and suspended in 0.9% NaCl to concentration of 2 x 10” per ml. Silica Gel 60 plates, KCN, tryptophan, KI and other reagents of analytical grade were purchased from Merck (Darmstadt, Fed. Rep. Germany). Rat neutrophilic granulocytes were obtained from Sephadex G-200-mediated acute subcutaneous inflammatory focus (Egger, 1984). The suspension of the cells contained 96% neutrophils and 4% lymphocytes. Human LDL was prepared according to the method of Esterbauer et al. (1989). The stock solution of LDL (8.9 mg/ml) contained 1 mg/ml of EDTA. The final concentration of LDL in oxidation experiments was 0.26 mg/ml in 0.8 mM EDTA. Liposomes were prepared by vortexing 3.5 mg asolectin (4.6 pmol of lipids) in 1 ml 0.06 M phosphate buffer, pH 6.0, containing 0.16 M NaCl. Ass&s for lipid peroxidation
Since it is desirable to assess lipid peroxidation by more than one method, the following assays have been chosen: (1) TBA-reactive substance test: 1 ml of sample was mixed with 0.375% 2-thiobarbituric acid in 0.25 M HCl, containing 1% Triton X-100, and heated for 15 min at 100°C. The absorption spectrum of the TBARS chromophore was monitored using a Perkin-Elmer 554 Spectrophotometer; the c value, used for calculation was 1.52 x lo5 M-’ cm-’ at 532nm (Placer et al., 1966). (2) Iodometric estimation of excess of H,O,, lipid peroxide and chloramines. An t value of 2.6 x lo4 M-’ cm-’ for KI, at 355 nm was used for calculations (Kimura et al., 1969). (3) 4-Hydroxy,2,3-trans-nonenal (HNE) estimation (Esterbauer et al., 1982; Esterbauer and Zollner, 1989): The sample (1 ml) was mixed with 1 ml of 2,4-dinitrophenylhydrazine reagent (50 mg/lOO ml 1 M HCl) and with 10 ~1 0.1% BHT, and after 2 hr extracted with dichloromethane. The concentrated extract was pre-separated on a Silica Gel 60 plate (20 x 20cm) into three classes, using dichloromethane as developer. Hydrazones of the first class (around R, 0.12) were scraped off, extracted with methanol and concentrated under N,. Then, hydrazones were separated on a Spherisorb ODS 2 5~ column with methanolacetonitrilewater (27: 10: 12) at a flow rate of 0.8 ml/min, with a 370 nm detector wavelength, at ambient temperature. Peak identification and quantification was achieved by comparison with a HNE hydrazone standard separated under identical conditions. (4) Monitoring the increase of the 234nm absorbance resulting from conjugated fatty acid hydroperoxide formation (Esterbauer et al., 1989).
ef al.
continuously at ambient temperature with the Perkin-Elmer Spectrophotometer 554 (Esterbauer et al., 1989). RESULTS AND DISCUSSION
Oxidation H20,-Cl-
of phospholipid system
liposomes
by the MPO-
Formation of HOC1 by the MPO-HrOr system is optimal when the concentration ratio of the substrates [H,O,]/[H+] x [Cl-] is maintained at 500-1000 (Zgliczynski et al., 1977). At pH 6.0 and at 0.16 M NaCl the optimal concentration of H,Oz, which is not inhibiting the enzyme, is 0.16mM. Therefore, the total amount of HrO, in a small sample is rather low. In order to achieve higher total amount, but low temporary concentration of H202, the liposomes were suspended with MPO in a small volume (0.5 ml) and filled into a dialysis bag, which was placed into a relatively large solution of H,O, (5.0 ml). H,O, could continuously diffuse into the bag and under these conditions its optimal concentration in the external fluid was 0.5 mM (Fig. 1). It was found that the consumption of H,Or from the external compartment was accompanied by the formation of non-diffusible iodide-oxidizing species, most likely lipid peroxide (Fig. 2). Simultaneously the TBARS test was performed either with the liposome fraction inside the bag (Fig. 2) or with the total mixture of the bag content and the external solution. Since part of the TBARS can diffuse through the bag, the estimation of their amount in the total solution gave results which were about 200% higher compared with the amounts inside the bag (not shown).
Incubation procedures
(1) Liposomes and the MPO system: liposomes (2 pmol of lipid) and MPO (2.0-30 pg i.e. 9.6-144 mu) in 0.5 ml of 0.06 M phosnhate buffer with 0.16 M NaCl were incubated inside a dialysis bag (0.8 cm in diameter) in 5 ml of an external solution containing 0.1-1.0 mM H202 (total amount of H,O,: 0.5-5.0 hmol). These conditions allow low temporal concentration of H,O, inside the bag, but high total amount of this substrate. The duration of incubation was l-2 hr at 37°C with continuous stirring. (2) Liposomes and neutrophils: liposomes (2pmol of lipid) were incubated with neutrophils (3 x 10’cells) in 0.066 M phosphate buffer, pH 6.7, containing 0.07 M NaCl and 5.5 mM glucose, in total volume of 1.1 ml. Neutrophils were stimulated with polystyrene particles (IO8 per sample). (3) LDL and MPO: LDL (0.26mg/ml) was mixed with MPO (5.3 pg i.e. 25 mu) in 2 ml of 0.06 M phosphate buffer containing 0.8 mM EDTA and 0.16 M NaCl. The reaction was started by addition of 0.5 pmol H,O, and monitored
500 Wavelength
550
600
(nm)
Fig. 1. Influence of the H,O, concentration on liposome oxidation by the MPO system. Absorption spectra of TBARS chromophore obtained by applying the TBA test to aliquots of the liposome suspension inside the bags (10 pg MPO + 2 pmol lipid in 0.5 ml) after 1 hr incubation at 37°C of the bags in external solutions (5 ml) containing H,O, at various concentrations: (0) no H,O,; (1) 0.2mM; (2) 0.5 mM and (3) 1.0 mM H,O, in 0.06 M phosphate buffer, pH 6.0, containing 0.16 M NaCl. Details of the experiment as described in Methods. Absorption spectra were measured against a sample containing liposomes only.
Myelo~ro~da~-rn~iat~
lipid peroxidation
123
r
Trp (mMt KCN (mM! Composition of internal solution
Taurine (mM) Lipid (~mol) -
MPO &I)
2.3
10.6
32.0
10.6
10.6
2.5
10.6
Internal solution
External solution
”
0
1
2
3 Sample
4
5
6
no:
Fig. 2. Effect of taurine, KCN and tryptophan on lipid peroxidation by the MPO-H,O&Isystem. Composition of solution inside the bag (0.5 ml) is shown in the figure: external solution (5 ml) contained 2.5 pmol H,O,. Bar 0 shows calculated distribution of H,O, between internal and external solution at equilibrium. Hydroperoxides were determined using the iodometric method in external q and internal & compartment separately, after 1 hr incubation at 37°C. The absorbance of KI, originating from KI oxidation by hydroperoxides was measured after 90 min incubation of the samples with 38 mM KI in 12.5% CH,COOH at ambient temperature, in the darkness. Absorbance of KIT formed by KI oxidation by N-chloramines (samples 3, 4, 5) Qj was measured immediately after mixing the samples with 38 mM KI in 12.5% CH,COOH. The formation of TBARS was assessed as described in Methods.
