8/4/2019 Rupture Erito

1/5

Volume 303, number 2,3, 154-158 FEBS 110800 1992 Federation of European Biochemical Societies 00145793/9Y%5.00June 1992

The interaction between ruptured erythrocytes and low-densitylipoproteinsGeorge Paganga, Catherine Rice-Evans, Rebecca Rule and David Leakeb

Divisio n of Biociretnist ry, Unircd Medical and Dettral Scltooh o/Guy s and Sr. Tlt otnuss Hospituh, Lantberh Pulace Road, Lond onSE1 TEH, UK and bDepctrtt nettf f Phy siology and Biochemist ry, Vt?iversiry of Reading, Reading. UK

Received 1 April 1992Low-density lipoproteins (LDL) are oxidatively modified on interaction with haem proteins. The interaction of ruptured erythrocytes with LDLinduces oxidative damage as detected by alterations in electrophorctic mobility and the peroxidation of the polyunsaturated fatty acyl chains.Difference spectroscopy reveals that the amplification of the oxidative process by the hacm protein is related to the transition of [he oxidation stateof the haemoglobin in the erythrocyte lysate from the oxy [x-Fei-O1] to the ferry1 [X-Fe =O] form. The incorporation of the lipid-solubleantioxidant, butylated hydroxy toluene, al specific time points during the LDL-erythrocyte interaction prolongs the lag phase to oxidation andeliminates the oxy-to.ferryl conversion of the haemoglobin. The timescale of this haem conversion is related WI he antioxidant status of the LDL.

Erythrocyte; Haemoglobin; Low-density lipoprotein (LDL); Fcrryl radical species; Iron release

1. INTRODUCTIONMany experimental approaches are currently beingapplied to the study of the susceptibility of low-densitylipoprotein (LDL) in vitro, to oxidative modification[l-4]. Specifically, it has been noted that all the systemsapplied involve at least the co-participation of transitionmetal ions or haem proteins. Thus, incubation in invitro systems with transition metal ions, especially cop-per [l], metmyoglobin or ferry1 myoglobin [5], and with

cellular systems such as cultured macrophages, smoothmuscle cells or endothelial cells [3,4,6] are capable ofmediating LDL oxidation. Furthermore, the macro-phage-induced oxidation ha: 29 absolute requirementfor the presence of iron in the culture medium [2]. Sinceoxidation of LDL is likely to be involved in the initialstages of an atherosclerotic lesion ([7,8], reviewed in [9]),it is relevant to consider a source of oxidants which maybe located, under specific circumstances, in the arterywall.

LDL peroxidation, is related to the time-range duringwhich oxyhaemoglobin [HX-Fe..O,J becomes acti-vated to the ferry1 state [HX-Fe=O]. The time of thehaem conversion is dependent on the antioxidant statusof the LDL. The incorporation of lipid-soluble antioxi-dants into the LDL at specific time points during theexposure to erythrocytes prolongs the lag phase ot oxi-dation, eliminates the oxy-to-ferry1 conversion of thehaemoglobin and delays the oxidative modification ofLDL.2. MATERIALS AND METHODS

We have explored the possibility of erythrocyte dis-ruption and subsequent oxidative changes inoxyhaemoglobin as a potential mediator of LDL oxida-tion. Thus we have investigated the ability of LDL tointeract with haemoglobin in lysates released from rup-tured erythrocytes, and the conditions under which sucherythrocytes induce oxidative modification of LDL.The results show that the onset of oxidative modifica-tion of LDL, as detected by altered surface charge and

