Free Radical Biology & Medicine 51 (2011) 1926–1936

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Free Radical Biology & Medicine

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Original Contribution

Fluorescent adducts formed by reaction of oxidized unsaturated fatty acids withamines increase macrophage viability

Maziar Riazy a,⁎, Marilee Lougheed a, Hans H. Adomat b, Emma S. Tomlinson Guns b, Guenter K. Eigendorf c,Vincent Duronio a, Urs P. Steinbrecher a,⁎a Department of Medicine, University of British Columbia, and Vancouver Coastal Health Research Institute, Vancouver, BC, Canada V6H 3Z6b Vancouver Prostate Centre, Vancouver General Hospital, Vancouver, BC, Canada V6H 3Z6c Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1

Abbreviations: HHE, 4-hydroxyhexenal; HNE, 4-hydacid; AOP-LDL, arachidonate oxidation product-modifiedbutylated hydroxytoluene; ESI, electrospray ionization;isolevuglandin; LA, linoleic acid; LDL, low-density lipoester; LOP-LDL, linoleate oxidation product-modified LDLMPM, mouse peritoneal macrophage; MS/MS, tandem mproduct; OP-LDL, oxidation product-modified LDL; oxLDL,choline; PE, phosphatidylethanolamine; p-HA, p-hydrphosphatidylinositol 3-kinase; PKB, protein kinase B; TPPbis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-⁎ Corresponding authors. Fax: +1 604 875 4497.

E-mail addresses: mriazy@interchange.ubc.ca (M. Riusteinbr@interchange.ubc.ca (U.P. Steinbrecher).

0891-5849/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.freeradbiomed.2011.08.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 March 2011Revised 22 August 2011Accepted 24 August 2011Available online 1 September 2011

Keywords:OxLDLAtherosclerosisAutoxidationIsolevuglandinUnsaturated fatty acidProtein modificationFree radicals

Macrophages are prominent components of human atherosclerotic lesions and they are believed to acceleratethe progression and/or complications of both early and advanced atherosclerotic lesions. We and others haveshown that oxidized low-density lipoprotein (oxLDL) induces growth and inhibits apoptosis in murine bonemarrow-derived macrophages. In this study, we sought to characterize the oxidative modification of LDL that isresponsible for this prosurvival effect. We found that both the modified lipid and the modified protein compo-nents of oxLDL can increase the viability of macrophages. The keymodification appeared to involve derivatizationof amino groups in apoB or in phosphatidylethanolamine by lipid peroxidation products. These reactive oxidationproducts were primarily unfragmented hydroperoxide- or endoperoxide-containing oxidation products of lino-leic acid or arachidonic acid. LC-MS/MS studies showed that some of the arachidonic acid-derived lysine adductswere isolevuglandins that contain lactam and hydroxylactam rings. MS/MS analysis of linoleic acid autoxidationadducts was consistent with 5- or 6-membered nitrogen-containing heterocycles derived from unfragmentedoxidation products. The amine modification by oxidation products generated a fluorescence pattern with an ex-citation maximum at 350 nm and emission maximum at 430 nm. This is very similar to the fluorescence spec-trum of copper-oxidized LDL.

roxynonenal; AA, arachidonicLDL; apo, apolipoprotein; BHT,FBS, fetal bovine serum; isoLG,protein; LME, L-lysine methyl; LPC, lysophosphatidylcholine;ass spectrometry; OP, oxidationoxidized LDL; PC, phosphatidyl-oxyphenylacetaldehyde; PI3K,, triphenylphosphine; XTT, 2,3-carboxanilide.

azy),

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© 2011 Elsevier Inc. All rights reserved.

Macrophages play a key role in atherosclerosis from its conceptionto complications [1]. In particular, macrophage burden correlates wellwith the rate of disease progression and risk of adverse clinical outcome[2]. Macrophage populations in the arterial intima are maintainedthrough monocyte recruitment and also by proliferation of macro-phages in lesions [3–5]. We and others have shown that at concentra-tions less than 75 μg/ml, oxidized LDL (oxLDL) is mitogenic andantiapoptotic for macrophages [6–11]. However, higher concentrationsof oxLDL are toxic or proapoptotic [10].

We previously reported that the antiapoptotic effect of oxLDL in-volves activation of protein kinase B (PKB) by the phosphatidylinositol3-kinase (PI3K) pathway [10] and reduction of ceramide levels byoxLDL-mediated inhibition of acid sphingomyelinase [11]. More recent-ly, we showed that oxLDL induced calcium oscillations in macrophagesand that calcium-dependentmechanisms also contribute to the prosur-vival effect of oxLDL [12].

OxLDL can also enhance macrophage growth [8]. However, weshowed that LDL modified by incubation with autoxidized arachidonicor linoleic acid (AOP and LOP, respectively) generated a product thatwas as potent as oxLDL in promoting growth of macrophages [8]. In ox-idation product-modified LDL (OP-LDL), unlike extensive modificationof LDLwith copper ion, endogenous lipids are not oxidized and LPC con-tent is not increased. Even though lysine residues of apoB become deri-vatized, the apoB is not fragmented [13–15]. Martens et al. showed thatOP-induced modifications of not only LDL but also structurally unre-lated proteins such as albumin or immunoglobulins confer mitogenicproperties toward macrophages [8], which suggested that modificationof a protein by autoxidized fatty acids was sufficient to cause growth inmacrophages. Immunochemical epitopes specific to this type ofmodifi-cation have been found in vivo and in LDL oxidized by other methods[16,17].

1927M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

A previous study showed that treatment of linoleic acid with soy-bean lipoxygenase generated a product that reacts with amino groupsto yield a fluorescent product, but the structure of this product hasnot been determined nor have its biological properties been investi-gated [18]. Arachidonic acid autoxidation can produce highly reactivespecies such as isolevuglandins that can quickly modify proteins [19].We hypothesized that the prosurvival effect of oxidized LDL is due, atleast in part, to adducts formed by reaction of linoleic acid (LA) orarachidonic acid (AA) oxidation products with amine groups in apoB.In this report, we compare the ability to increase macrophage viabilityand fluorescent characteristics of these adducts with those of oxLDL.We further characterize these adducts by nuclear magnetic resonance(NMR) andmass spectrometry (MS) and propose that adduct formationinvolves amine groups and unfragmented oxidized fatty acids yieldingnitrogen-containing heterocycles with an intact fatty acyl backbone. Anoverview of the experimental approach is presented in SupplementaryFig. 1.

Materials and methods

Materials

RPMI 1640 medium and gentamicin were from Canadian Life Tech-nologies (Burlington, ON, Canada). Defined fetal bovine serumwas sup-plied by Professional Diagnostics (Edmonton, AB, Canada). Arachidonicacid, linoleic acid, cholesterol, pyrrole, p-dimethylaminobenzaldehyde,diethylenetriaminepentaacetic acid, octyl glucoside, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT), N-methyldibenzopyrazine methyl sulfate, glucose oxidase,triphenylphosphine (TPP), and trinitrobenzenesulfonic acid were pur-chased from Sigma–Aldrich (Mississauga, ON, Canada). Deuterium oxide,CD3OD, CDCl3, and d6-dimethyl sulfoxide (d6-DMSO) were supplied byCambridge Isotope Laboratories (Andover, MA, USA). Egg phosphatidyl-choline (PC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(DPPE) were obtained from Northern Lipids (Vancouver, BC, Canada).Cayman Chemical (Ann Arbor, MI, USA) supplied 4-hydroxyhexenaland 4-hydroxynonenal. Limulus amebocyte lysate assay was from Bio-Whittaker (Walkersville, MD, USA). Glucose, L-tyrosine, sodium borohy-dride, and sodium hypochlorite were from Fisher Scientific (Vancouver,BC, Canada). All other chemicals were from VWR Canlab (Edmonton,AB, Canada).