Lipid peroxidation
in terms of TBARS
formation
was completely inhibited by 1 mM cyanide, which is not only a potent inhibitor of peroxidases but also a substrate of the MPO-I-&O&lchlorinating system (Zgliczybski and Stelmaszynska, 1979) (Fig. 2). A consumption of H20, in the KCN containing
6 (Fig. 2) indicates that cyanide acts as a substrate of the enzyme system. Taurine and tryptophan also prevented lipid from peroxidation (Fig. 2). Tryptophan is chlorinated and oxidized extensively by the MPO-H*O,-Clsystem (Zgliczynski and Stelmaszynska, 1988). sample
TERESASTELMASZY~SKA et
124
Taurine is chlorinated to N-chlorotaurine consuming more toxic HOCl, and due to this reaction is considered as buffer against oxidative damage by the MPO-chlorinating system (Slivka et al., 1980; Lin et al., 1988). Neutrophils contain high amount of taurine (McMenamy et al., 1960) and when stimulated release taurine chloramine (Weiss et al., 1983). Its accumulation in the environment proves that the rate of its formation exceeds consumption by components of the cells. The accumulation of N-chlorotaurine in the sample 5 (Fig. 2) proves that it does not react readily with lipids. Thus, it may be suggested that the kind of amine in a phospholipid (ethanolamine, serine or choline) may have influence on the susceptibility of fatty acid components of lipids to oxidation by the MPO-HI02Cl system. Indeed, Carlin and Djursster (1988) were unable to show peroxidation of liposomes by the MPO-H*O,-Cl- system. The liposomes used in their experiments contained 80-85% phosphatidyl serine. It is conceivable that all HOC1 formed by the enzyme system was consumed by chlorination of serine amino group to N-chloramine, the more so that the amount of H,Oz used was lower than the amount of lipids (Carlin and Djurslter, 1988). There was no lipid peroxidation in the absence of MPO at the H,02 concentration used (Figs 1 and 2). However, there was no linear dependence of the extent of TBARS formation on the MPO concentration (Fig. 3), in spite of increasing consumption of H,O, (Fig. 2). This may be caused by chlorination of the enzyme protein, competing with the lipid destruction process. Malondialdehyde-the main substrate for the TBARS chromophore can only be formed from fatty
O.‘O I
A
0.05
-
al.
acids with three or more double bonds, but not from linoleic acid which is the major polyunsaturated fatty acid (PUFA) in soybean phospholipid (55%). It seemed necessary to search for other aldehydes which are secondary products of lipid peroxidation. The estimation of aldehydes such as 4-hydroxynonenal can prove that lipid peroxidation has really occurred (Esterbauer and Zollner, 1989). Hydroxynonenal (HNE)-a product of o-6 PUFAs (linoleic acid) peroxidation was estimated as 2,4_dinitrophenylhydrazone according to Esterbauer et al. (1982) and Esterbauer and Zollner (1989). Preseparation by TLC of the hydrazones from sample 3 shown in Fig. 2 has revealed some carbonyl products in all three classes (zones) of different polarities (Esterbauer and Zollner, 1989). HNE was identified and quantified by subsequent HPLC separation (Fig. 4). It was found that in liposomes composed as sample 3 in Fig. 2, 0.23 nmol/ml of HNE was formed during 1 hr at 37°C. A more than lo-fold increase in HNE formation (3.2 nmol/ml) was observed after longer incubation (2 hr) at 37°C. In the control samples without MPO, but with H,Oz, there was only a trace of HNE (c 0.05 nmol/ml). All hydrazones found on the TLC plates except HNE are as yet unidentified. It is tempting to speculate that some of them are products chlorinated by the MPO system. Sepe and Clark (1985) suggested this new possibility-that phospholipid membrane injury caused by the MPO-H,O,-Clsystem may involve lipid halogenation. Oxidation of liposomes by stimulated neutrophils
Neutrophils obtained from rat subcutaneous exudate were stimulated by polystyrene latex granules to phagocytosis. The phagocytosing cells appear to be at the highest level of stimulation. The respiratory burst of the activated cells-production of superoxide and hydrogen peroxide is accompanied by leakage of azurophilic granules into the extracellular surroundings, most probably through phagocytic vacuoles open to the outside (Dewald et al., 1982). In this way myeloperoxidase and H,O, are released by the cells. Liposomes incubated with the stimulated neutrophils undergo oxidation. It was found that 3 x 10’ neutrophils/ml incubated with polystyrene granules and liposomes in buffer pH 6.7, with glucose and NaCl, produced about 1.6 nmol TBARS after 95 min (Fig. 5). The formation of TBARS was almost completely abolished by 1.5 mM KCN, which indicates involvement of MPO in this process. Peroxidation of human LDL by the MPO-H20, the MPO-H202-Clsystem
500 Wavelength
550
600
(nm)
Fig. 3. Influence of MPO concentration on liposome oxidation. Absorption spectra of the TBARS chromophore for aliquots of liposome suspension inside the bag (MPO + liposomes, 0.5 ml) after 1 hr incubation of the bags at 37°C in the 5 ml of external 0.5 mM H,O, solution. All in 0.06 M buffer, pH 6.0 0.16 M NaCI. (0) without MPO; (1) 2.3 pg MPO; (2) 10.6 pg MPO; (3) 32 pg MPO. Absorption spectra measured against sample containing only liposomes in buffer solution.
and
Peroxidation of LDL was measured by monitoring the change of the 234nm absorption resulting from formation of lipid conjugated diene hydroperoxide (Esterbauer et al., 1989) (Fig. 6). Kinetic pattern of the process indicates that MPO is an especially effective catalyst in the presence of H,O, and chloride suggesting participation of HOC1 in the reaction. In the absence of NaCl MPO catalyzes only mild oxidation of LDL by H,02 (Fig. 6). The propagation phase of LDL oxidation is seen in the presence of NaCl only. Taking into account an 6234= 29,500 M-’ cm-’ for conjugated lipid peroxides
Myelo~ro~da~-rn~iat~
lipid peroxidation
125
I P
P
c
2
4
6
8
10
12
14
0
Retention
time
2
4
6
8
10
12
14
16
(min)
Fig. 4. Separation of the dinitrophenylhydrazone of HNE by HPLC: (A) aliquot of the Iiposome suspension composed as sample 3 in Fig. 2; (B) standard HNE solution containing 80 pmol/20 ~1 injection sample. Details of the separation are described in Methods. HNE peak shown by arrow.
and assuming that the maximum increase of A = 2.49 originated from conjugated diene formation, it may be calculated that 336 nmoles of dienes/mg of LDL were formed under our experimental conditions
(Fig. 6). Since on the average LDL contains 556 nmol activated methylene groups per mg of LDL (Esterbauer er al., 1990), the majority of PUFAs (about
60% of activated methylene groups) have been peroxidized. Oxidation of LDL by the MPO-H20&1system was inhibited completely by I mM KCN-the inhibitor of hemeproteins, and was inhibited partially by 2.4mM taurine-the acceptor of HOCl. CONCLUDING REMARKS
0.08
I
‘~“.‘,:, , L
,
( , , ,
450
500
WaveLength
550
600
(nml
Fig. 5, Peroxidation of Iiposomes by rat neutrophils stimulated by polystyrene granules. Neutrophils (3 x IO’cells) were incubated for 95min at 37°C with: (I) liposomes (2 pmol pho~holipid), polystyrene granules (10s); (2) as 1 + KCN (1.5 mM at Iinal con~ntration); (3) only polystyrene granules (lOa). All samples in 0.066M phosphate buffer, pH 6.7 containing 0.07 M NaCl and 5.5 mM glucose in total volume 1.1 ml. The results are expressed as absorption spectra of the TBARS chromophore obtained in aliquot of the supernatant after centrifugation of the cells and granules. Absorption spectra were measured against supematant of the sample containing only liposomes and granules.