Fresh human blood was obtained from human volunteers (withinformed consent) and the plasma was separated after ccntrifugationfor use in the isolation of LDL. The huffy coat was removed anddiscarded and the packed cells were washed three timed with isotonicS mM phosphate-buffered saline, pH 7.4, by centrifugation at 4,000x g, 4% for 20 min. Erythrocytes were lysed with hypertonic phos-phate buffer, the membranes discarded and the oxyhaemoglobin con-centration of the supernatant detcrmincd from the extinction coeffiacients [IO] by measuring the absorbance in the Soret region. LDL wasprepared from the fresh plasma in a Kontron 2070 ultracentrifugcfitted with a fixed-angle Kontron rotor, according to the method ofChung el al. [II] for 3 h at 100,000 x g and lhen the LDL wasre-centrifuged for 14 hat 100,000xg to remove minor protein contaminanu. The concentration of protein was determined by a modifiedprocedure of Markwell et al. [I21 and the LDL was used at a finalconcentration of OS mg protein/ml,

Corrrspo~dcnce odrlrrss: C. l&e-Evans, Division of Biochemistry.UMDS St. Thomass Hospital, Lambeth Palace Road, London SEI7EH, UK, Fax: (44) (71) 928 3619.

LDL was incubated at 37C in the presence or the haemolysatc atfinal concentration of 20 PM oxyhaemoglobin. In some experimentshydrogen peroxide was added at a linal concentration of 25 PM.Oxidativc modification of the LDL was assessed by measuring thealtered surface charge. Samples were applied to agarosc gels (Beckmanparagon lipo clcctrophorcsis system) to osscss the increased relativeelectrophorctic mobility of the LDL. Lipoproteins were visualised bystaining with Sudan black B stain. Theextent of lipid peroxidation wasassayed using a modified thiobarbituric acid assay [13] with the ab.

IS4 Published by Elscsic: Science Pubiishcrs L1.V,

8/4/2019 Rupture Erito

2/5

Volume 303, number 2,3 FEBS LETTERS June 1992sorbance of the chtomaphore measured at 532 nm, corrcctcd forbackground absorbance at 580 nm due to possible contributions fromhaem compounds. Appropriate controls were incorporated accordingto Gutteridgc et al. [141.The visible spectra were recorded on a Beckman DU65 spcctro.photometer fitted with Quant 1 software and linked to an IBM PC/Z.Spectroscopic evaluation of the hacm oxidation states in terms of theconcentration of oxyhacmoglobin, mcthacmoglobin. ferry1 hacmo-globin and deoxyhaemoglobin were calculated by applying the milli-molar extinction coefficients of each of the baemoglobin species at theappropriate wavelengths [IS]. Observations of the effects of LDLoxidation on the state of the haemoglobin as a function of time weremeasured as dilference spectra with prior subtraction of the LDLspectrum. After interaction between haemolysate and LDL, totalhaemoklobin levels were measured after conversion to cyan-methaemoglobin with potassium fcrricynnide. or reduction to deoxy-haemoglobin by sodium dithionitc, and calculated by applying thestandard extinction coefficients of these different forms [I 51.

3. RESULTSLDL (0.5 mg/ml) was exposed to oxyhaemoglobin(20yM), in freshly prepared membrane-free erythorcyte

lysate, in the presence and absence of hydrogen perox-ide (25 yM) for a range of time periods up to 6 h at37C.The oxidative responses were studied by measuring(i) changes in the surface charge altering the electro-phoretic mobility of the LDL, (ii) the oxidation state ofthe haemoglobin by visible spectroscopy, (iii) the extentof lipid peroxidation.Tabel I shows the alteration in electrophoretic mobil-ity of LDL after treatment with membrane-free erythro-cyte lysate for up to 6 h. The additional presence ofhydrogen peroxide in the incubation medium inducesno further alteration in the surface charge. These resultsare consistent with the timescale for the erythrocyte-induced lipid peroxidation (Fig. I), as assessed by theformation of thiobarbituric acid-reactive substances.The membrane-free haemolysate in the absence andpresence of hydrogen peroxide elicited the same extentof oxidation to the polyunsaturated fatty acid side-chains of the lipids.Observations on the influence of LDL on the oxida-tion state of the haemoglobin in the haemolysate wereundertaken by scanning the difference spectrum of LDLin the presence of haemolysate, in the presence andabsence of exogenous hydrogen peroxide, with the sub-traction of the spectrum of LDL. The changes in thespectra of the oxidised forms of the haemoglobin withtime are illustrated in Fig .2. As the time proceeds, thespectra reveal that the oxyhaemoglobin, characterisedby peaks at 541 and 577 nm, declines slowly initially oninteraction with the LDL. After the slow phase a suddentransition to deoxyhaemoglobin occurs (at I = 210 minfor this LDL prep.), as shown on the spectrum, with thetransient appearance of a single peak at 555 nm, charac-teristic of deoxyhaemoglobin replacing the 541 and 577doublet of oxyhaemoglobin. This is followed by conver-sion to a spectrum, changing with time, with the appear-