Lipoprotein isolation and modification

LDL (d=1.019 to 1.063 g/ml)was isolated by sequential ultracentri-fugation of EDTA-anticoagulated fasting plasma obtained from healthynormolipidemic volunteers as described [10]. Before oxidation, LDLwas dialyzed to reduce EDTA concentration to 10 μM. Lipoprotein con-centration was expressed as mg/ml protein as measured with the PierceBCA assay. Copper-ion oxidationwas performedby incubating 200 μg/mlLDL for 24 h at 37 °Cwith 5 μMcopper sulfate. Oxidationwas stopped byaddition of 40 μM butylated hydroxytoluene (BHT) and 300 μM EDTA[10]. The modified LDL was then washed and concentrated to about1 mg/ml using Amicon Centriplus 20 ultrafilters. For modification ofLDL by fatty acid oxidation products, 10 mgneat arachidonic acid or lino-leic acid was autoxidized in a glass test tube by exposure to air at 40 °Cfor 72 h and the water-soluble component was incubated overnight atroom temperature with LDL in the presence of 300 μM EDTA and50 μM BHT [14]. p-Hydroxyphenylacetaldehyde (p-HA)-modified LDLwas generated by addition of 1 ml of 2 mMp-HA to 1 mgof LDL and incu-bation for 24 h at 37 °C. To generate p-HA, NaOCl (0.9 M)was sequential-ly added to L-tyrosine (2 mM) in ice-cold phosphate (20mM) buffercontaining 100 μM diethylenetriaminepentaacetic acid (pH 7.0) until afinal 1:1 molar ratio was achieved. The solution was then warmed for1 h at 37 °C and filter sterilized. Efficient conversion of L-tyrosine top-HA occurs under these conditions [20]. Hydroxyalkenal-modified

LDL was prepared by addition of 4-hydroxyhexenal (0.5 mg) or 4-hydroxynonenal (1.5 mg) to 1 mg of LDL dissolved in phosphate-buffered saline (PBS) containing 10 μM EDTA. These modified LDLpreparations were extensively dialyzed to remove unbound reactivematerial.

Lipoprotein characterization and analysis

LDL purity and extent of modificationwere evaluated by agarose gelelectrophoresis using a Titan kit (Helena Laboratories, Markham, ON,Canada). Lipoprotein bands were visualized by fat red staining. Electro-phoreticmigrationwas determined bymeasuring the distance betweenthemidpoint of origin and the leading edge of the LDL band. ApoBmod-ification was expressed as the migration of the modified LDL relative tothat of native LDL on the same gel.

Lipoprotein concentration was based on the amount of proteinmea-sured by the Lowry protein assay. Hydroperoxide contentwasmeasuredby a lipid hydroperoxide assay kit (Cayman Chemical Co.) according tothemanufacturer's protocol. Backgroundwas determined bymeasuringthe absorbance after treatment with (TPP) multiplied by 1.28 to correctfor the effect of (TPP) on the chromogen.

Modification of lysine methyl ester by oxidation products

Three milligrams of oxidation products of AA or LA dissolved in300 μl of PBS was added to 200 μl of lysine methyl ester (LME) from afreshly prepared aqueous solution of 10 mg/ml, pH 8.0 (molar ratio of1:1), and total volume was adjusted to 1 ml with PBS. The mixturewas incubated at room temperature overnight in airtight glass tubes.

Lipid extraction, liposome preparation and modification

One milligram of native or oxidized LDL in 0.5 ml PBS was acidifiedwith HCl (final conc. 10 mM), mixedwith ice-coldmethanol:chloroform(2:1, v/v), and vortexed. Then a solution containing 2 MKCl, 0.2 MH3PO4

(in water) mixed with chloroform (1:1, v/v) was added and vortexedagain. Centrifugation at 2000 rpm for 10 min yielded an upper aqueouslayer and a lower organic (lipid) layer with denatured protein at theinterface.

For liposomepreparation, the organic phase from the lipid extractionwas dried under nitrogen and the lipids were resuspended by vortexingin 1.5 ml of liposome buffer (150 mMNaCl containing 0.1 mMEDTA and10 mM Hepes, pH 7.5). The mixture was passed 10 times through a0.1-μm polycarbonate membrane at 37 °C under nitrogen flow using aLipexmini-extruder. Concentrations of liposomes are expressed accord-ing to the initial amount of LDL protein used for extraction. Phospholipidliposomeswere prepared bymixing 10 μmol of DPPE (for PE liposomes)or egg PC (for PC liposomes) with equimolar cholesterol and vortexingin 2 ml of liposome buffer and extruding as described above. Liposomeconcentrations are expressed in terms of initial concentration of phos-pholipid assuming 95% recovery after extrusion. The size of liposomeswas determined by a submicrometer particle sizer (Nicomp Model270). The mean diameter of PC liposomes was 136±20 nm, and thatof PE liposomes was 126±53 nm. Liposomes were modified by resus-pending OPs of AA or LA in 1 ml of PBS and incubating this with lipo-somes (2 mg total lipid) for 8 h at room temperature.

ApoB solubilization

After the lipid extraction of 0.5 mg LDL or oxLDL as described inthe previous section, the middle (protein) layer was washed with2 ml of ice-cold H2O and 2 ml of acetone. After a final wash withwater the apoB was solubilized in 0.5 ml of a 15 mg/ml aqueous solu-tion of octyl glucoside containing 10 mM NaOH. After 30 min at roomtemperature, excess detergent was removed by dialysis against PBS.

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Fig. 1. Lipid and protein fractions of oxLDL promote macrophage viability. Lipids and pro-teins of native (nLDL) or oxLDL were extracted using the Bligh–Dyer method. The frac-tions were subsequently reconstituted as described under Materials and methods.MPMs from CD-1 mice were isolated and seeded in 96-well plates at 10×104 cells/welland incubated for 4 days with the indicated concentrations of oxLDL or lipoprotein frac-tion. Viability was then assessed with the XTT reduction assay. The absorbance at 450 nmis shown on the y axis. The graph represents the means±SD of pooled data from threeindependent experiments, each done in quadruplicate. The difference between nLDLlipids and all other conditions at the corresponding concentration was statistically signif-icant (Pb0.05, calculated using Student's t test).

1928 M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

Cell culture and viability assay

Resident mouse peritoneal macrophages (MPMs) were collectedfrom CD-1 mice by peritoneal lavage with ice-cold PBS. Cells wereresuspended in RPMI 1640 medium supplemented with 10% FBS andadjusted to 1×105 cells per milliliter. For viability assays 100 μl of cellsuspension was added to each well of 96-well tissue culture platesand was incubated overnight at 37 °C in a humidified atmosphere of5% CO2 in air. At the start of experiments, the medium was replacedwith 100 μl RPMI 1640 medium containing 5% FBS with lipoproteinsor liposomes for 3–4 days without any further medium change. Macro-phage cell number was then estimated by the XTT formazan method,which measures the rate of reduction of the XTT dye by mitochondria.We have previously documented a close correlation between macro-phage number and formazan dye reduction [7]. At the indicated time,each well was supplemented with 50 μl of RPMI medium containing25 μM phenazine methosulfate and 1 mg/ml XTT. After 4 h of incuba-tion at 37 °C, absorbance was measured at 450 nm.

MS and liquid chromatography (LC)-MS experiments

For initial experiments, unit mass and tandem MS (MS/MS) datawere obtained on an Esquire ion trap instrument (Bruker Daltonics, Bil-lerica, MA, USA) and a Micromass LCT time-of-flight (TOF) system(Micromass, Manchester, UK). Samples (1 mg/ml in methanol) wereinjected into the electrospray ionization source at a flow rate of 10 μl/min. Subsequent LC-MS was carried out using a Waters AcquityUltra-Performance LC coupled with a photodiode array (PDA) detec-tor and a ZQ single-quadrupole or a Waters/Micromass Quattro Microtriple-quadrupole mass spectrometer (Waters Corp., Milford, MA,USA). Separations were carried out on a BEH C18 column (1.7 μm, 50or 100×2.1 mm; Waters). In initial runs, acetonitrile was ramped 20–100% (v/v in water) from 0.2 to 10 min at 0.8 ml/min with 50 mM am-monium acetate used as aqueous modifier, a column temperature of35 °C, and ~1/3 split to the MS. MS data were acquired in bothES+and ES−modes. In ES+mode, capillary was at 3 kV, cone volt-age 30 V, source and desolvation temperatures were 100 and250 °C, respectively, and desolvation gas flow was 800–1000 L/h. InES−mode, capillary was at 2.5 kV and the remaining parameters thesame as for ES+mode. OPs and OP-LME adducts were more efficientlyionized in ES+mode. An acetonitrile gradient (% by volume inwater) of0–0.2 min, 10%; 0.2–2.0 min, 10–15%; 2–25 min, 15–40%; 25–31 min,40–90% at 0.3 ml/min ultimately provided superior LC resolution neces-sary for these complex samples.

LC-MS data were also acquired using a Waters SYNAPT using theabove LC method and data collected in V mode (~10,000 resolution)using the lockspray option routinely, which provided mass accuraciesof 5–10 ppm. MS/MS data for m/z's of interest, with ions selected atunit resolution and collision ramps of 10–30 or 15–30 V (collisioncell 1) and 6, 12, or 16 V (collision cell 2) generated varying degreesof fragmentation. MassLynx software (4.1) was used to process allLC-MS data including the elemental composition analyses.