Experiments with the three model system usednamely purified MPO plus liposomes; purified MPO plus LDL; phagocytic cells, which can release MPO upon stimulation, plus liposomes-are in agreement with the assumption that the MPO-H,O,-Clsystem can promote lipid peroxidation in vitro. From these results several new questions arise. The mechanism of action of the MPO-H,O,-Clsystem on lipids is not known. It has been found that the enzyme system is continuously needed for the proceedings of lipid peroxidation presenting evidence against a free radical chain process (Sepe and Clark, 1985). The question arises whether the MPO-mediated lipid pet-oxidation has any biological relevance. It is very probable in oivo that MPO is released in inflammatory foci during phagocytosis (Dewald et al., 1982) or from disrupted neutrophils, which have a very short lifespan during the period of intensive ingress (Egger et al., 1988). The MFQ system may also act if neutrophils are in close contact with target cell membranes as in the antibody-de~ndent attack on the target celis. Then, at least part of the cytotoxic action of neutrophiis may originate from the reaction of HOC1 (formed by the MPO-catalyzed oxidation of ubiquitous Cl- ions by the product of the respiratory burst-H,Oz) with lipids of membranes. Another point is to which extent ammo compounds present in inflammatory foci are alternative
126
TFRESA
&ELMASZYikKAet d.
2.5
0.:
IO
20
30
40 Time
50
60
TO
80
(mint
Fig. 6. Time course of LDL oxidation by the MPO-H,O, and the MPO-H,Or-Cl- system. Change of the 234 nm absorbance was measured in the samples containing: MPO (5.3 pg i.e, 25.4 mu), 0.8 mM EDTA, LDL (0.52 mg) in 2 ml of 0.06 M phosphate buffer, pH 6.0, with 0.16 M NaCl x x , 0-O or without NaCl x---x , O---O; A.. .A the same as 0-O + 2.4 mM taurine; O---O the same , as O---- 0 + 1 mM KCN. Reaction was started with one portion of H20, (1 pmol) x ---x , x-x or with two portions of H,O, (2 x OS@mol) O---O, O-0, indicated by arrows. The reference sample contained LDL and MPO in the same buffer. There was no change of the 234 nm absorbance in the samples containing H,O, but without the enzyme. Other details were described in Methods.
for HOCI. Recently it has been shown by Sharonov and Covorova (1990) that serum proteins compete with erythrocyte membranes for HOCI. reactants
SUMMARY
The possible involvement of myeloperoxidase (MPO) was studied in several model systems of lipid pet-oxidation.
Exposure of liposomes to the MPO-H,02-Clsystern results in consumption of H,O,, formation of iodide-oxidizing species (lipid hydroperoxides?) and of thiobarbituric acid reactive substrates (TBARS). Oxidation does virtually not occur in the absence of either MPO or H20,. The extent of lipid peroxidation is optimal in narrow concentration range of MPO (20-60 pg/ml) and H,O, (0.2-I mM) under the experimental conditions used, where the liposomes
Myeloperoxidase-media lted lipid peroxidation
and the enzyme were separated initially from the substrate-H,Oz by a semipermeable membrane. Addition of KCN, an inhibitor of peroxidases, or taurine or tryptophan, alternative rectants for HOCl, inhibits lipid destruction. 4-Hydroxynonenal, a characteristic and one of the most cytotoxic products of lipid peroxidation, was identified and quantified by HPLC as a further product of the reaction of the MPO-H,O&system with liposomes. Rat neutrophils stimulated by polystyrene granules oxidized liposomes as shown by the TBARS test. This process was also inhibited by KCN, indicating the possible involvement of MPO in the neutrophilmediated lipid peroxidation. Human low density lipoprotein (LDL) was rapidly oxidized by the MPO-H,O&system. The kinetics of the oxidation was monitored continuously by measuring the 234 nm conjugated diene absorption. In the absence of chloride LDL was mildly oxidized, but in the presence of chloride a propagation phase with a rapid increase of the 234nm absorption was observed. Acknowledgemenrs-This paper was supported in part by Grant CPBP 04.01.2.08 from the Polish Academv of Sciences and by a grant from the Association for International Cancer Research, St Andrews. The authors are indebted to Professor 1. Olsson for a generous gift of human myeloperoxidase, to Professor H. Esterbauer for kind advice, to Mgr H. Puhl for a sample of human LDL and to Dr S. Supanz for technical assistance. REFERENCES
Carlin G. (1985) Peroxidation of hnolenic acid promoted by human polymorphonuclear leukocytes. J. Free Rud. Biol. Med. 1, 255-262. Carlin G. and Djurslter R. (1988)Peroxidation of phospholipids promoted by myeloperoxidase. Free Rad. Res. Commun. 4, 251-257.
Cathcart M. K., Morel D. W. and Chisholm G. M. (1985) Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J. Leukocyte Biol. 38, 341-350.
Clark R. A. and Klebanoff S. J. (1975) Neutrophil-mediated tumor cell cytotoxicity. Role of the peroxidase system. J. exp. Med. 141, 1442-1455. Claster S., Chin D. T.-Y., Quintauilha A. et al. (1984) Neutrophils mediate lipid peroxidation in human red cells. Blood 64, 1079-1084. Dewald B., Bretz U. and Baggiolini M. (1982) Exocytosis induced in neutrophils by chemotactic agents and other stimuli. In Leukocyte Lokomotion and Chemoraxis (Edited by Keller H. U. and Till G. 0.). Ann Arbour. Egger G. (1984) The Sephadex Model of acute inflammation. In Blood Cells in Nuclear Medicine. Part II. Migratory Blood Cells, pp. 103-I 16. Boston. Egger G. (1988) Characteristic of ingress and life span of neutrophils at a site of acute inflammation, determined with the Sephadex model in rats. Expl Puthol. 35, 209-218.
Esterbauer H., Cheeseman K. H., Dianzani M. U., Poli G. and Slater T. F. (1982) Separation and characterization of the aldehydic products of lipid peroxidation stimulated bv ADP-Fe*+ in rat liver microsomes. Biochem. J. 208. 129-140. Esterbauer H., Striegl G., Puhl H. and Rotheneder M. (1989) Continuous monitoring of in oitro oxidation of human low density lipoprotein. Free Rad. Res. Commun. 6, 67-75.
127
Esterbauer H. and Zollner H. (1989) Methods for determination of aldehydic lipid peroxidation products. Free Rad. Biol. Med. 7, 197-203.