0 1 2 3 4 STime(h)Fig. I. The time dependency ofthecxtent of lipid peroxidation of LDL(0.5 mS/ml protein) induced by etythrocyte lysate(20,uM) + hydrogenperoxide (2.5 PM). as thiobarbituric acid-reactiveompounds.

ante characteristic of mixed spectral forms of met, ferry1and oxy haemoglobin. Calculation of the proportion ofoxy, met and ferry1 haemoglobin by applying the extinc-tion coefficients (Winterbourn [35]) allows the deterrni-nation of the concentration of each oxidised form, ex-pressed in Fig. 3 as the percentage of the original haemconcentration. The production of ferry1 haemoglobin isconfirmed by the characteristic shifts of the Soret band,the red shift from 414 to 420 nm representing fcrrylformation and the blue shift to 403 nm representing themet state (Fig. 2). Concomitantly the propagation oflipid peroxidation is amplified after progressionthrough the lag phase (as seen in Fig. 1).Assessment of the effects of adding a lipid-solubleantioxidant to the LDL on the time to oxidative transi-tion of the oxyhaemoglobin was undertaken (Fig. 4).Incorporation of butylated hydroxytoluene (BHT) intothe LDLIerythrocyte incubations 30 and 90 min afterthe initiation of the interaction suppresses the oxidisingeffects of the haemoglobin system on the LDL (withsuppression of lipid peroxidation) and this is reflectedin the oxidation state of the haem protein, as shown inthe spectra (Fig. 4). Addition of BHT attenuates the lossof oxyhaemoglobin as shown in panel 6 compared to

TableThectTects ofcrythrocytc lysate(20pM) 4 hydrogen peroxidc(2SluM)on the relative electrophorctic mobility (REM) of LDL (OS mS/mlprotein) as a function of timeTime (h) REM0 I0.5 I1 I .062 I.203 1.404 I so5 1.606 I .68

155

8/4/2019 Rupture Erito

3/5

Volume 03, umber .3 FEBS LETTERS June 1992

405 460 515 510 625 660 sdo SSO 6dO Ski0

Fig. 2. Difference spectra as a function of time of oxyhaemoglobin in erythrocyte lysatc 2 hydrogen peroxide (25 PM) as it oxidises LDL, withthe subtraction of the LDL, (Panel A) The Sorer region: (1) SOmin. 100 min. (2) 2SOmin. (3) 300 min, (4) 450 min. The arrows indicate the directionof the absorbance changes. (Panel B) Visible longwave spectral region: the arrows indicate the direction of the absorbance changes at time pointsof 50, 100, 150, 200, 250, 300. 350, 400 and 450 min.

panel A; the later addition of the BHT, within the timeperiod during which the pronounced loss of oxyhaemo-globin would have occurred in its absence, sustains theoxyhaemoglobin level even further and inhibits the oxi-dation of LDL by the haemoglobin,In the absence of BHT (Fig. 5) the oxyhaemoglobinlevel slowly declines as oxidation occurs, the slow phasebeing considerably shorter for this LDL preparationcompared to that in Fig. 3 due to the well-documentedvariation in the susceptibility of LDL isolated from dif-ferent donors to oxidation [16,17]. At 100 min there isa sudden rapid transition to the deoxy form, as de-scribed above for the LDL preparations shown in Fig.3. In the presence of BHT, the deoxyhaemoglobin con-version is prevented and the formation of ferrylhaemo-globin is inhibited, with the oxyhaemoglobin levelslowly and progressively declining with time (Fig. 5).The maintenance of the oxyhaemoglobin state is verypronounced with the later addition of the antioxidant,within the timescale prior to the induction of the pro-nounced oxidative activation of the haemoglobin. Thedata show no loss of haem and no release of iron fromthe haemoglobin during the period of the reaction, Upto 100 min (Fig. 5) there is no distinction between theslow rates of oxidation of the oxyhaemoglobin whetherthe BHT is present or not. As the duration of the inter-action between the LDL and the haemolysate is pro-longed, the BHT prevents the oxidative transition of156