A Waters Alliance LC coupled with a PDA and 2475 multiwave-length fluorescence detector was used for detection of fluorescent ad-ducts (excitation/emission set at 350 nm/430 nm). A 2.1×150 XterraMS C18 5-μm column with an acetonitrile gradient (% by volume in20 mM ammonium acetate) of 0.2–2.0 min, 10–15%; 2–25 min, 15–40%; 25–31 min, 40–90%; 34.25–34.5 min, 90–100%; 100%; 36.25–36.5 min, 100–10%, total runtime 40 min, flow rate 0.3 ml/min, andcolumn temperature 35 °C was used for separation.

Statistical analysis

Results in Figs. 1 and 2 represent MPM viability expressed asmeans±SD of pooled data from at least three experiments. Comparisonof twomeanswas done using a two-tailed Student t test.Whenmultiple

means were compared, P values were further confirmed by one-wayANOVA followed by Tukey's test. ANOVA and Tukey's analysis were car-ried out with GraphPad Prism 5 software (La Jolla, CA, USA).

NMR experiments

NMRmeasurementswere done on anAvance 400NMRspectrometer(Bruker Spectrospin, Fallanden, Switzerland) operating at 400 MHz.Samples were prepared at a concentration of 5 mg/ml in d6-DMSO orin a 1:1 mixture of CDCl3 and CD3OD. Chemical shifts with reference totetramethylsilane are given in ppm; for multiplet signals the center po-sition is reported.

Results

Amine modification by OPs is sufficient to increase macrophage viability

Sakai et al. had suggested that LPC in oxLDL is important in oxLDL-mediated survival [6]. Martens et al. subsequently challenged thiswith data showing that in MPMs, the effect of oxLDL on macrophagegrowth is not due to its LPC content [8]. In contrast, they found thatamino group modification of apoB is essential, as prior methylation offree amino groups in LDL abolished growth-promoting propertieswhen this methylated LDL was subsequently oxidized. To determinewhether the lipid or protein moiety of LDL contributes to the oxLDL-mediated increase in macrophage viability, we extracted the lipid andreconstituted the lipid and protein moieties of oxLDL separately. Theability of these fractions to promote macrophage viability was com-pared. Fig. 1 shows that both lipid and protein fractions can increase vi-ability of MPMs.

Previous reports from other groups have documented that oxi-dized phospholipids bound to apoB and the same oxidized phospho-lipid incorporated into the outer monolayer of LDL both contributedto binding to COS-7 cells transiently transfected with CD36 [21]. Inthat study, binding to CD36 was apparently mediated by 1-palmitoyl2-(5′-oxovaleroyl)-PC. We hypothesized that the increase in macro-phage viability might be due to the modification of amine-containingphospholipids by OPs. To explore this possibility, PE-containing lipo-somes were modified by OPs from arachidonic and linoleic acid. Fig. 2A

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Fig. 2. Amine modification by borohydride-reducible functional groups other than alkenals is sufficient for the enhancement of viability by OPs. MPMs were isolated and seeded in96-well plates at 10×104 cells/well and incubated with the indicated concentrations of LDL or various liposome or lipoprotein modifications and viability was assessed after 4 daysby the XTT reduction assay. The absorbance at 450 nm is shown on the y axis. All graphs represent the viability of MPMs expressed as means±SD of pooled data from at least threeindependent experiments, each done in triplicate. Statistical analyses were performed as described under Materials and methods. The level of significance of difference is indicatedin the graphs as follows: * or #P≤0.05, ** or ##P≤0.01. (A) Phosphatidylethanolamine liposomes were modified by thermally oxidized arachidonic acid and linoleic acid (AOP-PEand LOP-PE, respectively). Modification of phosphatidylcholine-containing liposomes, which lack the reactive amine head group, by oxidized arachidonic acid (AOP-PC) was used asa control. * or # denotes the level of significance between control (AOP-PC) and AOP-PE or LOP-PE, respectively. There was no difference between AOP-PC group and no-treatmentor nLDL-treated controls (not shown). (B) Sodium borohydride (100 mM) was used to reduce arachidonic acid oxidation products before or after LDL modification ((AOP+BH4)LDL and (AOP-LDL)+BH4, respectively). * and # denote the level of significance between (AOP+BH4) LDL and AOP-LDL or (AOP-LDL)+BH4, respectively. (C) LDL was modifiedwith 4-hydroxyhexenal or 4-hydroxynonenal (HHE-LDL and HNE-LDL, respectively) as described under Materials and methods. * or # denotes the level of significance betweenoxLDL and HHE-LDL or HNE-LDL, respectively.

1929M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

shows that OP-PE liposomes are capable of increasingMPM viability. Onthe other hand, the product of incubation of OPs with PC liposomes,which lack the free amino group, did not increase viability (Fig. 2A). Toconfirm that the increased macrophage viability in the formazan-basedassay is not spurious secondary to an induction of phagocytic burst ormitochondrial enzymes, we conducted parallel experiments in whichformazan reduction results were confirmed by counting the number ofcells in both mouse peritoneal and bone marrow-derived macrophages(Supplementary Figs. 2 and 3).

Borohydride-reducible functional groups other than alkenals are essen-tial for the induction of viability by OPs

As shown in Fig. 2B, pretreatment of OPs with borohydride pre-vents the formation of a growth-promoting product when mixedwith LDL. This figure also shows that borohydride treatment after

LDL modification does not diminish the ability to increase viability.This suggests that borohydride-reducible functional groups are im-portant for prosurvival adduct formation, whereas such functionalgroups are not required aftermodification. The electrophoretic mobilityof LDL was reduced by only 50% with borohydride pretreatment (notshown), which suggests some reactive OPs are not amenable to reduc-tion by borohydride. Borohydride-reducible functional groups includereactive aldehydes. Aldehydes are capable of modifying lysine residuesand creating fluorescent adducts. In fact, aldehydic reactive products arereportedly the main components of oxidized fatty acids responsible forlysinemodification of apoB [22,23]. Therefore, the susceptibility to reduc-tion by borohydride would be consistent with an aldehyde moiety in ox-idized fatty acids reacting with LDL amino groups. To test this possibility,we modified LDL with 4-hydroxynonenal (HNE) or 4-hydroxyhexenal(HHE) to generate HNE-LDL and HHE-LDL, respectively. Fig. 2C showsthat HNE-LDL and HHE-LDL had a much smaller effect on macrophage

1930 M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

growth than AOP-LDL. As HNE and HHE are the major fragmented alde-hydic products of arachidonic acid oxidation [23], it seemed unlikelythat these aldehydes accounted for the mitogenic modification of LDLby fatty acid oxidation products. Borohydride-reducible functionalgroups also include hydroperoxides and endoperoxides. We thereforeincubated OPs with a selective reducing agent, triphenylphosphine(TPP), before LDL modification. A partial inhibition was observed withTPP preincubation (Supplementary Table 1), suggesting that at leastpart of the induction of viability by OPs can be explained by hydro-peroxides or endoperoxides.

OP modification of lysine and apoB generates fluorescent adducts

We and others have reported that oxidized LDL has characteristicfluorescence properties, with maximum excitation at 350–360 nmand maximum emission at 430 nm [24]. To see if OP modification ofLDL generates similar fluorescent products, we compared the fluores-cence of AOP-LDL and LOP-LDL with that of oxLDL. In addition to nativeLDL (nLDL), acetylated LDLwas used as a control for nonoxidative lysinemodification. OxLDL, AOP-LDL, and LOP-LDL all exhibited a more than20-fold increase in fluorescence intensity (excitation/emission of350 nm/430 nm) compared to nLDL. In contrast, acetylation of LDL pro-duced no increase influorescence intensity (Fig. 3A).Wenextmeasuredthe fluorescence of adducts formed by modification of LME with OPs.Lysine methyl ester contains two potentially reactive amino groups.However, the α amine of lysine is six times less reactive compared tothe ε amine [25] and it is believed that esterification of the carboxylgroup further reduces reactivity of the α amine. Hence, although theα amine is not protected, at least 90% of adducts are anticipated to beon the ε amino group. Fluorescence scanning of LOP-LME (Fig. 3B)revealed a fluorescence spectrum almost identical to what has beenreported for oxLDL [22], whereas therewas nofluorescence in OP alone.