Esterbauer H., Dieber-Rotheneder M., Waeg G., Striegl G. and Jiirgens G. (1990) Biochemical, structural and functional properties of oxidized low-density lipoprotein. Chem. Res. Toxieol. 3, 77-92.
Harrison J. E. and Seed F. A. (1981) Acetoacetate is an electron donor to myeloperoxidase and a promoter of myeloperoxidase-catalyzed fatty acid peroxidation. Biochem. Med. 26, 339-355.
Kagawa Y. and Racker E. (1966) Partial resolution of the enzymes catalyzing oxidative phosphorylation. J. biol. Chem. 241, 2467-2474. Kimura M., Murayama K., Nomoto M. and Fuyita Y. (1969) Calorimetric detection of peptides with tert-butyl hypochlorite and potassium iodide. J. Chromatogr. 4, 458-461.
Lin Y. Y., Wright C. E., Zagorski M. and Nakanishi K. (1988) 13-C-NMR study of taurine and N-chlorotaurine in human cells. Biochim. biophys. Acta 969, 242-250. McMenamy R. W., Lund C. C., Neville G. J. er al. (1960) Studies of unbound amino acids distributions in plasma, erythrocytes, leukocytes and urine of normal human subjects. J. clin. Invest. 39, 1675-1687. Odajima T. (1971) Myeloperoxidase of the leukocyte of normal blood. II. The oxidation-reduction reaction mechanism of the myeloperoxidase system. Biochim. biophys. Acta 235, 52-60.
Placer Z. A., Cushman L. L. and Johnson B. C. (1966) Estimation product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analyt. Biochem. 16, 359-364.
Putter J. and Becker R. (1986) Peroxidases. In Methods of Enzymatic Analysis (Edited Bergmeyer H. U., Bergmeyer J. and Grazzl M.), pp. 286-293. Vol. III. Schaur R. J. (1989) Aktivierter Sauerstoff und Lipidperoxidation bei der akuten Entzundung. Wissenschaftliche 2. Humboldt
Universitiii
Berlin 38, 95-99.
Sepe S. M. and Clark R. A. (1985) Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a hposome model and injury by myeloperoxidase, hydrogen peroxide and halides. J. Immunol. 134, 1888-1895. Sharonov B. P. and Govorova N. Y. (1990) Ability of serum proteins to inhibit MPO-dependent oxidation of red cells. Free Rad. Biol. Med. 9, Suppl. 1, abstract 24. Shvka A., LoBuglio A. F. and Weiss S. J. (1980) A potential role for hypochlorous acid in granulocyte-mediated tumor cell cytotoxicity. Blood 55, 347-355. Stossel T. P., Mason R. J. and Smith A. L. (1974) Lipid peroxidation by human blood phagocytes. J. clin. Invest. 54, 638-645.
Svensson B. E. (1988) Abilities ofperoxidases to catalyze peroxidaseoxidase oxidation of thiols. Biochem. J. 256, 757. Thomas M. J., Shirley P. S., Hedrick C. C. et al. (1986) Role of free radical processes in stimulated human polymorphonuclear leukocytes. Biochemistry 25, 8042-8048.
Weiss S. J. and Shvka A. (1982) Monocyte and granulocytemediated tumor cell destruction. A role for the hydrogen peroxide-myeloperoxidase-chloride system. J. c/in. Invest. 69, 255-262. Weiss S. J., Lampert M. B. and Test S. T. (1983) Long-lived oxidants generated by human neutrophils: Characterization and bioactivity. Science 222, 625-628. Winterbourn C. C. (1990) Neutrophil oxidants: Production and reaction. In Oxygen Radicals. Systemic Events and Disease Processes (Edited by Das D. K. and Essman W. B.), pp. 31-70. Kargel, Basel. Zgliczynski J. M., Selvaraj R. J., Paul B. B., Stelmaszynska T., Poskitt P. K. F. and Sbarra A. J. (1977) Chlorination by the myeloperoxidase-H,O,-Clantimicrobial system
128
TFXESA
?iTELht~sZnkzK~
at acid and neutral pH. Proc. Sot. expl. Biol. Med. 154, 418-422. Zgliczynski J. M. and Stelmasxynska T. (1979) Hydrogen cyanide and cyanogen chloride formation by the myeloperoxidase-H,O,-Clsystem. Biochim. biophys. Acta 567, 309-314.
et al.
Zglicxynski J. M. and Stehnasxytiska T. (1988) The respiratory burst of neutrophilic granulocytes and its influence on infected tissues: Indirect consequences. In The Respiratory Burst and Its Physiological Significance (Edited by Sbarra A. J. and Strauss R. R.), pp. 315-338. Plenum Press, New York.
Copyright
0
0020-7 11X/92 $5.00 + 0.00 1991 Pergamon Press plc
POSSIBLE INVOLVEMENT OF MYELOPEROXIDASE LIPID PEROXIDATION
IN
TRRRSASTRLMASZYI+SKA,~* ELISABETRKUKOVETZ,’ G. EGGRR~ and R. J. SCHADR’ ‘Institute of Biochemistry, University of Graz Schubertstrasse 1, A-8010 Graz, Austria and *Institute of Functional Pathology, University of Graz, Mozartgasse 14/D, A-8010 Graz, Austria (Received 28 January 1991) Abstract-l. Exposure of liposomes to the MPO-H,O,-Clsystem results in oxidation of lipids. Malondialdehyde and 4-hydroxynonenal are formed. 2. Oxidation of liposomes by stimulated rat neutrophils, assessed by malondialdehyde formation, is inhibited by KCN. This indicates involvement of MPO in the process. 3. The MPO-H,O, system oxidizes mildly LDL but in the presence of chloride a propagation phase, with a rapid increase of conjugated diene formation, was observed.
