Proc. Natl. Acad. Sci. USAVol. 90, pp. 8742-8746, September 1993Biochemnstry

Immunochemical detection of 4-hydroxynonenal protein adducts inoxidized hepatocytesKoJi UCHIDA*, LUKE I. SZWEDA, Ho-ZOON CHAE, AND EARL R. STADTMANtLaboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Contributed by Earl R. Stadtman, June 11, 1993

ABSTRACT We report here the development of an immu-nochemical procedure that uses an antibody specific to the4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specflc antibody was prepared byimmuning rabbits with a HNE-keyhol limpet hemocyaninconjugate and purifying the rabbit serum on an affnity gelprepared by covalent attachment of a HNE-conjugated hep-tapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent ofHNE-histidine residues per subunit were obtained by ilncubat-ing samples of glyceraldehyde-3-phosphate dehydrogenasewith inreased amounts ofHNE and subjected to immunoblot-ting with the HNE-specific antibody, the intensities of the blotswere directly proportional to the number of HNE-hlstidineadducts as measured dirdy by amino add analysis. Bindingof the HNE-conjugated glyceraldehyde-3-phosphate dehydro-genase to the HNE-specific antibody could be completelyinhibited by HNE-N-acetylhlstidine, HNE-N-acetyllysine, orHNE-glutathlone, suggesting that the antigenic determinntrecognized by the antibody Is the HNE moiety, not the HNE-amino acid conjugates, such as ENE-histidine, HNE-Iysine,and HNE-cysteine. The utiliy of the HNE-specific antibodywas demonstrated by its ability to react selectively with anumber of HNE-protein adducts in Immunoblot analyses ofcrude homogenates of rat liver hepatocytes that had beenexposed to HNE or oxidative stresses with tert-butylhydroper-oxide or metal-ion-catalyzed oxidation systems.

4-Hydroxynonenal (HNE), a major aldehydic product oflipidperoxidation (1-3), is believed to be largely responsible forcytopathological effects observed during oxidative stress (4).It has been proposed that HNE exerts these effects becauseof its facile reactivity with biological materials, particularlysulfhydryl groups of proteins (5). The reaction ofHNE withsulfhydryl groups leads to the formation of thioether adductsthat further undergo cyclization to form hemiacetals (4, 6).HNE also reacts with histidine and lysine residues ofproteinsto form stable Michael addition-type adducts (Fig. 1, refs.7-9). It is therefore crucial to develop sensitive and specificmethods for the detection of HNE-modified protein.A procedure has been developed to detect HNE-histidine

and -lysine adducts by amino acid analysis after stabilizationwith NaBH4 (7, 8). This method does not, however, allow forthe direct characterization of proteins modified by HNE. Inthe present paper, we describe the preparation of antibodiesthat specifically recognize HNE-protein adducts. The abilityof these antibodies to detect HNE derivatives of proteins inrat liver hepatocytes after treatment with HNE or exposureto oxygen free-radical-generating systems is described.

MATERIALS AND METHODSMaterials. The stock solution of trans-4-hydroxy-2-

nonenal was prepared by acid treatment (1 mM HCl) ofHNE

Tthe publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

diethylacetal, which was synthesized according to the pro-cedure of De Montarby et al. (10). The concentration of theHNE stock solution was determined by measurement ofUVabsorbance at 224 nm (11). Keyhole limpet hemocyanin(KLH) and 1-3-(3-dimethylaminopropyl)carbodiimide conju-gation kits were obtained from Pierce. Horseradish peroxi-dase-linked anti-rabbit IgG immunoglobulin, 125I-labeled pro-tein A (35 mCi/mg; 1 Ci = 37 GBq), and enhanced chemilu-minescence (ECL) immunoblotting detection reagents wereobtained from Amersham. Affi-Gel 10 was obtained fromBio-Rad. Na-Acetyl-L-histidine, Na-acetyl-L-lysine, glu-tathione, bovine serum albumin (BSA), tert-butylhydroper-oxide (t-BHP), and collagenase (type IV) were obtained fromSigma. Ala3-His-Ala3 methyl ester was obtained from Re-search Genetics (Huntsville, AL). Rabbit muscle glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) was obtainedfrom Calbiochem. 2-Thiobarbituric acid and malonaldehydebis(dimethyl acetal) were obtained from Baker and Aldrich,respectively. The protein concentration was measured byusing the BCA protein assay reagent obtained from Pierce.RNE-GAPDH Adducts. To generate HNE-GAPDH ad-