Autoxidation of arachidonic and linoleic acids produces mainlyunfragmented oxygenated species

The above results indicate that autoxidized linoleic acid or arachidonicacid can derivatize amino groups on proteins or phospholipids, and theproducts promote survival and growth of macrophages. To characterizethese oxidation products, we subjected arachidonic acid and linoleicacid to air oxidation at 40 °C for 24, 48, or 72 h and analyzed samplesfrom each time point by electrospray ionization mass spectrometry

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Fig. 3. OP modification of lysine and apoB generates fluorescent adducts. (A) Fluorescence inacetylation (oxLDL, AOP-LDL, LOP-LDL, and Ac-LDL, respectively) was measured with excitfrom at least three preparations expressed as fold increase relative to nLDL. (B) Fluorescen

(ESI-MS) in positive-ion mode and proton NMR. Each sample was alsotested for its ability to modify apoB by measuring electrophoretic mobil-ity. Table 1 presents proportionate peak intensities for themajor ions ob-served in each sample. With increased degree of oxidation, more than90% of AA or LA is consumed while the relative peak intensities for OPsincrease. ESI-MS of AOP shows the emergence of new masses in therangem/z 325 to 420 and with LOP in the rangem/z 315 to 400. A repre-sentativemass spectrum of unoxidized arachidonic or linoleic acid and ofcorresponding samples after 72 h oxidation is shown in SupplementaryFig. 4. The m/z values of the most abundant products are consistentwithmajor ions forming a serieswithmass increments of 16 Da, suggest-ing addition of oxygen atoms without fragmentation of the hydrocarbonbackbone. Thiswas confirmedby the elemental composition of these ionsderived from accurate mass measurements. Table 1 also shows the num-ber of original olefinic protons calculated fromprotonNMRanalysis usingthe methyl protons as standards (there are 8 olefinic protons in unoxi-dized arachidonic acid). This table also includes predicted structural as-signments based on each m/z. These studies suggest that by 24 h, mostof the arachidonic acid has been oxidized by the addition of two mole-cules of oxygen, consistent with the formation of two hydroperoxidesor epidioxides. This is supported by the olefinic proton number in theNMR studies (see below and Table 2). Subsequently, there is an increasein the proportion of fatty acid molecules with lower degrees of oxygen-ation, presumably reflecting secondary rearrangement or decomposi-tion of hydroperoxides, perhaps to epoxides, ketones, and hydroxides.As well, Table 1 shows the effect of each oxidized fatty acid sample onthe electrophoretic mobility of LDL, suggesting that the increase in rel-ative abundance of OPs correlates with the ability to modify LDL. TheNMR data (Table 2) show that oxidation of arachidonic acid for 72 h re-sults in the loss of 6.3 of the 8 original unconjugated olefinic protonsand 7.3 of 10 protons on carbons originally adjacent to double bonds.Oxidized arachidonic acid had a total of 29.2 protons using the methylprotons as a reference, so about 3 of the original 32 protons in arachido-nic acid are not accounted for in the analysis of the oxidized products.Approximately 3 new protons appear as exchangeable downfield pro-tons (hydroxyl, hydroperoxyl protons), 2.6 appear as nonexchangeabledownfield protons (e.g., on carbons binding with hydroxyl, dioxetane,hydroperoxide, or endoperoxide), 4 appear as low-polarity or epoxideprotons, and about 1.5 appear as conjugated olefinic protons. Oxidationof linoleic acid resulted in a loss of 3.4 of the original 4 unconjugatedolefinic protons and 3.5 of 6 protons on carbons originally adjacent todouble bonds. These were fully accounted for with the appearance of

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tensity of 25 μg/ml nLDL or LDL that had been modified by copper sulfate, AOP, LOP, oration/emission wavelengths set at 350 nm/430 nm. The results represent pooled datace spectra of LOP and LOP-LME.

Table 1Time course analysis of polyunsaturated fatty acid autoxidation products.

Assignment Unoxidized 24 h 48 h 72 h

Arachidonic acidm/z 305 +H 27.5 0.0 0.0 0.0m/z 327 +Na 72.5 26.1 8.3 2.2m/z 343 +Na+

oxygen0.0 6.9 9.6 9.0

m/z 345 +Na+H2O 0.0 1.0 4.6 10.0m/z 359 +Na+2

oxygen0.0 4.9 6.4 8.6

m/z 375 +Na+3oxygen

0.0 8.4 19.7 28.3

m/z 391 +Na+4oxygen

0.0 49.3 45.9 35.8

m/z 407 +Na+5oxygen

0.0 1.0 3.7 4.3

m/z 423 +Na+6oxygen

0.0 2.5 1.8 1.8

Original olefinic protons (chemshift 5.30 ppm)

7.8 2.7 1.3 1.00

LDL modification, electrophoreticmobility (mm)

5 30 35 36

Linoleic acidm/z 303 +Na 18.7 29.7 7.5 2.8m/z 319 +Na+

oxygen0.0 19.5 27.8 30.9

m/z 325 +2 Na−H 81.3 0. 0.0 0.0m/z 335 +Na+2

oxygen0.0 42.4 53.5 55.2

m/z 351 +Na+3oxygen

0.0 5.9 8.6 8.8

m/z 367 +Na+4oxygen

0.0 2.5 2.7 2.2

Original olefinic protons (chemshift 5.30 ppm)

4.0 1.3 1.0 0.5

LDL modification, electrophoreticmobility (mm)

5 11.5 12.0 12.5

9 mg of neat arachidonic acid or linoleic acid was incubated for 24–72 h in an airatmosphere at 40 °C. At each time point, a sample of oxidized fatty acid was analyzedby mass spectrometry. A parallel sample was dissolved in CD3OD/CDCl3 and protonNMR spectra were collected. A third aliquot was dissolved in PBS and incubated for24 h with LDL. Agarose gel electrophoresis was then performed to assess the extentof LDL modification. Results are representative of three independent experiments.

Table 2NMR quantification of exchangeable protons in autoxidation products of arachidonicacid or linoleic acid.

Arachidonicacid protonidentification

Chemicalshift (ppm)

No. of protons

Unoxidized 72 h ox(4054)

72 h ox+D2O

C20 (reference) 0.84 3 3 3C2 2.24 2.1 2.3 2.0C3 1.54 2.1 2.0 2.0C4,16 2.04 4.0 1.7 1.5C7,10,13 2.80 6.0 1.0 0.7C17,18,19 1.30 6.0 6.0 6.0C5,6,8,9,11,12,14,15 5.30 7.8 1.5 1.5Conj. olefinic protons 5.4–6.0 0 1.5 1.5COOH 12 0.9 0.75 0HCO 11.5 0 0.03 0All other downfield H 4.2–11 0 5.4 2.6New low-polarityprotons

1.4–2.4 0 3.5 2.9

Epoxide protons 2.6–3.4 0 0.5 0.5Total protons 31.9 29.2 24.2

Linoleic acid protonidentification

Unoxidized(3956)

72 h ox(14,822)

72 h+D2O

C18 (reference) 0.85 3 3 3C2 2.20 2.1 2.3 2.4C3 1.47 2.0 4.1 3.7C4–7,15–17 1.24 14.2 14.4 14.6C8,14 2.01 4.0 1.1 1.2C11 2.73 1.9 0.4 0.4C9,10,12,13 5.30 4.0 0.6 0.9Conj. olefinic protons 5.4–6.0 0 1.5 1.2COOH 12 1.0 0.9 0HCO 11.5 0 0.2 0.1All other downfield H 4.2–11 0 2.6 0.5New low-polarityprotons

1.4–2.4 0 1.2 1.2

Epoxide protons 2.6–3.4 0 0.3 0.3Total protons 32.5 32.6 29.5

Each fatty acidwas autoxidized for 72 has describedunderMaterials andmethods and in thefootnote to Table 1. The oxidized fatty acids were dissolved in deuterated DMSO and NMRspectra were obtained. D2O was then added to permit identification of exchangeableprotons, and a second set of NMR spectra was collected. The methyl protons were used asthe internal standard for integration. Results are representative of three independentexperiments. For the interpretation of NMR data please refer to the text.

1931M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

2.1 exchangeable downfield protons, 0.6 nonexchangeable downfieldprotons, 3.6 low-polarity or epoxide protons, and 1.5 conjugated olefinicprotons. Therefore, the NMR data corroborate the MS data, suggestingthat unfragmented oxidation products predominate after 72 h ofautoxidation.

Unfragmented linoleic acid autoxidation products can modify lysinemethyl ester

Modification of LME by LOPwas chosen as the initial adduct to studybecause there are fewer potential products thanwith AOPmodification.LOPwas used tomodify LME as described underMaterials andmethodsand the LOP-LME reaction mixture (equivalent to 10 μg of LA) wasinjected into the ionization source of an Esquire ion trap instrument.Fig. 4 shows a representative mass spectrum analysis of LOP-LME inpositive-ion mode. In addition to LME (m/z 161) and masses known toexist in LOP, new products were found that existed only in the LOP-LME reaction mixture. The m/z for these products (such as 455, 457,477) were all within the 450–510 range, suggesting that adducts areformed by reaction of LME with unfragmented linoleic acid containingone to four atoms of oxygen. The inset in Fig. 4 lists the predicted andobserved m/z of LOP-LME adducts.