INTRODUCTION Myeloperoxidase (MPO)-an enzyme of neutrophilic granulocytes is released from the azurophilic granules after stimulation of the cells. The list of the activities of the enzyme is long: it peroxidizes classical peroxidase substrates such as phenols and aromatic amines;
in combination with H,02 and chloride ions it produces HOC1 which chlorinates many substrates such as amines, amino acids, proteins and other biologically active substances (for review see Zgliczynski and Stelmaszynska, 1988). This enzyme also exerts oxidase-peroxidase activity towards such substrates as NADH (Odajima, 1971), some thiols (Svensson, 1988) and acetoacetate (Harrison and Seed, 1981). MPO may develop its activity in uivo after stimulation of neutrophils, which triggers the respiratory burst producing hydrogen peroxide among other oxygen radicals, i.e. superoxide anion and probably hydroxyl radicals (for review see Winterbourn, 1990). Considerable interest is focused on the role of the neutrophil respiratory burst products in tumor cell cytotoxicity. The mechanisms of cytotoxic action of neutrophils are various and their effectiveness depends in great part on the target cell resistance. The cytotoxic action of neutrophils is partly based on the MPO-halogenating system, at least towards some cell lines (Slivka et al., 1980; Clark and Klebanoff, 1975; Weiss and Slivka, 1982). The question arises, as to what extent this cytotoxicity is dependent on membrane lipid peroxidation and formation of its secondary cytotoxic products. There is some information concerning the contribution of activated neutrophils to lipid peroxidation (Stossel et al., 1974; Claster et al., 1984; Carlin, 1985; Thomas et al., 1986). Recently it was found that in a model of acute inflammation activated neutrophils
*On leave from the Institute of Medical Biochemistry, Medical Academy, ul. Kopernika Poland. Bc 24,1--H
7, 31-034 Krakow,
form 4-hydroxynonenal (HNE )-one of the most cytotoxic products of lipid peroxidation (Schaur, 1989). Few investigations focus on MPO involvement in lipid pet-oxidation. It has been found that cofactors such as acetoacetate (Harrison et al., 1981) or chelated iron and iodide (Carlin and Djursiiter, 1988) are needed for MPO-mediated lipid peroxidation, assessed by thiobarbituric acid-reactive substance test (TBARS). Sepe and Clark (1985) have shown that the MPO-chlorinating system can lyse phospholipid liposomes. The aim of this study was to ascertain whether the MPO-H,O,-Clsystem acts on phospholipid liposomes and is responsible for the production of cytotoxic secondary products of lipid peroxidation (HNE). Also, this work was performed because of its possible relevance to atherosclerosis, which is now connected with oxidative modification of low density lipoprotein (Esterbauer et al., 1989). It is known that stimulated phagocytes of blood-neutrophils and monocytes oxidize low density lipoprotein making it cytotoxic to vascular cells (Cathcart et al., 1985). Therefore an attempt was made to show that the MPO-H,O, system and predominantly the MPOHrO&lsystem is involved in peroxidation of LDL.
MATERIALS AND METHODS Asolectin (phosphatidylcholine from soybean) containing 55% linoleid-acid and 8.2% linolenic acid (Kagawa and Racker, 1966). taurine and butvlated hvdroxvtoluene (BHT) were obtained from Sigma Chemical-Company (St iouis; U.S.A.). 2,2’azino-di-(3-ethyl-benzothiazohne-(6)-sulfonic acid (ABTS) and thiobarbituric acid (TBA) were from Serva (Fed. Rep. Germany). Myeloperoxidase (MPO) was a generous gift from Professor Inge Olsson (Lund University, Sweden). The enzyme activity was measured with ABTS as a substrate (Piitter and Becker, 1986). H,O, solutions were prepared from 30% H,O, (Merck, Fed. Rep. Germany) and standardized manganometrically. Polystyrene particles (0.8-l pm in diameter) were prepared in the Institute of
122
TERESA STELMA~ZY~SKA
Catalysis and Surface Chemistry, PAN, Krakow, Poland. The particles were washed thoroughly with water and suspended in 0.9% NaCl to concentration of 2 x 10” per ml. Silica Gel 60 plates, KCN, tryptophan, KI and other reagents of analytical grade were purchased from Merck (Darmstadt, Fed. Rep. Germany). Rat neutrophilic granulocytes were obtained from Sephadex G-200-mediated acute subcutaneous inflammatory focus (Egger, 1984). The suspension of the cells contained 96% neutrophils and 4% lymphocytes. Human LDL was prepared according to the method of Esterbauer et al. (1989). The stock solution of LDL (8.9 mg/ml) contained 1 mg/ml of EDTA. The final concentration of LDL in oxidation experiments was 0.26 mg/ml in 0.8 mM EDTA. Liposomes were prepared by vortexing 3.5 mg asolectin (4.6 pmol of lipids) in 1 ml 0.06 M phosphate buffer, pH 6.0, containing 0.16 M NaCl. Ass&s for lipid peroxidation
Since it is desirable to assess lipid peroxidation by more than one method, the following assays have been chosen: (1) TBA-reactive substance test: 1 ml of sample was mixed with 0.375% 2-thiobarbituric acid in 0.25 M HCl, containing 1% Triton X-100, and heated for 15 min at 100°C. The absorption spectrum of the TBARS chromophore was monitored using a Perkin-Elmer 554 Spectrophotometer; the c value, used for calculation was 1.52 x lo5 M-’ cm-’ at 532nm (Placer et al., 1966). (2) Iodometric estimation of excess of H,O,, lipid peroxide and chloramines. An t value of 2.6 x lo4 M-’ cm-’ for KI, at 355 nm was used for calculations (Kimura et al., 1969). (3) 4-Hydroxy,2,3-trans-nonenal (HNE) estimation (Esterbauer et al., 1982; Esterbauer and Zollner, 1989): The sample (1 ml) was mixed with 1 ml of 2,4-dinitrophenylhydrazine reagent (50 mg/lOO ml 1 M HCl) and with 10 ~1 0.1% BHT, and after 2 hr extracted with dichloromethane. The concentrated extract was pre-separated on a Silica Gel 60 plate (20 x 20cm) into three classes, using dichloromethane as developer. Hydrazones of the first class (around R, 0.12) were scraped off, extracted with methanol and concentrated under N,. Then, hydrazones were separated on a Spherisorb ODS 2 5~ column with methanolacetonitrilewater (27: 10: 12) at a flow rate of 0.8 ml/min, with a 370 nm detector wavelength, at ambient temperature. Peak identification and quantification was achieved by comparison with a HNE hydrazone standard separated under identical conditions. (4) Monitoring the increase of the 234nm absorbance resulting from conjugated fatty acid hydroperoxide formation (Esterbauer et al., 1989).
ef al.
continuously at ambient temperature with the Perkin-Elmer Spectrophotometer 554 (Esterbauer et al., 1989). RESULTS AND DISCUSSION
Oxidation H20,-Cl-
of phospholipid system
liposomes
by the MPO-
Formation of HOC1 by the MPO-HrOr system is optimal when the concentration ratio of the substrates [H,O,]/[H+] x [Cl-] is maintained at 500-1000 (Zgliczynski et al., 1977). At pH 6.0 and at 0.16 M NaCl the optimal concentration of H,Oz, which is not inhibiting the enzyme, is 0.16mM. Therefore, the total amount of HrO, in a small sample is rather low. In order to achieve higher total amount, but low temporary concentration of H202, the liposomes were suspended with MPO in a small volume (0.5 ml) and filled into a dialysis bag, which was placed into a relatively large solution of H,O, (5.0 ml). H,O, could continuously diffuse into the bag and under these conditions its optimal concentration in the external fluid was 0.5 mM (Fig. 1). It was found that the consumption of H,Or from the external compartment was accompanied by the formation of non-diffusible iodide-oxidizing species, most likely lipid peroxide (Fig. 2). Simultaneously the TBARS test was performed either with the liposome fraction inside the bag (Fig. 2) or with the total mixture of the bag content and the external solution. Since part of the TBARS can diffuse through the bag, the estimation of their amount in the total solution gave results which were about 200% higher compared with the amounts inside the bag (not shown).