ducts with different degrees of modification, GAPDH (1 mg)was treated with variable HNE concentrations ranging from0 to 2mM in 1 ml of50mM sodium phosphate buffer, pH 7.2.After incubation for 2 hr at 37°C, the mixtures were dividedinto two portions. One portion was treated with NaBH4, andthe HNE-histidine content in the reduced HNE-GAPDHadducts was determined by amino acid analysis, as described(8, 9). The other portion was treated with Laemmli samplebuffer (12) at 100°C for immunoblot analysis.

Hepatocyte Isolation. Hepatocytes were isolated by thecollagenase method of Seglen (13) from 6-month-old Fischer344/Brown Norway rats. Hepatocytes were suspended in 10mM Hepes buffer, pH 7.4/137 mM NaCl/4.6 mM KCl/1.1mM KH2PO4/0.6 mM MgSO4 and frozen at -70°C at aprotein concentration of -40 mg/ml for later use.

Modification of Hepatocytes. HNE-modified hepatocyteswere prepared by incubation of hepatocytes (1.7 mg/ml) withvarious concentrations (0-1.0 mM) ofHNE in 0.5 ml of50mMMops, pH 7.2, for 2 hr at 37°C. Lipid peroxidation of hepa-tocytes was routinely performed by incubation hepatocytes(1.7 mg/ml) with iron-dependent free-radical-generating sys-tems [iron/ascorbate, iron/15-hydroperoxyeicosatetraenoicacid (15-HPETE), iron/ascorbate/15-HPETE], or with I-BHP0.5 ml of 50 mM Mops, pH 7.2. All reagents were freshlyprepared just before incubation. After addition of HNE oroxidants, samples were shaken at 37°C for the durationsindicated in the figure legends.

Abbreviations: HNE, 4-hydroxynonenal; KLH, keyhole limpethemocyanin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;BSA, bovine serum albumin; 15-HPETE, 15-hydroperoxyeicosatet-raenoic acid; t-BHP, tert-butylhydroperoxide.*Present address: Department of Food Science and Technology,School of Agriculture, Nagoya University, Nagoya 464-01, Japan.tTo whom reprint requests should be addressed at: National Insti-tutes of Health, 9000 Rockville Pike, Building 3, Room 222, Be-thesda, MD 20892.

8742

Proc. Natl. Acad. Sci. USA 90 (1993) 8743

A

B

C

NH-

OH

NH-

A C-0

OH

FIG. 1. Structures of the Michael addition-type HNE adducts.(A) HNE-istidine. (B) HNE4ysine. (C) HNE-cysteine.

Lipid Peroxidation Assay. Lipid peroxidation of hepato-cytes was determined in terms of 2-thiobarbituric acid-reactive substances according to the methods ofMasaki et al.

(14). Hepatocytes incubated with and without oxidant in 0.1ml of reaction mixtures were treated with 0.5 ml of 2.8%(wt/vol) trichloroacetic acid and 0.5 ml of 1% 2-thiobarbituricacid/0.05 M NaOH and then boiled for 20 min. After cooling,the sample was centrifuged (11,000 x g, 3 min), and theabsorbance of the supernatant was measured at 534 nm.Malonaldehyde bis(dimethyl acetal), which yields malon-aldehyde (MDA) with acid treatment, was used as a standard.

Preparation of Ala3-H-Alsa3 Heptapeptide ConningHNE-Hstldine Adduct. The reaction mixture contained 5 mgofpeptide and6mMHNE in 1 mlof50mM sodium phosphatebuffer, pH 7.2. After incubation for 20 hr at 37°(, HNE-modified peptide was isolated on a TSK-Gel octadecylsilylsilica-80TM column (TOSO HAAS, 0.46 x 25 cm) by apply-ing a linear gradient of 0-20% acetonitrile (vol/vol) in 0.05%trifluoroacetic acid for 20 min. The HNE-histidine adduct inthe isolated peptide was identified by amino acid analysis.