To further characterize the adducts in LOP-LME, major ions uniqueto LOP-LMEwere analyzed by ESI-MS/MS. Table 3 presents the putativeparent ion composition and the major daughter ions observed for each.Fig. 5A presents the MS/MS plot of m/z 455 (the empiric formula:

C25H46N2O5). Fruebis et al. had suggested that OPs of unsaturatedfatty acids could modify proteins through a concerted reaction involv-ing the amine group of lysine residues and reactive functions (such ashydroperoxy groups) on OPs [18]. According to this model, adductswith nitrogen-containing heterocycles (such as pyrrole, pyrroline, orpyridine) are produced. Fukuzawa et al. have shown that keto-oleicacid can modify amine-containing amino acids without fragmentation,leading to the generation of hydroxylactam-containing adducts withthe same fluorescence properties we observed in OP-LME [26]. Basedon the reaction mechanisms proposed by Fruebis et al. [18] andFukuzawa et al. [26], one can propose several putative structures forthe parent adduct that could produce the observed daughter ionsupon fragmentation (Fig. 5B, schemes (a) through (e)).

Fragmentation of the carboxylic acid-containing side chain adjacentto the heterocycle accounts for the 144, 312, and 296 daughter ions. Thegreater abundance ofm/z 142 relative to 144 can be explained by desa-turation of the aliphatic chain upon fragmentation (Fig. 5B, scheme (c))or by the existence of anunsaturated bondwithin the aliphatic chain be-tween the heterocyclic structure and the carboxyl (Fig. 5B, scheme (e)).Additionally, a charge-remote fragmentation [27] can explain how adaughter fragment can undergo dehydrogenation (Fig. 5B, scheme (d)).

Lou et al. have reported conditions under which tertiary amines canundergo spontaneous dehydrogenation during fragmentation [28].Scheme (e) in Fig. 5B illustrates this reaction, which explains the exis-tence of m/z 311 and 295 daughter ions. Table 3 shows the major

144. 2

161. 2

293. 4

319 .3

335. 3

351.3

477.4

461.3489. 3

505. 4

0.0

0.5

1.0

1.5

x105

Intens.

100 150 200 250 300 350 400 450 500 550 m/z

LME+LA +H +Na +H-H2O +Na -H2O+1 oxygen (457) 479 439 (461)+2 oxygen (473) 495 (455) (477)+3 oxygen (489) 511 471 493+4 oxygen (505) 527 487 509

LOP-LME ES+

Fig. 4. ESI-MS analysis of adducts formed by modification of lysine methyl ester withoxidized linoleic acid. The mass spectrum (100–600 range) of LOP-LME was acquiredon an Esquire ion trap instrument. m/z of 161 corresponds to LME and 300bm/zb370corresponds to LOP. New masses (420bm/zb510) were found exclusively in the LOP-LME reaction mixture. The inset table shows the predicted masses of given assign-ments assuming no fragmentation of the acyl chain. Masses in parentheses representthe most abundant masses found in our analyses.

1932 M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

daughter ions for other adducts, including m/z 457. The ion with m/z457 may be a hydrogenated form of 455. The detection of core frag-ments 312 and 296 as well as the 144 fragment representing the car-boxyl side chain (see Fig. 5A) is consistent with this assumption. Theemergence of fragments withm/z of 391 and 341, however, could be asign of the existence of other isobaric compounds within the LOP-LMEmixture. Another less likely explanation could be that saturation ofthe double bond is accompanied by intramolecular rearrangementsleading to a different pattern of fragmentation.

Species with higher m/z followed the same fragmentation patternand commonly generated fragments with m/z 335 and 319, which arepossibly the sodated forms of core structures (m/z 312 and 296, respec-tively) that were observed in adducts withm/z 455 and 457.

These results are therefore consistent with adducts containing 5- or6-membered nitrogenheterocycles,most likely a pyrrole, a pyrroline, ora derivative thereof. To determine if pyrrole derivatives might explainthe fluorescence at 350 nm/430 nm wavelengths, we compared thefluorescence of pyrrole with that of its derivatives 3-pyrroline and 3-hydroxypyrrolidine. No fluorescence was seen at 350 nm/430 nmwith 3-pyrroline but there was with the other two compounds, andthe fluorescence intensity of 3-hydroxypyrrolidine was about fourfoldhigher than that of pyrrole (data not shown).

Unfragmented arachidonic acid autoxidation products can modify lysinemethyl ester

AA autoxidation yields farmore products compared to LA. For exam-ple, isolevuglandins alone,which are found inAOP but not LOP, comprise64 isomeric structures. Our initial analysis of adducts formed by AOP andby AOP-LME reactionwas done by LC coupled to a ZQ single-quadrupole

Table 3ESI-MS/MS analysis and possible assignments of adducts of lysine methyl ester withautoxidized linoleic acid.

Parent ion composition assignment Daughter ions

455 LME+LA+OOH−H2O+H+ 437, 423, 378, 312, 296, 144, 142457 LME+LA+O+H+ 438, 391, 379, 341, 312, 296, 144473 LME+LA+OOH+H+ 455, 419, 413, 357, 335, 319, 144477 LME+LA+OOH−H2O+Na+ 459, 335, 317489 LME+LA+2OOH−H2O+H+ 471, 453, 335, 317, 195491 LME+LA+2OOH−2H2O+Na+ 473, 335, 319, 211, 195505 LME+LA+OOH+OOH+H+ 487, 405, 351, 333, 317, 211, 195

spectrometer. The total ion current of the LC eluent in positive-ionmodereflects very complex samples (Supplementary Fig. 5A). By selection ofmass spectra in 30-s windows of the AOP-LME chromatogram com-pared with the AOP and AA controls, masses with m/z of 475, 491,493, and 509 were found to be unique to AOP-LME (SupplementaryFig. 5B). Fig. 6 shows the results of single-ion recordings (SIRs) of theabove-mentioned masses as described under Materials and methods.Each plot depicts an overlay of SIRs of one m/z for the AOP and for theAOP-LME mixture, which clearly demonstrates these masses existonly in the reactionmixture. Eachmass produced at least a dozen chro-matographic peaks probably reflecting a large number of structural iso-mers. Unoxidized arachidonic acid eluted at 30.5 min (not shown),whereas adducts eluted between 3 and 23 min.

The isotopic distribution of these masses and SIR scans in positiveand negative scan modes indicated that these species are simply pro-tonated in ES+mode (Supplementary Fig. 6). Elemental compositionanalysis of adducts suggested the following empirical formulae as themost likely candidates, C27H43N2O5 (for m/z 475), C27H43N2O6 (for m/z491), C27H45N2O6 (for m/z 493), and C27H45N2O7 (for m/z 509), withthe same predicted empirical formula for all the peaks of a givenm/z. El-emental composition analysis of one representative peak for eachm/z isshown in the supplementary data (Supplementary Table 2).

AOP-LME adducts include isolevuglandin-modified LME

MS/MS studies on AOP-LME adducts were also carried out with theSYNAPT. Fig. 7A depicts the fragment plots for m/z 509 and 491 usingpooled data from all chromatographic peaks from each mass. Thesefragmentation patterns are quite similar, suggesting related molecularstructures with m/z 509 carrying an additional H2O. However, they areindeed distinct analytes as demonstrated by their lack of alignment inFig. 6. An identical fragmentation pattern has been reported before byBrame et al. for hydroxylactam adducts of isolevuglandin-modified pro-teins [19]. Schemes (c) to (f) of Fig. 7B represent the structure of thesedaughter ions. Anm/z of 84.1 is consistent with that species being gen-erated from fragmentation followed by cyclization of the lysyl portionof the molecule as has been previously observed [19]. An m/z of 475probably represents the dehydrated form of m/z 493 (Fig. 8). The frag-mentation pattern of these adducts was identical to those of lactam-containing adducts formed bymodification of lysine by isolevuglandins(Fig. 8B and [19]). LC-MS/MS studies of AOP-LME showed that majordaughter ions (such as 84.1) existed in all peaks of a single species(see Supplementary Fig. 7 for m/z 493). Because these fragments arepresent regardless of which isolevuglandin the adduct is derived from,we conclude that the adducts are derived fromdifferent isolevuglandins(i.e., isoLG, iso[4]LG, iso[7]LG, and iso[10]LG). We further studied thefluorescence properties (excitation/emission: 350 nm/430 nm) ofAOP-LME adducts using a Waters Alliance LC system coupled with a2475 multiwavelength fluorescence detector system. Fluorescentpeaks were found between 10 and 25 min, which corresponds verywell to the region where adducts are eluted. An alignment of the AOP-LME fluorescence peaks and chromatogram for m/z 491 is given as anexample in the supplementary data (Supplementary Fig. 8).