Incubation procedures
(1) Liposomes and the MPO system: liposomes (2 pmol of lipid) and MPO (2.0-30 pg i.e. 9.6-144 mu) in 0.5 ml of 0.06 M phosnhate buffer with 0.16 M NaCl were incubated inside a dialysis bag (0.8 cm in diameter) in 5 ml of an external solution containing 0.1-1.0 mM H202 (total amount of H,O,: 0.5-5.0 hmol). These conditions allow low temporal concentration of H,O, inside the bag, but high total amount of this substrate. The duration of incubation was l-2 hr at 37°C with continuous stirring. (2) Liposomes and neutrophils: liposomes (2pmol of lipid) were incubated with neutrophils (3 x 10’cells) in 0.066 M phosphate buffer, pH 6.7, containing 0.07 M NaCl and 5.5 mM glucose, in total volume of 1.1 ml. Neutrophils were stimulated with polystyrene particles (IO8 per sample). (3) LDL and MPO: LDL (0.26mg/ml) was mixed with MPO (5.3 pg i.e. 25 mu) in 2 ml of 0.06 M phosphate buffer containing 0.8 mM EDTA and 0.16 M NaCl. The reaction was started by addition of 0.5 pmol H,O, and monitored
500 Wavelength
550
600
(nm)
Fig. 1. Influence of the H,O, concentration on liposome oxidation by the MPO system. Absorption spectra of TBARS chromophore obtained by applying the TBA test to aliquots of the liposome suspension inside the bags (10 pg MPO + 2 pmol lipid in 0.5 ml) after 1 hr incubation at 37°C of the bags in external solutions (5 ml) containing H,O, at various concentrations: (0) no H,O,; (1) 0.2mM; (2) 0.5 mM and (3) 1.0 mM H,O, in 0.06 M phosphate buffer, pH 6.0, containing 0.16 M NaCl. Details of the experiment as described in Methods. Absorption spectra were measured against a sample containing liposomes only.
Myelo~ro~da~-rn~iat~
lipid peroxidation
123
r
Trp (mMt KCN (mM! Composition of internal solution
Taurine (mM) Lipid (~mol) -
MPO &I)
2.3
10.6
32.0
10.6
10.6
2.5
10.6
Internal solution
External solution
”
0
1
2
3 Sample
4
5
6
no:
Fig. 2. Effect of taurine, KCN and tryptophan on lipid peroxidation by the MPO-H,O&Isystem. Composition of solution inside the bag (0.5 ml) is shown in the figure: external solution (5 ml) contained 2.5 pmol H,O,. Bar 0 shows calculated distribution of H,O, between internal and external solution at equilibrium. Hydroperoxides were determined using the iodometric method in external q and internal & compartment separately, after 1 hr incubation at 37°C. The absorbance of KI, originating from KI oxidation by hydroperoxides was measured after 90 min incubation of the samples with 38 mM KI in 12.5% CH,COOH at ambient temperature, in the darkness. Absorbance of KIT formed by KI oxidation by N-chloramines (samples 3, 4, 5) Qj was measured immediately after mixing the samples with 38 mM KI in 12.5% CH,COOH. The formation of TBARS was assessed as described in Methods.
Lipid peroxidation
in terms of TBARS
formation
was completely inhibited by 1 mM cyanide, which is not only a potent inhibitor of peroxidases but also a substrate of the MPO-I-&O&lchlorinating system (Zgliczybski and Stelmaszynska, 1979) (Fig. 2). A consumption of H20, in the KCN containing
6 (Fig. 2) indicates that cyanide acts as a substrate of the enzyme system. Taurine and tryptophan also prevented lipid from peroxidation (Fig. 2). Tryptophan is chlorinated and oxidized extensively by the MPO-H*O,-Clsystem (Zgliczynski and Stelmaszynska, 1988). sample
TERESASTELMASZY~SKA et
124
Taurine is chlorinated to N-chlorotaurine consuming more toxic HOCl, and due to this reaction is considered as buffer against oxidative damage by the MPO-chlorinating system (Slivka et al., 1980; Lin et al., 1988). Neutrophils contain high amount of taurine (McMenamy et al., 1960) and when stimulated release taurine chloramine (Weiss et al., 1983). Its accumulation in the environment proves that the rate of its formation exceeds consumption by components of the cells. The accumulation of N-chlorotaurine in the sample 5 (Fig. 2) proves that it does not react readily with lipids. Thus, it may be suggested that the kind of amine in a phospholipid (ethanolamine, serine or choline) may have influence on the susceptibility of fatty acid components of lipids to oxidation by the MPO-HI02Cl system. Indeed, Carlin and Djursster (1988) were unable to show peroxidation of liposomes by the MPO-H*O,-Cl- system. The liposomes used in their experiments contained 80-85% phosphatidyl serine. It is conceivable that all HOC1 formed by the enzyme system was consumed by chlorination of serine amino group to N-chloramine, the more so that the amount of H,Oz used was lower than the amount of lipids (Carlin and Djurslter, 1988). There was no lipid peroxidation in the absence of MPO at the H,02 concentration used (Figs 1 and 2). However, there was no linear dependence of the extent of TBARS formation on the MPO concentration (Fig. 3), in spite of increasing consumption of H,O, (Fig. 2). This may be caused by chlorination of the enzyme protein, competing with the lipid destruction process. Malondialdehyde-the main substrate for the TBARS chromophore can only be formed from fatty
O.‘O I
A
0.05
-
al.
acids with three or more double bonds, but not from linoleic acid which is the major polyunsaturated fatty acid (PUFA) in soybean phospholipid (55%). It seemed necessary to search for other aldehydes which are secondary products of lipid peroxidation. The estimation of aldehydes such as 4-hydroxynonenal can prove that lipid peroxidation has really occurred (Esterbauer and Zollner, 1989). Hydroxynonenal (HNE)-a product of o-6 PUFAs (linoleic acid) peroxidation was estimated as 2,4_dinitrophenylhydrazone according to Esterbauer et al. (1982) and Esterbauer and Zollner (1989). Preseparation by TLC of the hydrazones from sample 3 shown in Fig. 2 has revealed some carbonyl products in all three classes (zones) of different polarities (Esterbauer and Zollner, 1989). HNE was identified and quantified by subsequent HPLC separation (Fig. 4). It was found that in liposomes composed as sample 3 in Fig. 2, 0.23 nmol/ml of HNE was formed during 1 hr at 37°C. A more than lo-fold increase in HNE formation (3.2 nmol/ml) was observed after longer incubation (2 hr) at 37°C. In the control samples without MPO, but with H,Oz, there was only a trace of HNE (c 0.05 nmol/ml). All hydrazones found on the TLC plates except HNE are as yet unidentified. It is tempting to speculate that some of them are products chlorinated by the MPO system. Sepe and Clark (1985) suggested this new possibility-that phospholipid membrane injury caused by the MPO-H,O,-Clsystem may involve lipid halogenation. Oxidation of liposomes by stimulated neutrophils
Neutrophils obtained from rat subcutaneous exudate were stimulated by polystyrene latex granules to phagocytosis. The phagocytosing cells appear to be at the highest level of stimulation. The respiratory burst of the activated cells-production of superoxide and hydrogen peroxide is accompanied by leakage of azurophilic granules into the extracellular surroundings, most probably through phagocytic vacuoles open to the outside (Dewald et al., 1982). In this way myeloperoxidase and H,O, are released by the cells. Liposomes incubated with the stimulated neutrophils undergo oxidation. It was found that 3 x 10’ neutrophils/ml incubated with polystyrene granules and liposomes in buffer pH 6.7, with glucose and NaCl, produced about 1.6 nmol TBARS after 95 min (Fig. 5). The formation of TBARS was almost completely abolished by 1.5 mM KCN, which indicates involvement of MPO in this process. Peroxidation of human LDL by the MPO-H20, the MPO-H202-Clsystem
500 Wavelength
550
600
(nm)
Fig. 3. Influence of MPO concentration on liposome oxidation. Absorption spectra of the TBARS chromophore for aliquots of liposome suspension inside the bag (MPO + liposomes, 0.5 ml) after 1 hr incubation of the bags at 37°C in the 5 ml of external 0.5 mM H,O, solution. All in 0.06 M buffer, pH 6.0 0.16 M NaCI. (0) without MPO; (1) 2.3 pg MPO; (2) 10.6 pg MPO; (3) 32 pg MPO. Absorption spectra measured against sample containing only liposomes in buffer solution.