Antibody Preparation. The HNE-modified KLH (HNE-KLH) immunogen was prepared by reaction of 5 mg of KLHwith 8.7mM HNE in 1 ml of50mM sodium phosphate buffer,pH 7.2, for 2 hr at 37°C. The yield of HNE-histidine adductgenerated in the HNE-KLH was 272.4 nmol/mg ofprotein asdetermined by amino acid analysis.HNE-modifled KLH (1 mg/ml) was emulsified in the same

volume of Freund's complete adjuvant and incubated intra-dermally into several sites in New Zealand White rabbits.After 4, 7, and 11 weeks, s.c. booster injections in incompleteFreund's adjuvant were repeated. Antibody response wasmonitored by immunoblots using HNE-modified BSA as theantigen.

Antibodies were partially purified by affinity chromatog-raphy using a HNE-histidyl peptide column. Affi-Gel 10 wasderivatized by incubation of the gel slurry with HNE-conjugated Ala3-His-Ala3 methyl ester in 0.1 M Hepes, pH8.0, for 20 hr at 40C. The antiserum (2 ml) was diluted to 20ml with 0.1 M Hepes buffer, pH 8.0, and passed three timesover a column containing 1 ml of affinity resin. Unboundproteins were washed with 5 ml of 0.1 M Hepes buffer, pH8.0, followed by 5 ml of 100 mM NaCl, and bound antibodieswere eluted with 5 ml of 0.1M glycine, pH 2.5. The eluate was

neutralized immediately with 1 M Tris-HCl, pH 8.0, andstored at -70°C.ELISA. A coating antigen was prepared by incubating 1 mg

of GAPDH with 2 mM HNE in 1 ml of 50 mM sodiumphosphate buffer, pH 7.2, for 2 hr at 37C. The reaction wasquenched by addition of 100 ,1 of 10 mM N-acetylcysteinefollowed by addition of 800 jl of 8 M guanidine hydrochlo-ride/133 mM Tris/13 mM EDTA, pH 7.2, to obtain a homo-geneous protein solution. A 100-pl aliquot of the antigensolution was added to each well of a 96-well microtiter plateand incubated for 20 hr at 4°(. The antigen solution wasremoved, and the plate was washed with Tris buffer saline(TBS) containing 10% Tween 20 (TBS/Tween). Each wellwas incubated with 200 ,u of 1% BSA in Tris/Tween for 30min at 370C in a moist chamber to block the unsaturatedplastic surface. The plate was then washed once with TBS/Tween.The haptens for HNE-specific antibody were prepared by

incubating Na-acetylhistidine, Na-acetyllysine, and glu-tathione with 8 mMHNE in 50mM sodium phosphate buffer,pH 7.2, for 24 hr at 37°C. The adducts were purified byreversed-phase HPLC on a Develosil octadecylsilyl silica-5column (8 x 25 cm): HNE-N-acetylhistidine and HNE-glutathione were eluted with 25% acetonitrile in 0.1% tin-fluoroacetic acid, and HNE-N-acetyllysine was eluted with20% (vol/vol) acetonitrile in 0.1% trifluoroacetic acid at aflow rate of 2.5 ml/min. The adducts were chemically char-acterized by fast atom bombardment-mass spectrometry. Ahapten was incubated with HNE-specific antibody at 40C for20 hr to yield hapten/antibody mixtures containing antibodyat 50 ng/ml and variable concentrations of hapten. A 100-plaliquot of the hapten was added to each well and incubatedfor 1 hr at 37°C. After discarding the supernatants andwashing three times with Tris/Tween, a 100-,4 aliquot of a 5x 104 dilution of goat anti-rabbit IgG conjugated to horse-radish peroxidase in TBS/Tween was added. After incuba-tion for 1 hr at 3rC, the supernatant was discarded, and theplates were washed three times with TBS/Tween. Enzyme-linked antibody bound to the well was observed by adding a10-A4 aliquot of 1,2-phenylenediamine in 0.1 M citrate/phosphate buffer, pH 5.0, and 0.003% H202 to each well. Thereaction was terminated by addition of 50 1d of 2 M sulfuricacid, and absorbance at 490 was read on a micro-ELISA platereader. Results were expressed as B/BO, calculated as [ex-perimental OD - background OD (no antibody)]/[total OD(no competitor) - background OD].Immunoblot Analysis. For estimation of HNE-protein ad-