oxy, via deoxy, to ferryl. The addition of the antioxidantduring the lag phase (prior to the time at which the rapiddecrease in the oxyhaemoglobin levels occurs) sup-presses lipid peroxidation as assessed by measurementsof electrophoretic mobility and inhibition of the pro-duction of thiobarbituric acid-reactive substances.

00 100 200 300Tirpn f m i n iI. . \ ...a..,

Fig, 3. The proportions of oxy-, met- and ferry1 haemoglobin generatedduring the interaction oferythrocyte lysate (20_~Maemoglobin) withLDL (0.5 mg/ml protein).

8/4/2019 Rupture Erito

4/5

Volume 303, number 2,3 FEBS LETTERS June 1992

.I......,....,....,.,..,..,,,.........., ..,..I..,.,...,... * ,.., . ..,. . ..,, - a... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T525 560 595 630 635 525 560 595 630 6&- 625 560 595 639 665Fig. 4. Difference spectra of the effects of the antioxidant, butylated hydroxytoluene, on the spectral transition of oxyhaemoglobin in crythrocytelysate as it reacts with LDL. with the subtraction of the LDL. (Panel A) LDL (0.5 mdml protein), erythroWe iysate (20pM hamOg!Obin). hndB) Addition of BHT (lOOtiM final concentration) 30 min after the initialion of the reaction. (Panel C) Addrtionof BHT 90 min after the initiationof the interaction, The arrows show the direction of the absorbance changes at time points of 3, 30.60, 120, 150. 180, 210 and 300 min.

4. DISCUSSIONIt is well known that haemoglobin possesses peroxi-dase activity [18] and that the protein in both its oxy-and met forms (as well as free haemin) catalyses theoxidation of unsaturated fatty acids [19-211. Addition-

ally, the haem proteins, haemoglobin and myoglobin,activated by hydrogen peroxide can initiate the oxida-tion of cell membranes and of LDL [5,22,23]. Elevatedlevels of hydrogen peroxide .or organic hydroperoxidesinduce destabilisation of the haem ring and release ironfrom haemoglobin 124,251.Similar effects of excess hy-drogen peroxide on myoglobin have been observed[23,26]. Previous work from this group has shown thatruptured cardiac myocytes under conditions of oxida-tive stress [27] generate free radicals, the identity ofwhich can be assigned to the ferry1 myoglobin radicalspecies. Giulivi and Davies [28] have demonstrated thatcontinual exposure of intact erythrocytes to a flow ofhydrogen peroxide leads to the formation of ferry1haemoglobin; their proposal is that the synproportiona-tion reaction rapidly reduces the ferry1 haemoglobin tomethaemoglobin, a mechanism which has also been de-scribed in myoglobin/hydrogen peroxide systems inwhich the mole ratio of the haem protein concentrationwas in excess of that of the oxidising component [29].The resu!ts described in this paper clemonstra?e thatmembrane-free red cell haemoiysatcs mediate the oxida-tive modification of LDL without addition of ex-ogenous hydrogen peropide; transition of the oxidation

state of the oxyhaemoglobin il) the haemolysate signalsthe enhancement of lipid peroxidation and the alteredsurface charge on the LDL.The mode of action of transition metals or haemproteins, such as habmoglobin and myoglobin, in the

1 0 0

6060

000 100 200 300Timejmln)

Fig. 5. The decline in the oxyhaemoglobin levels as a function oftimei n crythrocyte lysate during interaction with LDL, in the presence andabsence of BHT, (0) No BHT addition; (0) BHT added 30 min afterthe initiation of the interaction; (& BHT added 90 min after theinitiation of the interaction.