Discussion

Protein modification by lipid peroxidation products is a commonevent in many age-related and pathological conditions. In this paperwe report new observations regarding a particular form of aminogroup modification by unfragmented autoxidation products of linoleicor arachidonic acid. Our NMR and MS studies indicate that this metal-free method for autoxidizing polyunsaturated fatty acids causes mini-mal fragmentation of the carbon backbone despite the fact that nearlyall of the fatty acid molecules are oxidized and these oxidized fattyacids are capable of forming covalent adducts with free amino groupson proteins or phosphatidylethanolamine. Fluorescence and MS/MS

142. 1

296. 0

312. 1

419.1

437 .2

0

200

400

600

Intens.

100 200 300 400 500 m/z

LOP-LME MS/MS 455 (ES+)

N

OH

CH3

OOH

N

O

LME

CH3

OOH

144312-O296

144

OOHN

LME

OHCH3

144

(282/174)

O

OHN

LMEO

CH3

(284/172)

A C

BMolecular Formula = C25H46N2O5 Molecular weight = 454.3

144.1

312-O

296

312-O 296

312-O 296

LME

a

295.0

N OOH

O

HOCH3

m/z = 469.3

N O

OHO

CH3

Hm/z = 455.3 144

312-O

296b

c

d

e

LMELME

N

O

CH3

OOH

LME

142O

OHN

O

LME

CH3

312-O

296

N

O

CH3

OOH

LME

142O

OHN

OH

LME

CH3

314-H2O

296

O

OHN

LMEOH

CH3OOHN

LMEOH

CH3

142311

-O295

+

144

(m/z)

Fig. 5. MS/MS analysis and putative structures ofm/z 455 in LOP-LME. (A) LOP-LME was analyzed by ESI-MS/MS in ES+mode scan, which reveals two pseudoparent ions ([M−H2O]+

and [M−2H2O]+) plus major daughter ions. (B) The empirical formula and molecular weight ofm/z 455. Scheme (a) depicts several putative structures based on the concerted reactionsuggested by Fruebis et al. [18] and scheme (b) is based on a pathway proposed by Fukuzawa et al. [26] in which a hydroxylactam (m/z 469) is produced by modification of lysine withketo-oleic acid. A derivative of that compound withm/z 455 and daughter ions withm/z 144 and 296 is also depicted. Scheme (c) explains how a daughter ion with an unsaturated C–Cbond can account for them/z 142 fragment. Scheme (d) shows charge-remote fragmentation, which is commonly seen in MS/MS analysis of fatty acids and their derivatives leading tocreation of unsaturated bonds with a loss of hydrogen. Scheme (e) depicts dehydrogenation and generation of a C=N double bond yieldingm/z 311 and 295 daughter ions.

%

0

100SIR of 475.3 (ES+)

19.98

12.06

9.56

16.30AOP-LMEAOP

12.06

10.66

6.833.68

14.80

17.14

SIR of 493.3 (ES+) AOP-LMEAOP

(min)2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50

10.73

7.04

3.77

14.48

18.25

SIR of 509.3 (ES+) AOP-LMEAOP

8.75

14.48

18.45

AOP-LMEAOP

SIR of 491.3 (ES+)

A

B

C

D

%

0

100

%

0

100

%

0

100

Fig. 6. Them/z 475, 491, 493, and 509 species are unique to the AOP-LME reaction mix-ture. AOP and AOP-LME (equivalent to 10 μg AA) were resolved on a BEH C18 column(1.7 μm, 50×2.1 mm). An acetonitrile gradient (10–90%, by volume in water) was usedwith a total run time of 35 min, including the 100% acetonitrile flush and reequilibra-tion. The eluents were directed to a ZQ single-quadrupole mass spectrometer. Single-ion recordings in ES+mode for m/z 475, 491, 493, and 509 were compared betweenAOP and AOP-LME; only the latter showed peaks with these m/z values. The y axis rep-resents peak intensities relative to the highest peak of (A) 1.83×105, (B) 3.74×105,(C) 3.03×105, and (D) 1.43×105.

1933M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

studies strongly suggest the formation of pyrrole-type adducts withamino groups.

The biologic relevance ofmodified aminophospholipids is supportedby reports showing that oxidized phospholipids (not covalently associ-ated with apoB) are recognized by the scavenger receptor CD36 [21,29]and are also biologically active [30]. Our results with OP-modified PE li-posomes confirm that prosurvival activity requires a relatively smallstructure, namely a small molecular domain containing an aminogroup coupled to an oxidized fatty acid by a pyrrole-type adduct.

Our studies do not identify a unique structural adduct that increasesviability upon modifying apoB. Autoxidized fatty acid preparations areexpected to contain a large number of reactive products includinglipid peroxides, epoxides, 2-alkenals, 4-hydroxyalkenals, and, in thecase of arachidonic acid, other biologically active compounds such asisoprostanes and isolevuglandins [19,31–33]. Fragmented aldehydessuch as HNE and HHE are widely recognized for their ability to modifyproteins and DNA; however, in our hands HNE- or HHE-modified LDLdid not promote survival in macrophages. This is in line with the obser-vation by Fruebis et al. that these hydroxyaldehydes cannot account forthe fluorescence observed with adducts of lysine and linoleic acid oxi-dation products [18]. Similarly, our MS and NMR analyses of OPsshowed no detectable formation of fragmented aldehydes despite oxi-dation of more than 90% of the starting polyunsaturated fatty acid.These results together with susceptibility to NaBH4 and TPP reductionpoints to unfragmented aldehydic products, hydroperoxides, and/or en-doperoxides as the main peroxidation products responsible for confer-ring a prosurvival effect on modified LDL.

The MS study of LOP-LME showed only very low intensity new ionsin the region betweenm/z 250 andm/z 343, where one might expect tofind adducts of lysine methyl ester with fragmented aldehydes derivedfrom linoleic acid hydroperoxide. However, there were several prom-inent new ions in the range m/z 434–505 that could represent lysineadducts with linoleate-derived oxidation products having an intact18-carbon backbone. It has been proposed that linoleic acid-derivedhydroperoxides [18] and keto-oleic acid [26] can modify lysine resi-dues to produce 5-membered pyrrole, lactam, or hydroxylactam(and possibly 6-membered pyridine) heterocycles with fluorescence

100 150 200 250 300 350 400 450 500 550

AOP-LMETOF MSMS 509.30 ES+ 491.2886

473.2778

84.0797455.2651

142.0753 413.2594

330.1889

509.30

510.30m/z

100 150 200 250 300 350 400 450 500

%

0

100 84.0797491.29

473.2859

142.0797

144.0992 330.2024455.2808

492.28413.1804

%

0

100 AOP-LMETOF MSMS 491.30 ES+

Molecular Formula = C27 H44 N2 O7Formula Weight = 508.3

CH3

N

O

O

OH CH3

OH

LME

OH

A

B

CH3

N

O

O

OH CH3

OH

LME+

a

b

c

d

e

f-H O m/z = 491.3

CH3

N

O

O

OH CH3LME+

-2H2O m/z = 473.3

CH3

N

O

O

OH CH3

+

m/z = 330

HNH2+

m/z = 84.1

ONH3

CH2CH3

O

+

m/z = 144

NH2

CH3

N

O

O

OH CH3

+

m/z = 413m/z = 509.3

2

m/z = 509.3

Fig. 7. The m/z 509 and 491 species are isolevuglandin-derived hydroxylactam-containing adducts. (A) MS/MS analysis of AOP-LME (equivalent of 10 μg of AA) acquired on a SYNAPTsystem operating in TOF mode for m/z 509 and 491. Plots represent the pooled data from all the chromatographic peaks of each mass. Intensities are normalized to the highest peak(226 for m/z 491 and 1.42×103 for m/z 509). (B) Molecular weight, empirical formula, and structures of parent ions (m/z 491 and 509) and their fragmentation products. In schemes(a) through (f), the iso[4]LG-derived adduct is used as an example.

1934 M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

spectra similar to what we found. The MS/MS analysis of LOP-LMEadducts is consistent with products suggested by both mechanisms.We postulated that the existence of m/z 142/144 doublet and m/z 312daughter ions is consistent with fragmentation of the hydrocarbon

A

B Formula We ight = 49

CH3

N

O

O

OH CH3

OH

LME

CH3

N

O

O

OH CH3LME

a

b

c

d-H O m/z = 475.3H3

N+

HONH3

CH2CH3

O

+

m/z = 144

NH3

CH3

N

O+

m/z = 415m/z = 493.3

2

m/z = 493.3

100 150 200 250 300 350 400 450 500

%

0

100

%

0

10084.0797

475.30

457.2965

144.0841332.2082

415.2777

493.30

AOP-LMETOF MSMS 493.30 ES+

+2

Fig. 8. The m/z 493 and 475 species are isolevuglandin-derived lactam-containing adducts.tem operating in TOF mode for m/z 493 and 475. Plots represent the pooled data from all th(884 and 119 for m/z 493 and 475, respectively). (B) Molecular weight, empirical formula,iso[4]LG-derived adduct is used as an example for schemes (a) through (d).

chain adjacent to the heterocycle (Fig. 5). However, a fragmentationof LME, just below N of the heterocycle, is also consistent with thesedaughter ions. This is similar to the fragmentation pattern seen inisoLG-LME adducts.