ducts in the homogenates of hepatocytes treated with HNEor oxidants, proteins were precipitated with 109o trichloro-acetic acid, rinsed twice with acetone, and treated withLaemmli sample buffer (12) for 3-5 min at 1000(. Protein wasthen separated in duplicate on SDS/10%6PAGE gels. One gelwas stained with Coomassie brilliant blue, and the other wasused for immunoblot analysis. Proteins were transferred tonitrocellulose membranes, incubated with 2% BSA in TBS/Tween for blocking, washed, and treated with HNE-specificantibody (2 ug/ml). This procedure was followed by theaddition of horseradish peroxidase conjugated to goat anti-rabbit IgG immunoglobulin and ECL reagents. The bandswere visualized by autoradiography. Alternatively immunecomplexes were quantified with 125I-labeled protein A usinga Phospholmager (Molecular Dynamics).

RESULTSAntibodies Specific to the HNE Moiety. GAPDH treated

with HNE was used to assess the ability of anti-HNE-KLHantibody to detect HNE-modified protein. When GAPDHwas incubated (2 hr, 37°C) with HNE, there was a progressiveincrease (from 0 to 7 mol/mol) in the number of HNE-

Biochemistry: Uchida et aL

8744 Biochemistry: Uchida et al.

histidine adducts formed as the concentration ofHNE in thereaction mixture was varied from 0 to 2 mM. This result wasaccompanied by a progressive increase in the reactivity oftheprotein with anti-HNE-KLH antibody (Fig. 2A). Antibodyreactivity appeared proportional to the yield of HNE-histidine adduct (Fig. 2B). Fig. 3 shows that the HNE adductsof N-acetylhistidine, N-acetyllysine, and glutathione can allinhibit the binding of antibody to HNE-modified GADPH. Ina control study the non-HNE-derivatized forms of thesecompounds exhibited no effect (data not shown).

Determination of HNE-Proteins in HNE-Treated Hepato-cytes. To determine whether the antibody could detect HNEadducts in tissue proteins, proteins from rat liver hepatocytestreated with various concentrations (0-1.0 mM) ofHNE weresubjected to immunoblot analysis with the HNE antibody.The immunoreactivity of the various protein bands clearlydepended upon the HNE concentration used (Fig. 4B): Littleor no immunoreactivity was detected with proteins from thecells treated with 0-1.0 ,uM HNE (lanes 1 and 2); cells treatedwith 10 pLM HNE revealed a 48-kDa protein that exhibited astrong reactivity with the antibody (lane 3); and cells treatedwith 100 and 1000 IuM HNE produced multiple proteins withstrong immunoreactivity (lanes 4 and 5). Treatment of thecells with increased concentrations of HNE up to 1000 ,uMdid not affect the pattern of electrophoretic mobilities ofproteins detected by Coomassie blue staining (Fig. 4A).

In addition to HNE, several other highly reactive aide-hydes produced during lipid peroxidation can form covalentbonds with proteins (4). Among these, malonaldehyde, amajor product of arachidonic and linoleic acid oxidation, hasreceived much attention because it reacts readily with lysineresidues of proteins (15). In contrast to results obtained withHNE-treated hepatocytes, the protein from malonaldehyde-treated hepatocytes exhibited no cross-reactivity with the

A HNE (mM)0 0.1 0.2 0.4 1 2

B >

C._._

um

0._

6._0

m

700-

600

500-

400 -

0300-

200-

100 ;

0 4

0 1 2 3 4 5 6 7

HNE-histidine adduct(mol/mol subunit)

FIG. 2. Stoichiometry of the yield of HNE-histidine adduct andreactivity of anti-HNE-KLH antibody with HNE-modified protein.(A) HNE-modified GAPDH with antibody. (B) Correlation betweenyield of HNE-histidine adduct and reactivity of antibody withHNE-modified GAPDH. GAPDH (1 mg) in 1 ml of 50 mM sodiumphosphate buffer, pH 7.2, was treated with HNE (0-2 mM) for 2 hrat 3rC. After incubation, the mixtures were divided into twoaliquots. One aliquot was treated with NaBH4, and the yield ofHNE-histidine adduct was determined by amino acid analysis.Another aliquot was treated with Laemmli sample buffer at 100°C for3-5 min, and proteins (0.5 pg) were loaded on SDS/12% PAGE slabgels. The gels were transblotted to nitrocellulose membranes andtreated with antibody (2 ug/ml). Membranes were then washed andtreated with 125I-labeled protein A for determination ofimmunoglob-ulin bound to HNE-histidine adduct generated in the HNE-modifiedGAPDH.