and
Peroxidation of LDL was measured by monitoring the change of the 234nm absorption resulting from formation of lipid conjugated diene hydroperoxide (Esterbauer et al., 1989) (Fig. 6). Kinetic pattern of the process indicates that MPO is an especially effective catalyst in the presence of H,O, and chloride suggesting participation of HOC1 in the reaction. In the absence of NaCl MPO catalyzes only mild oxidation of LDL by H,02 (Fig. 6). The propagation phase of LDL oxidation is seen in the presence of NaCl only. Taking into account an 6234= 29,500 M-’ cm-’ for conjugated lipid peroxides
Myelo~ro~da~-rn~iat~
lipid peroxidation
125
I P
P
c
2
4
6
8
10
12
14
0
Retention
time
2
4
6
8
10
12
14
16
(min)
Fig. 4. Separation of the dinitrophenylhydrazone of HNE by HPLC: (A) aliquot of the Iiposome suspension composed as sample 3 in Fig. 2; (B) standard HNE solution containing 80 pmol/20 ~1 injection sample. Details of the separation are described in Methods. HNE peak shown by arrow.
and assuming that the maximum increase of A = 2.49 originated from conjugated diene formation, it may be calculated that 336 nmoles of dienes/mg of LDL were formed under our experimental conditions
(Fig. 6). Since on the average LDL contains 556 nmol activated methylene groups per mg of LDL (Esterbauer er al., 1990), the majority of PUFAs (about
60% of activated methylene groups) have been peroxidized. Oxidation of LDL by the MPO-H20&1system was inhibited completely by I mM KCN-the inhibitor of hemeproteins, and was inhibited partially by 2.4mM taurine-the acceptor of HOCl. CONCLUDING REMARKS
0.08
I
‘~“.‘,:, , L
,
( , , ,
450
500
WaveLength
550
600
(nml
Fig. 5, Peroxidation of Iiposomes by rat neutrophils stimulated by polystyrene granules. Neutrophils (3 x IO’cells) were incubated for 95min at 37°C with: (I) liposomes (2 pmol pho~holipid), polystyrene granules (10s); (2) as 1 + KCN (1.5 mM at Iinal con~ntration); (3) only polystyrene granules (lOa). All samples in 0.066M phosphate buffer, pH 6.7 containing 0.07 M NaCl and 5.5 mM glucose in total volume 1.1 ml. The results are expressed as absorption spectra of the TBARS chromophore obtained in aliquot of the supernatant after centrifugation of the cells and granules. Absorption spectra were measured against supematant of the sample containing only liposomes and granules.
Experiments with the three model system usednamely purified MPO plus liposomes; purified MPO plus LDL; phagocytic cells, which can release MPO upon stimulation, plus liposomes-are in agreement with the assumption that the MPO-H,O,-Clsystem can promote lipid peroxidation in vitro. From these results several new questions arise. The mechanism of action of the MPO-H,O,-Clsystem on lipids is not known. It has been found that the enzyme system is continuously needed for the proceedings of lipid peroxidation presenting evidence against a free radical chain process (Sepe and Clark, 1985). The question arises whether the MPO-mediated lipid pet-oxidation has any biological relevance. It is very probable in oivo that MPO is released in inflammatory foci during phagocytosis (Dewald et al., 1982) or from disrupted neutrophils, which have a very short lifespan during the period of intensive ingress (Egger et al., 1988). The MFQ system may also act if neutrophils are in close contact with target cell membranes as in the antibody-de~ndent attack on the target celis. Then, at least part of the cytotoxic action of neutrophiis may originate from the reaction of HOC1 (formed by the MPO-catalyzed oxidation of ubiquitous Cl- ions by the product of the respiratory burst-H,Oz) with lipids of membranes. Another point is to which extent ammo compounds present in inflammatory foci are alternative
126
TFRESA
&ELMASZYikKAet d.
2.5
0.:
IO
20
30
40 Time
50
60
TO
80
(mint
Fig. 6. Time course of LDL oxidation by the MPO-H,O, and the MPO-H,Or-Cl- system. Change of the 234 nm absorbance was measured in the samples containing: MPO (5.3 pg i.e, 25.4 mu), 0.8 mM EDTA, LDL (0.52 mg) in 2 ml of 0.06 M phosphate buffer, pH 6.0, with 0.16 M NaCl x x , 0-O or without NaCl x---x , O---O; A.. .A the same as 0-O + 2.4 mM taurine; O---O the same , as O---- 0 + 1 mM KCN. Reaction was started with one portion of H20, (1 pmol) x ---x , x-x or with two portions of H,O, (2 x OS@mol) O---O, O-0, indicated by arrows. The reference sample contained LDL and MPO in the same buffer. There was no change of the 234 nm absorbance in the samples containing H,O, but without the enzyme. Other details were described in Methods.
for HOCI. Recently it has been shown by Sharonov and Covorova (1990) that serum proteins compete with erythrocyte membranes for HOCI. reactants
SUMMARY
The possible involvement of myeloperoxidase (MPO) was studied in several model systems of lipid pet-oxidation.
Exposure of liposomes to the MPO-H,02-Clsystern results in consumption of H,O,, formation of iodide-oxidizing species (lipid hydroperoxides?) and of thiobarbituric acid reactive substrates (TBARS). Oxidation does virtually not occur in the absence of either MPO or H20,. The extent of lipid peroxidation is optimal in narrow concentration range of MPO (20-60 pg/ml) and H,O, (0.2-I mM) under the experimental conditions used, where the liposomes
Myeloperoxidase-media lted lipid peroxidation
and the enzyme were separated initially from the substrate-H,Oz by a semipermeable membrane. Addition of KCN, an inhibitor of peroxidases, or taurine or tryptophan, alternative rectants for HOCl, inhibits lipid destruction. 4-Hydroxynonenal, a characteristic and one of the most cytotoxic products of lipid peroxidation, was identified and quantified by HPLC as a further product of the reaction of the MPO-H,O&system with liposomes. Rat neutrophils stimulated by polystyrene granules oxidized liposomes as shown by the TBARS test. This process was also inhibited by KCN, indicating the possible involvement of MPO in the neutrophilmediated lipid peroxidation. Human low density lipoprotein (LDL) was rapidly oxidized by the MPO-H,O&system. The kinetics of the oxidation was monitored continuously by measuring the 234 nm conjugated diene absorption. In the absence of chloride LDL was mildly oxidized, but in the presence of chloride a propagation phase with a rapid increase of the 234nm absorption was observed. Acknowledgemenrs-This paper was supported in part by Grant CPBP 04.01.2.08 from the Polish Academv of Sciences and by a grant from the Association for International Cancer Research, St Andrews. The authors are indebted to Professor 1. Olsson for a generous gift of human myeloperoxidase, to Professor H. Esterbauer for kind advice, to Mgr H. Puhl for a sample of human LDL and to Dr S. Supanz for technical assistance. REFERENCES
Carlin G. (1985) Peroxidation of hnolenic acid promoted by human polymorphonuclear leukocytes. J. Free Rud. Biol. Med. 1, 255-262. Carlin G. and Djurslter R. (1988)Peroxidation of phospholipids promoted by myeloperoxidase. Free Rad. Res. Commun. 4, 251-257.