157

8/4/2019 Rupture Erito

5/5

Volume 303, number 2,3 FEBS LETTERS June 1992oxidation of LDL is through the catalysis of the propa-8ation of the oxidation of preformed hydroperoxides[30,31]:

LOOH t M(+)+ -> LOO* i- MLOOH + M _> LO + M(+l)fLOO + LH -=r LOOH I- LLO + LH ->LOH + LL- I- 02 -->LOOLOO + LH ->LOOH + L=The data here suggest that after an initial slow phasecorresponding to the antioxidant capacity of the LDL,hydroperoxides can interact with haemoglobin in a sim-ilar manner to hydrogen peroxide, forming ferry1haemoglobin which is then rapidly reduced to mixturesconsisting mainly of oxy- and met forms, possibly by thesynproportionation reaction [28],Incorporation of the chain-breaking antioxidant ar-rests the rapid transition from the oxy to the ferry1 form

and inhibits LDL oxidation, supporting the idea that itis the interaction between lipid hydroperoxides andoxyhaemoglobin which is essential for the haemo-globin-mediated modification to LDL to take place.Enhancement of the antioxidant status of the LDL in-creases the resistance of LDL to oxidation and to oxida-five damage induced by erythrocyte lysate; thus the an-tioxidant capacity of the LDL is a controlling factor inthe oxidation of oxyhaemoglobin to more reactive,damaging forms.Balla et al. [32] have recently reported the destructionof haem and the release of iron on interaction of LDLwith haemin/hydrogen peroxide mixtures (ten- ortwenty-fold molar excess of peroxide). Our studiesclearly show that during interaction between LDL anderythrocyte lysate, LDL oxidation and oxidative activa-tion of the oxyhaemoglobin occur with no requirementfor exogenous oxidants, such as hydrogen peroxide, andinvolving no haem destruction nor iron release duringthe timescale studied. Any added hydrogen peroxidewould clearly be removed by catalase action in thehaemolysate.It has been shown that probucol or BHT lower thefrequency of occurrence of atherosclerotic lesions inanimals [33,34]. Our examination of the incorporationof the lipid-soluble antioxidant, butylated hydroxytolu-ene, into the EDL/haem protein systems reveals a delayin the onset of the oxidative conversion of theoxyhaemoglobin, the abolition of the transition to theferryl- and met state and the inhibition of LDL oxida-tion. Thus haem proteins leaking from ruptured cellsmay be capable of enhancing the oxidation of LDLwhich has penetrated the endothelium of the coronaryvessels. This may occur by hacm protein-mediated de-composition of preformed peroxides in LDL which hasalready been minimally oxidised by contact with neigh-bouring cells or the enzymatic activity of lipoxygenases..,158

Acktlo~v(crlgwlmrs: We wish to thank the British Heart Foundation,St. Thomas Endowments Trust, the British Technology Group andthe Medical Research Council for financial support, We also acknowl-edge Dr. Victor Darley-Usmar and Dr. .I. Fee for stimulating discus-sions.REFERENCES[l] Esterbauer, J., Striegl, G., Puhl, H. and Rothendet, M. (1989)