0mm

1.0-

0.8

0.6-

0.4-

0.2-

o.0o .10 6 10o5 Io4 1i03 10-2 10O1 100

Competitor (mM)

FiG. 3. ELISA competition curves for anti-HNE-KLH antibody.Assays were done as described and used HNE-modified GAPDH asthe absorbed antigens. Number on abscissa indicates the concentra-tion ofcompetitors when antibody was preincubated with competitorsat 4°C for 20 hr. Competitors were as follows: *, HNE-N-acetylhistidine; A, HNE-N-acetyllysine; *, HNE-glutathione; o,N-acetylhistidine; t, N-acetyliysine; o, glutathione. B/Bo, calculatedas [experimental OD - background OD (no antibody)]/[total OD (nocompetitor) - background OD].

HNE-KLH antibody as judged by immunoblotting tech-niques (data not shown).

Detection ofEiNE-Proteins in Oxidized Hepatocytes. Becauseorganic hydroperoxides such as t-BHP can induce lipid per-oxidation and membrane damage in cultured hepatocytes (16),we examined the ability of t-BHP to provoke formation ofHNE epitopes of the cellular proteins. Incubation of hepato-cytes with 1 mM t-BHP (2 hr; 37C) markedly increased theoxidation of endogenous lipids to yield malonaldehyde (12.5nmol/mg of protein), as determined by the production ofthiobarbituric acid-reactive substances. Coomassie blue stain-ing of the protein bands obtained upon SDS/PAGE analysisshowed little difference between the t-BHP-treated samplesand untreated controls, except, perhaps, that there weresignifcantly more high-molecular-mass proteins in oxidizedsamples (Fig. 5A). Nevertheless, proteins from the t-BHP-treated hepatocytes contained significantly more HNEepitopes than the untreated controls (Fig. SB). A similarincrease in the level of HNE epitopes was seen in the protein

A1 2 3 4 5

KDa97-66-

45-

31-

21-

B1 2 3 4 5

KDa97-

66-

45-

31 -

21-

FIG. 4. SDS/PAGE and subsequent immunoblot analysis ofHNE-modified hepatocyte proteins. (A) SDS/PAGE. (B) Immuno-blot. Hepatocytes (1.7 mg/ml) were treated with HNE [0mM (lanes1), 10-3 mM (lanes 2), 10-2 mM (lanes 3), 10-1 mM (lanes 4), 1 mM(lanes 5)] in 0.5 ml of 50 mM Mops buffer, pH 7.2, for 2 hr at 3rC.Total proteins (30 ug) from HNE-treated hepatocytes were loaded onSDS/PAGE and transblotted to nitrocellulose membrane. Subse-quently, the membrane was incubated with the antibody (2 i.g/ml).

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 8745

A1 2

.... "I, KDa

..... 97-

66-

B1 2

...4:.: ....f'

H.;45 -

21- .::

21 :::

preparation used in these experiments exhibits a low level ofbinding to a number of proteins in the hepatocyte extracts.This is a characteristic of most polyclonal antibodies andlikely reflects nonspecific binding of the antibodies to one ormore ofa multiplicity ofepitopes present in most proteins. Toexamine the specificity of the antibody reaction, proteinsfrom untreated hepatocytes and from hepatocytes that hadbeen treated (oxidized) with t-BHP were subjected to immu-noblot analysis in both the absence and presence of acompeting Gly3-HNE-His-Gly3 methyl ester. In the presenceof 10 ,uM HNE-peptide competitor, the immunoblottingpatterns of the unoxidized and oxidized samples are almostidentical and are indistinguishable from the pattern obtainedfor the control sample in the absence of inhibitor (data notshown).