Cathcart M. K., Morel D. W. and Chisholm G. M. (1985) Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic. J. Leukocyte Biol. 38, 341-350.
Clark R. A. and Klebanoff S. J. (1975) Neutrophil-mediated tumor cell cytotoxicity. Role of the peroxidase system. J. exp. Med. 141, 1442-1455. Claster S., Chin D. T.-Y., Quintauilha A. et al. (1984) Neutrophils mediate lipid peroxidation in human red cells. Blood 64, 1079-1084. Dewald B., Bretz U. and Baggiolini M. (1982) Exocytosis induced in neutrophils by chemotactic agents and other stimuli. In Leukocyte Lokomotion and Chemoraxis (Edited by Keller H. U. and Till G. 0.). Ann Arbour. Egger G. (1984) The Sephadex Model of acute inflammation. In Blood Cells in Nuclear Medicine. Part II. Migratory Blood Cells, pp. 103-I 16. Boston. Egger G. (1988) Characteristic of ingress and life span of neutrophils at a site of acute inflammation, determined with the Sephadex model in rats. Expl Puthol. 35, 209-218.
Esterbauer H., Cheeseman K. H., Dianzani M. U., Poli G. and Slater T. F. (1982) Separation and characterization of the aldehydic products of lipid peroxidation stimulated bv ADP-Fe*+ in rat liver microsomes. Biochem. J. 208. 129-140. Esterbauer H., Striegl G., Puhl H. and Rotheneder M. (1989) Continuous monitoring of in oitro oxidation of human low density lipoprotein. Free Rad. Res. Commun. 6, 67-75.
127
Esterbauer H. and Zollner H. (1989) Methods for determination of aldehydic lipid peroxidation products. Free Rad. Biol. Med. 7, 197-203.
Esterbauer H., Dieber-Rotheneder M., Waeg G., Striegl G. and Jiirgens G. (1990) Biochemical, structural and functional properties of oxidized low-density lipoprotein. Chem. Res. Toxieol. 3, 77-92.
Harrison J. E. and Seed F. A. (1981) Acetoacetate is an electron donor to myeloperoxidase and a promoter of myeloperoxidase-catalyzed fatty acid peroxidation. Biochem. Med. 26, 339-355.
Kagawa Y. and Racker E. (1966) Partial resolution of the enzymes catalyzing oxidative phosphorylation. J. biol. Chem. 241, 2467-2474. Kimura M., Murayama K., Nomoto M. and Fuyita Y. (1969) Calorimetric detection of peptides with tert-butyl hypochlorite and potassium iodide. J. Chromatogr. 4, 458-461.
Lin Y. Y., Wright C. E., Zagorski M. and Nakanishi K. (1988) 13-C-NMR study of taurine and N-chlorotaurine in human cells. Biochim. biophys. Acta 969, 242-250. McMenamy R. W., Lund C. C., Neville G. J. er al. (1960) Studies of unbound amino acids distributions in plasma, erythrocytes, leukocytes and urine of normal human subjects. J. clin. Invest. 39, 1675-1687. Odajima T. (1971) Myeloperoxidase of the leukocyte of normal blood. II. The oxidation-reduction reaction mechanism of the myeloperoxidase system. Biochim. biophys. Acta 235, 52-60.
Placer Z. A., Cushman L. L. and Johnson B. C. (1966) Estimation product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analyt. Biochem. 16, 359-364.
Putter J. and Becker R. (1986) Peroxidases. In Methods of Enzymatic Analysis (Edited Bergmeyer H. U., Bergmeyer J. and Grazzl M.), pp. 286-293. Vol. III. Schaur R. J. (1989) Aktivierter Sauerstoff und Lipidperoxidation bei der akuten Entzundung. Wissenschaftliche 2. Humboldt
Universitiii
Berlin 38, 95-99.
Sepe S. M. and Clark R. A. (1985) Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a hposome model and injury by myeloperoxidase, hydrogen peroxide and halides. J. Immunol. 134, 1888-1895. Sharonov B. P. and Govorova N. Y. (1990) Ability of serum proteins to inhibit MPO-dependent oxidation of red cells. Free Rad. Biol. Med. 9, Suppl. 1, abstract 24. Shvka A., LoBuglio A. F. and Weiss S. J. (1980) A potential role for hypochlorous acid in granulocyte-mediated tumor cell cytotoxicity. Blood 55, 347-355. Stossel T. P., Mason R. J. and Smith A. L. (1974) Lipid peroxidation by human blood phagocytes. J. clin. Invest. 54, 638-645.
Svensson B. E. (1988) Abilities ofperoxidases to catalyze peroxidaseoxidase oxidation of thiols. Biochem. J. 256, 757. Thomas M. J., Shirley P. S., Hedrick C. C. et al. (1986) Role of free radical processes in stimulated human polymorphonuclear leukocytes. Biochemistry 25, 8042-8048.
Weiss S. J. and Shvka A. (1982) Monocyte and granulocytemediated tumor cell destruction. A role for the hydrogen peroxide-myeloperoxidase-chloride system. J. c/in. Invest. 69, 255-262. Weiss S. J., Lampert M. B. and Test S. T. (1983) Long-lived oxidants generated by human neutrophils: Characterization and bioactivity. Science 222, 625-628. Winterbourn C. C. (1990) Neutrophil oxidants: Production and reaction. In Oxygen Radicals. Systemic Events and Disease Processes (Edited by Das D. K. and Essman W. B.), pp. 31-70. Kargel, Basel. Zgliczynski J. M., Selvaraj R. J., Paul B. B., Stelmaszynska T., Poskitt P. K. F. and Sbarra A. J. (1977) Chlorination by the myeloperoxidase-H,O,-Clantimicrobial system
128
TFXESA
?iTELht~sZnkzK~
at acid and neutral pH. Proc. Sot. expl. Biol. Med. 154, 418-422. Zgliczynski J. M. and Stelmasxynska T. (1979) Hydrogen cyanide and cyanogen chloride formation by the myeloperoxidase-H,O,-Clsystem. Biochim. biophys. Acta 567, 309-314.
et al.
Zglicxynski J. M. and Stehnasxytiska T. (1988) The respiratory burst of neutrophilic granulocytes and its influence on infected tissues: Indirect consequences. In The Respiratory Burst and Its Physiological Significance (Edited by Sbarra A. J. and Strauss R. R.), pp. 315-338. Plenum Press, New York.