Free Rad. Res, Commun. 6, 67-75.[2] Leake, D.S. and Rankin, SM. (1990) Biochem. I. 270.741-748,[3] Henriksen, T., Mahoney, E.M. and Steinberg, D. (1983) Arterio-sclerosis 3, 149-159.[4] Henriksen, T., Mahoney, E.M. and Steinberg, D. (1981) Proc.Natl. Acad. Sci. USA 78, 6499-6503,[S] Dee. G.. Rice-Evans, C., Obeyesekera, S., Meraji, S., Jacobs, M,and Bruckdorfer. K.R. (1991) FEBS Lett. 294.3842.[6] Parthasarathy, S., Printz, DJ.. Boyd, D.. Joy, L. and Steinberg,D, (1986) Arteriosclerosis 6, 505-510.[7] Steinbrecher, U.P., Zhang, H. and Lougheed, M. (1990) FreeRad. Biol. Med. 9, 155-168.[a] Yla-Herttuala, S.. Palinski, W., Rosenfeld, M.E., Parthasarathy,S., Carrw, T.E.. Butler, S..Witztum,J.L. andSteinberg,D. (1989)J. Clin. Invest. 84, 1086-1095.[9] Rice-Evans, C, and Bruckoorfer, K.R. (1992) Mol. Aspects Med.13, l-11!.[lo] Bencsch. R.E., Benesch, R, and Yung. S. (1973) Anal. Biochem,55, 245-248.[II] Chung, B.H,, Wilkinson. T., Geer, J.C. and Segrest, J.P. (1980)J, Lipid Res. 22 1, 284-3 17.[l?] Markwell, M,A.. Haas, SM., Bieber, L,L. and Tobert, NE.(1978) Anal. Biochem. 87, 106-210.&its, R.. Kumar, KS. and Hochstein. I? (1976) Arch. Biochem.Biophys. 172,463-&S.Guttcridge, J.M.C. (1986) Free Rad. Res. Commun. 1, 173-184.Winterbourn, CC., McGrath, B.M. and Carrell, R.W. (1976)Biochcm. J. I55,493-502,Dicber-Rothencder, M., Puhl, H.. Waeg, Ci., Striegl, G. andEsterbuuer, H. (1991) J. Lipid Res. 32, 1325-1332.Babiy, A.V., Gebicki. J.M. and Sullivan, D.R. (1990) Athero-sclerosis 8 1, 175-182.Keilin, D. and Hartree, E.F. (1950) Nature 166, 513-514.Tap@, A.L. (1953) Arch, Biochem, Biophys. 44, 378-395.Tappet, A.L. (1955) J. Biol. Chem. 217.721-733.Gutteridge, J.M.C. (1987) Biochitn. Biophys. Acta 917,219-223.Harel, S, and Kanner, J, (1988) Free Rad. Res. Commun. 5,21-23,Rice-Evans, C., Okunade, G. and Khan, R, (1989) Free Rad. Rcs,Commun. 7, 45-54.Guueridge, J.M.C. (1976) FEBS Lett. 201, ?91--295.Puppo, A. and Halliwcll, 6. (1988) Biochem. J. 249, 185-190.Hare), S,, Salan, M,A. and Kanner, 1. (1988) Free Rad. Rcs.Commun. 5, 1 -19.Turner, J.J.O., Rice-Evans, C., Davies, M. and Newman, E,S.R.(1991) Biochem. J. 277, 833-837.Giulivi, C. und Davies, K. (1990) J. Biol. Chem. 265, 19453-19460,Whitburn, K.D. (1987) Arch. Biochem. Biophys. 253,419-430.Labeque, R. and Marnett, L. (1988) Biochemistry 27,7960-7970.OBrien, P.J. (1969) Can. J. Biochem. 47, 485492.Balla, G., Jacob, H.S., Eaton, J.W., Belcher, J.D. and Verccllotti,G.M. (1991) Arteriosclerosis Thrombosis 11, 1700-1711.Carew, T.E,, Schwenke, DC. and Steinberg, D. (1987) Proc.Natl. Acad. Sci. USA 84, 7725-7729.Bjorkhem. I., Henriksson-Freyschuss, A., Breuer, 0.. Diczfalusy,LT.,!krghund, L. and Henriksson, P. (1991) ArteriosclerosisThrombosis 11, IS-22.[35] Winterbourn, CC. (1985) in: HandboG ofbE~&ds forOxygenRadical Research (Crecnwald, R.A. rd.) pp. 137-141, CRCPress, Baton Rouge.