FIG. 5. SDS/PAGE and subsequent immunoblot analysis ofprotein fractions from hepatocytes treated with t-BHP. (A) SDS/PAGE. (B) Immunoblot. Hepatocytes (1.7 mg/ml) were treated with1 mM t-BHP (lanes 2) in 0.5 ml of 50 mM Mops buffer, pH 7.2, for2 hr at 37°C. Total proteins (100 pg) from control (lanes 1) andoxidized (lanes 2) hepatocytes were loaded on SDS/PAGE andtransblotted to nitrocellulose membrane. Subsequently, the mem-brane was incubated with the antibody (2 pg/ml).

from hepatocytes exposed to various iron-dependent free-radical-generating systems-i.e., systems composed of eitheriron/ascorbate, iron/15-HPETE, or iron/ascorbate/15-HPETE (Fig. 6). It is generally accepted thatHNE is producedmainly as a product of oxidation of arachidonic acid inreactions catalyzed by metal ions (17), in which 15-HPETEmay be an intermediate.

It is evident upon examination of the immunoblots of thecontrol (untreated) samples in the HNE experiments (Fig.4B), the t-BHP experiment (Fig. SB), and iron-dependentfree-radical experiment (Fig. 6B) that the polyclonal antibody

A

KDa97-

66-

1 2 3 4 5 6123456

KDa97-

B

1 2 3 4 5 6,*,it A t .

66-

45-45-

31-

21- 31-

21-_ ..r... *

FIG. 6. SDS/PAGE and subsequent immunoblot analysis ofprotein fractions from hepatocytes treated with iron-dependent free-radical-generating systems. (A) SDS/PAGE. (B) Immunoblot. He-patocytes (1.7 mg/ml) were treated with iron-dependent free-radical-generating systems in 0.2 ml of 50mM Mops buffer, pH 7.2, for 3 hrat 37C. Total proteins (100 mg) from control (lanes 1) and hepato-cytes oxidized by treatment with iron/ascorbate (lanes 2), iron/15-HPETE (0.75 pM) (lanes 3), iron/15-HPETE (1.5 pM) (lanes 4),iron/ascorbate/15-HPETE (0.75 pM) (lanes 5), or iron/ascorbate/15-HPETE (1.5 pM) (lanes 6) were loaded on SDS/PAGE andtransblotted to nitrocellulose membrane. Subsequently, the mem-brane was incubated with the antibody (2 pg/ml). Final concentra-tions ofiron, ascorbate, and 15-HPETE were 50 pM, 1 mM, and 0.75JAM (or 1.5 pM), respectively.

DISCUSSIONOur study shows that immunization of rabbits with HNE-KLH conjugate leads to the production of anti-HNE-specificantibodies. The amount of antibody that binds specifically toHNE-modified GAPDH is proportional to the number ofHNE-histidine epitopes present (Fig. 2). The fact thatHNE-glutathione, HNE-N-acetyllysine, HNE-N-acetylhistidine(Fig. 3), and HNE-histidine-modified heptapeptide can allcompete for the binding ofantibodies to HNE-treated proteinindicates that the HNE moiety of the amino acid conjugate isa common antigenic determinant. It is evident from theresults presented here that antibody raised to HNE-treatedKLH can be used for the sensitive and specific detection ofHNE-modified protein in cell preparations. The fact thatiron-dependent free-radical-generating systems and t-BHPcan provoke the generation of HNE-protein adducts in ratliver hepatocytes shows that these oxidation systems facili-tate the oxidation of endogenous polyunsaturated fatty acidsto HNE.Treatment of rat liver hepatocytes with a low concentration

(10 ,uM) ofHNE leads to the highly selective modification ofa single protein of =48 kDa (Fig. 4B, lane 3). This resultinvites speculation that this protein could be a critical targetfor HNE cytotoxicity or, to the contrary, that it may be aspecific scavenger of HNE and, therefore, protect cellsagainst lipid peroxide-mediated toxicity. Clearly, the identi-fication of this protein and a determination of the biologicalconsequences of its interaction with HNE merit immediateattention. The failure ofextremely low concentrations (1 AM)ofHNE (Fig. 4, lane 2) or low levels of lipid peroxidation togenerate HNE adducts in hepatocyte proteins may be fromthe protective action of glutathione, which is widely distrib-uted in cells. The ability ofHNE to undergo rapid nonenzy-mic Michael addition-type ofreaction with glutathione is wellestablished (1, 5). Moreover, this spontaneous conjugation ofHNE to glutathione can be enhanced by at least two ordersof magnitude by rat liver cytosolic glutathione-S-transferase(18-20). Indeed, the exposure of hepatocytes to HNE leadsto a rapid loss of endogenous glutathione (21). In addition, ithas been noted that the reduction of the aldehyde moiety ofHNE to the corresponding hydroxy derivative by the NADH-dependent liver alcohol dehydrogenase would convert it to arelatively unreactive form (21, 22).