Molecular Formula = C27 H44 N2O62.3

b

C

O

O

OH CH3

m/z = 332

NH2+

m/z = 84.1O

OH CH3

100 150 200 250 300 350 400 450 500 550

84.0831475.31

457.3123

259.1458

144.1036332.2216

439.2922

476.33

m/z

AOP-LMETOF MSMS 475.30 ES+

(A) MS/MS analysis of AOP-LME (equivalent of 10 μg of AA) acquired on a SYNAPT sys-e chromatographic peaks of each mass. Intensities are normalized to the highest peakand structures of parent ions (m/z 475 and 493) and their fragmentation products. An

1935M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

Our LC-MS analysis of AOP-LME revealed species withm/z ratios andfragmentation patterns very consistent with isolevuglandin-derivedlactam and hydroxylactam-containing adducts. A pyrrole-containingadduct of LME with any of the isolevuglandins will have an m/z of 477in positive-ion mode (which was not found in our system) but furtheroxygenation of pyrrole ring produces lactam- (+16, m/z 493) andhydroxylactam- (+32, m/z 509) containing adducts, which werefound in our reaction mixture. This fits very well with previous reportsshowing that, unless air is strictly excluded, all of the lysyl-pyrrole ad-ducts of levuglandins and isolevuglandins will be oxidized to lactamand hydroxylactam [19]. The presence of dehydrated lactamand hydro-xylactam adducts (withm/z 475 and 491, respectively) is similarly con-sistentwith previous reports that lysyl-lactam and lysyl-hydroxylactamcan exist as sodated, protonated, and dehydrated species [34]. The rela-tive intensities of our major daughter ions were similar to studies byBrame et al. [19]. One difference we observed was the existence of them/z 144 daughter ion in both the lactam and the hydroxylactam ad-ducts. This ion was not found by Brame et al., most probably becausethey used lysine (instead of LME) as the amine.

Although there are no reports of fluorescence spectra forisolevuglandin-modified LDL, our chromatographic studies suggest thatlysyl-isolevuglandin adducts could contribute to the fluorescence ofoxLDL, as the excitation/emission maxima are at 350 nm/430 nm,identical to oxLDL, and there is evidence that oxidized LDL containsisoLG-lysyl adducts [34]. It is also known that LG-modified LDL behavessimilar to oxLDL in that it is taken up avidly but degraded poorly bymacrophages. Interestingly, the LG-modified LDL uptake is competitivelyinhibited by excess oxLDL [35], suggesting detection by similar patternrecognition receptors. On the other hand, because linoleic acidautoxidation cannot generate isolevuglandins, it is clear that the isoLG-lysine structure is not essential for generation of fluorescence orincreasing macrophage viability.

Although it is quite possible for autoxidation products of arachidonicacid to proceed with the same chemical reactions proposed for linoleicacid, as explained, the major masses identified in AOP-LME are consis-tent with isoLG-LME adducts. Considering the high reactivity of isoLG's,it is expected that they would modify amine groups before most otheroxidation products. It is still likely that other adducts are present in themixture but have not been identified because of lower intensities. It iswell known that higher numbers of double bonds (triene systems andbeyond) increase the likelihood of producing bicyclic endoperoxidesthat are prostaglandin-like precursors [36] and can generateγ-ketoalde-hydes (such as isoLG's) by a spontaneous cleavage of the endoperoxidemoiety. However, Pryor et al. [36] have proposed amechanismbywhichhydroperoxides of a diene system (e.g., linoleic acid) may produce suchbicyclic endoperoxides. Zhang et al. [37] reported the production of dou-bly allylic dihydroperoxide from linoleic acid using singlet oxygen. Thissuggests that LOP may contain γ-ketoaldehydes with reactivity and be-havior similar to those of isoLG's. However, to our knowledge, there areno reports of unfragmented γ-ketoaldehydes being produced from lino-leic acid autoxidation. Additionally, adducts produced from γ-linolenicacid (18:3) but not linoleic acid are recognized by anti-iso[4]LG-lysineantibody [34], which suggests that prostaglandin-like precursors do notexist in LOP. The structures we propose here for LOP-LME adducts maycontain a pyrrole, lactam, or hydroxylactam core, a feature that is sharedby isoLG-lysine. However, the hydrocarbon side chains in the LOP-LMEadducts are on positions 2 and 5 of the heterocycle, whereas in theisoLG adducts, they are on positions 3 and 4. Salomon et al. [34] haveshown that the length of residues on positions 3 and 4 is themajor deter-minant for recognition by anti-LG-lysine and anti-iso[4]LG-lysine anti-bodies, whereas differences on positions 2 and 5 are not recognized.

In bone marrow-derived macrophages, oxLDL inhibits apoptosis byactivation of the PI3K/PKB axis [10], blocking ceramide generation [11]and inducing calcium oscillations and activation of calcium-dependentkinases such as eF2K [12]. Similarly, in peritoneal macrophages, oxLDLinduces growth by activation of the PI3K/PKB axis [7]. We plan future

studies in our laboratory to determine if oxLDL and OP-LDL use thesame intracellular signaling pathways for inducingmacrophage survivaland to look for specific interacting proteins on the cell surface.

The present findings have potential importance not only for athero-sclerosis but also for any other condition with increased oxidativestress. For example, advanced lipid peroxidation-modified proteinshave been reported to correlate significantly with periodontal disease(inflammatory disease of the gingiva and adjacent soft tissue) [38]and lipid-induced glomerular disease [39]. Our results with adducts ofunfragmented oxidation products demonstrate that they can activateprosurvival pathways in macrophages. Depending on the stage of theplaque, an increase inmacrophage numbers could have beneficial or ad-verse effects on plaque size and stability. Thus reagents that can detectthese unfragmented adducts in vivomight lead to novel screening toolsfor diseases related to lipid peroxidation. Likewise, strategies to modu-late their generation might be of therapeutic value.

Acknowledgments

This workwas supported by the Heart and Stroke Foundation of Brit-ish Columbia and Yukon (HSFBCY to U.P.S. and V.D.) and the CanadianInstitutes of Health Research (GrantMT8630 toU.P.S.).We acknowledgevaluable discussions and advice from Dr. Antonio Gomez-Munoz andDr. Joseph L. Witztum.

Appendix A. Supplementary data

Supplementary data to this article can be found online at doi:10.1016/j.freeradbiomed.2011.08.029.

References

[1] Li, A. C.; Glass, C. K. The macrophage foam cell as a target for therapeutic interven-tion. Nat. Med. 8:1235–1242; 2002.

[2] Moreno, P. R.; Falk, E.; Palacios, I. F.; Newell, J. B.; Fuster, V.; Fallon, J. T. Macro-phage infiltration in acute coronary syndromes: implications for plaque rupture.Circulation 90:775–778; 1994.

[3] Berliner, J. A.; Parhami, F.; Fang, Z. T.; Fogelman, A. M.; Territo, C. Regulation ofmonocyte and neutrophil entry into the vessel wall. Behring Inst. Mitt. 92:87–91; 1993.

[4] Katsuda, S.; Coltrera, M. D.; Ross, R.; Gown, A. M. Human atherosclerosis. IV. Im-munocytochemical analysis of cell activation and proliferation in lesions of youngadults. Am. J. Pathol. 142:1787–1793; 1993.

[5] Rekhter, M. D.; Gordon, D. Active proliferation of different cell types, includinglymphocytes, in human atherosclerotic plaques. Am. J. Pathol. 147:668–677;1995.

[6] Sakai, M.; Miyazaki, A.; Hakamata, H.; Sasaki, T.; Yui, S.; Yamazaki, M.; Shichiri,M.; Horiuchi, S. Lysophosphatidylcholine plays an essential role in the mitogeniceffect of oxidized low density lipoprotein on murine macrophages. J. Biol. Chem.269:31430–31435; 1994.

[7] Martens, J. S.; Reiner, N. E.; Herrera-Velit, P.; Steinbrecher, U. P. Phosphatidylino-sitol 3-kinase is involved in the induction of macrophage growth by oxidized lowdensity lipoprotein. J. Biol. Chem. 273:4915–4920; 1998.

[8] Martens, J. S.; Lougheed, M.; Gomez-Munoz, A.; Steinbrecher, U. P. A modificationof apolipoprotein B accounts for most of the induction of macrophage growth byoxidized low density lipoprotein. J. Biol. Chem. 274:10903–10910; 1999.