In a recent paper Esterbauer and coworkers (23) demon-strated that antiserum raised against HNE-modified lowdensity lipoprotein recognizes various proteins treated withHNE in vitro. The epitopes recognized by this antiserum maybe similar to those for the antibody described in this paper.In fact, antibody raised against HNE-modified KLH hasrevealed clear cross-reactivity with oxidized low densitylipoprotein (K.U. and E.R.S., unpublished data).

We thank Dr. L. Tsai of this laboratory for the synthesis ofHNE

KDa

97-

66 -

45

31-

21-

Biochemistry: Uchida et aL

8746 Biochemistry: Uchida et al.

diethylacetal. We also thank Dr. Tsukasa Matsuda of NagoyaUniversity (School of Agriculture) for his helpful advice and discus-sions concerning the ELISA study.

1. Esterbauer, H., Cheeseman, K. H., Dianzani, M. U., Poli, G.& Slater, T. F. (1982) Biochem. J. 206, 129-140.

2. Benedetti, A., Comporti, M. & Esterbauer, H. (1980) Biochim.Biophys. Acta 620, 281-296.

3. Benedetti, A., Pompella, A., Fulceri, R., Ramani, A. & Com-porti, M. (1986) Biochim. Biophys. Acta 876, 658-666.

4. Esterbauer, H., Schaur, R. J. &Zollner, H. (1991)FreeRadicalBiol. Med. 11, 81-128.

5. Esterbauer, H., Zollner, H. & Scholz, N. (1975)Z. Naturforsch.C 30, 466-473.

6. Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp.67, 225-244.

7. Uchida, K. & Stadtman, E. R. (1992) Proc. Natl. Acad. Sci.USA 89, 4544-4548.

8. Szweda, L. I., Uchida, K., Tsai, L. & Stadtman, E. R. (1993)J. Biol. Chem. 268, 3342-3347.

9. Uchida, K. & Stadtman, E. R. (1993) J. Biol. Chem. 268,6388-6393.

10. De Montarby, L., Mosset, P. & Gree, R. (1988) TetrahedronLett. 29, 3895.

Proc. Natl. Acad. Sci. USA 90 (1993)

11.

12.13.14.

15.16.

17.

18.

19.

20.

21.

22.

23.

Schauenstein, E., Taufer, M., Esterbauer, H., Kylianek, A. &Seelich, T. (1971) Monatsh. Chem. 102, 517-529.Laemmli, U. K. (1970) Nature (London) 227, 680-685.Seglen, P. 0. (1976) Methods Cell. Biol. 13, 29-83.Masaki, N., Kyle, M. E. & Farber, J. L. (1989)Arch. Biochem.Biophys. 269, 390-399.Chio, K. S. & Tappel, A. L. (1969) Biochemistry 8, 2827-2832.Von Ruecker, A. A., Han-Jeon, B. G., Wild, M. & Bidling-maier, F. (1989) Biochem. Biophys. Res. Commun. 163, 836-842.Prior, W. A. & Porter, N. A. (1990) Free Radical Biol. Med. 8,541-543.Jensson, H., Guthenberg, C., Alin, P. & Mannervik, B. (1986)FEBS Lett. 203, 207-209.Alin, P., Danielson, U. H. & Mannervik, B. (1985) FEBS Lett.179, 267-270.Danielson, U. H., Esterbauer, H. & Mannervik, B. (1987)Biochem. J. 247, 707-713.Cadenas, E., Muller, A., Brigelius, R., Esterbauer, H. & Sies,H. (1983) Biochem. J. 214, 479-487.Sellin, S., Holmquist, B., Mannervik, B. & Vallee, B. (1991)Biochemistry 30, 2514-2518.Chen, Q., Esterbauer, H. & Jurgens, G. (1992) Biochem. J. 288,249-254.