[9] Hamilton, J. A.; Myers, D.; Jessup, W.; Cochrane, F.; Byrne, R.; Whitty, G.; Moss, S.Oxidized LDL can induce macrophage survival, DNA synthesis, and enhanced pro-liferative response to CSF-1 and GM-CSF. Arterioscler. Thromb. Vasc. Biol. 19:98–105; 1999.

[10] Hundal, R. S.; Salh, B. S.; Schrader, J.W.; Gomez-Munoz, A.; Duronio, V.; Steinbrecher,U. P. Oxidized low density lipoprotein inhibits macrophage apoptosis through activa-tion of the PI 3-kinase/PKB pathway. J. Lipid Res. 42:1483–1491; 2001.

[11] Hundal, R. S.; Gomez-Munoz, A.; Kong, J. Y.; Salh, B. S.; Marotta, A.; Duronio, V.;Steinbrecher, U. P. Oxidized low density lipoprotein inhibits macrophage apopto-sis by blocking ceramide generation, thereby maintaining protein kinase B activa-tion and Bcl-XL levels. J. Biol. Chem. 278:24399–24408; 2003.

[12] Chen, J. H.; Riazy, M.; Smith, E. M.; Proud, C. G.; Steinbrecher, U. P.; Duronio, V.Oxidized LDL-mediated macrophage survival involves elongation factor-2 kinase.Arterioscler. Thromb. Vasc. Biol. 29:92–98; 2009.

[13] Lougheed, M.; Lum, C. M.; Ling, W.; Suzuki, H.; Kodama, T.; Steinbrecher, U. Highaffinity saturable uptake of oxidized low density lipoprotein by macrophagesfrom mice lacking the scavenger receptor class A type I/II. J. Biol. Chem. 272:12938–12944; 1997.

[14] Steinbrecher, U. P.; Lougheed, M.; Kwan, W. C.; Dirks, M. Recognition of oxidizedlow density lipoprotein by the scavenger receptor of macrophages results from

1936 M. Riazy et al. / Free Radical Biology & Medicine 51 (2011) 1926–1936

derivatization of apolipoprotein B by products of fatty acid peroxidation. J. Biol.Chem. 264:15216–15223; 1989.

[15] Zhang, H.; Yang, Y.; Steinbrecher, U. P. Structural requirements for the binding ofmodified proteins to the scavenger receptor of macrophages. J. Biol. Chem. 268:5535–5542; 1993.

[16] Magil, A. B.; Frohlich, J. J.; Innis, S. M.; Steinbrecher, U. P. Oxidized low-densitylipoprotein in experimental focal glomerulosclerosis. Kidney Int. 43:1243–1250; 1993.

[17] Palinski, W.; Horkko, S.; Miller, E.; Steinbrecher, U. P.; Powell, H. C.; Curtiss, L. K.;Witztum, J. L. Cloning of monoclonal autoantibodies to epitopes of oxidized lipo-proteins from apolipoprotein E-deficient mice: demonstration of epitopes of ox-idized low density lipoprotein in human plasma. J. Clin. Invest. 98:800–814; 1996.

[18] Fruebis, J.; Parthasarathy, S.; Steinberg, D. Evidence for a concerted reaction be-tween lipid hydroperoxides and polypeptides. Proc. Natl. Acad. Sci. U. S. A. 89:10588–10592; 1992.

[19] Brame, C. J.; Salomon, R. G.; Morrow, J. D.; Roberts II, L. J. Identification of ex-tremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the iso-prostane pathway and characterization of their lysyl protein adducts. J. Biol. Chem.274:13139–13146; 1999.

[20] Hazen, S. L.; Hsu, F. F.; Heinecke, J. W. p-Hydroxyphenylacetaldehyde is the majorproduct of L-tyrosineoxidationby activatedhumanphagocytes: a chloride-dependentmechanism for the conversion of free amino acids into reactive aldehydes by myelo-peroxidase. J. Biol. Chem. 271:1861–1867; 1996.

[21] Boullier, A.; Gillotte, K. L.; Horkko, S.; Green, S. R.; Friedman, P.; Dennis, E. A.;Witztum, J. L.; Steinberg, D.; Quehenberger, O. The binding of oxidized low den-sity lipoprotein to mouse CD36 is mediated in part by oxidized phospholipidsthat are associated with both the lipid and protein moieties of the lipoprotein.J. Biol. Chem. 275:9163–9169; 2000.

[22] Steinbrecher, U. P. Oxidation of human low density lipoprotein results in deriva-tization of lysine residues of apolipoprotein B by lipid peroxide decompositionproducts. J. Biol. Chem. 262:3603–3608; 1987.

[23] Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid peroxidation andantioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13:341–390;1992.

[24] Quehenberger, O.; Koller, E.; Jurgens, G.; Esterbauer, H. Investigation of lipid peroxida-tion in human low density lipoprotein. Free. Radic. Res. Commun. 3:233–242; 1987.

[25] DeCaprio, A. P.; Olajos, E. J.; Weber, P. Covalent binding of a neurotoxic n-hexanemetabolite: conversion of primary amines to substituted pyrrole adducts by 2,5-hexanedione. Toxicol. Appl. Pharmacol. 65:440–450; 1982.

[26] Fukuzawa, K.; Kishikawa, K.; Tokumura, A.; Tsukatani, H.; Shibuya, M. Fluorescentpigments by covalent binding of lipid peroxidation by-products to protein andamino acids. Lipids 20:854–861; 1985.

[27] Cheng, C.; Gross, M. L. Applications and mechanisms of charge-remote fragmen-tation. Mass Spectrom. Rev. 19:398–420; 2000.

[28] Lou, X.; Spiering, A. J.; deWaal, B. F.; van Dongen, J. L.; Vekemans, J. A.; Meijer, E. W.Dehydrogenation of tertiary amines in matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry. J. Mass Spectrom. 43:1110–1122; 2008.

[29] Podrez, E. A.; Hoppe, G.; O'Neil, J.; Hoff, H. F. Phospholipids in oxidized LDL notadducted to apoB are recognized by the CD36 scavenger receptor. Free Radic.Biol. Med. 34:356–364; 2003.

[30] Watson, A. D.; Subbanagounder, G.;Welsbie, D. S.; Faull, K. F.; Navab, M.; Jung, M. E.;Fogelman, A. M.; Berliner, J. A. Structural identification of a novel pro-inflammatoryepoxyisoprostane phospholipid in mildly oxidized low density lipoprotein. J. Biol.Chem. 274:24787–24798; 1999.

[31] Nakamura, T. Red pigment-forming substances from autoxidized linolenate:identification of prostaglandin-like substances. Lipids 20:180–186; 1985.

[32] Harvilla, C. M.; Hachey, D. L.; Porter, N. A. Coordination (Ag+) ion spray–massspectrometry of peroxidation products of cholesterol linoleate and cholesterolarachidonate: high-performance liquid chromatography–mass spectrometryanalysis of peroxide products from polyunsaturated lipid autoxidation. JACS122:8042–8055; 2000.

[33] Spiteller, G. Lipid peroxidation in aging and age-dependent diseases. Exp. Geron-tol. 36:1425–1457; 2001.

[34] Salomon, R. G.; Sha, W.; Brame, C.; Kaur, K.; Subbanagounder, G.; O'Neil, J.; Hoff,H. F.; Roberts II, L. J. Protein adducts of iso[4]levuglandin E2, a product of the iso-prostane pathway, in oxidized low density lipoprotein. J. Biol. Chem. 274:20271–20280; 1999.

[35] Hoppe, G.; Subbanagounder, G.; O'Neil, J.; Salomon, R. G.; Hoff, H. F.Macrophage rec-ognition of LDL modified by levuglandin E2, an oxidation product of arachidonicacid. Biochim. Biophys. Acta 1344:1–5; 1997.

[36] Pryor, W. A.; Stanley, J. P.; Blair, E. Autoxidation of polyunsaturated fatty acids. II.A suggested mechanism for the formation of TBA-reactive materials fromprostaglandin-like endoperoxides. Lipids 11:370–379; 1976.

[37] Zhang, W.; Sun, M.; Salomon, R. G. Preparative singlet oxygenation of linole-ate provides doubly allylic dihydroperoxides: putative intermediates in thegeneration of biologically active aldehydes in vivo. J. Org. Chem. 71:5607–5615; 2006.

[38] Su,H.; Gornitsky,M.; Velly, A.M.; Yu,H.; Benarroch,M.; Schipper, H.M. Salivary DNA,lipid and protein oxidation in nonsmokers with periodontal disease. Free Radic. Biol.Med. 46:914–921; 2009.

[39] Iacobini, C.; Menini, S.; Ricci, C.; Scipioni, A.; Sansoni, V.; Mazzitelli, G.; Cordone,S.; Pesce, C.; Pugliese, F.; Pricci, F.; Pugliese, G. Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptor-mediatedmechanisms. J. Pathol. 218:360–369; 2009.